**The Essential Role of anxA2 in Langerhans Cell Birbeck Granules Formation**

### **Shantae M. Thornton 1, Varsha D. Samararatne 1, Joseph G. Skeate 1, Christopher Buser 2, Kim P. Lühen 3, Julia R. Taylor 1, Diane M. Da Silva 3,4 and W. Martin Kast 1,3,4,\***


Received: 5 March 2020; Accepted: 12 April 2020; Published: 15 April 2020

**Abstract:** Langerhans cells (LC) are the resident antigen presenting cells of the mucosal epithelium and play an essential role in initiating immune responses. LC are the only cells in the body to contain Birbeck granules (BG), which are unique cytoplasmic organelles comprised of c-type lectin langerin. Studies of BG have historically focused on morphological characterizations, but BG have also been implicated in viral antigen processing which suggests that they can serve a function in antiviral immunity. This study focused on investigating proteins that could be involved in BG formation to further characterize their structure using transmission electron microscopy (TEM). Here, we report a critical role for the protein annexin A2 (anxA2) in the proper formation of BG structures. When anxA2 expression is downregulated, langerin expression decreases, cytoplasmic BG are nearly ablated, and the presence of malformed BG-like structures increases. Furthermore, in the absence of anxA2, we found langerin was no longer localized to BG or BG-like structures. Taken together, these results indicate an essential role for anxA2 in facilitating the proper formation of BG.

**Keywords:** anxA2; Birbeck granules; Langerhans cell; A2t

### **1. Introduction**

Birbeck granules (BG) are cytoplasmic organelles which resemble tennis rackets in two-dimensional (2D) cross-sections. Since their discovery nearly 60 years ago, BG have largely remained elusive in derivation, composition, and function [1]. BG exist in a wide variety of morphologies when imaged through transmission electron microscopy (TEM), however, characteristic images typically contain a translucent "head" portion attached to a "rod" containing 5 to 10 nm linear striations through its center [2]. Three-dimensionally, BG "rods" are composed of two adjacent, superimposed zippered membranes forming a flat, circular disk or cytomembrane sandwiching structure (CMS) connected to the "head", a vesicular lobe on the outer edge [3,4].

BG are the hallmark structures found in Langerhans cells (LC), which are the antigen presenting cells (APC) of the mucosal epithelium [5]. LC are highly specialized cells of the innate immune system which have the primary function of sampling their environment for foreign antigens [6]. Langerin (CD207), a type-II transmembrane c-type lectin, is the primary protein comprising BG and is a requisite for formation [7,8]. Collective evidence has demonstrated a function for langerin and BG in antigen binding, uptake, and processing through a nonclassical pathway [9–12]. In this process, langerin acts as a cell surface pattern recognition receptor (PRR) and is trafficked through the endosomal recycling compartment (ERC) under the control of Rab11a where it accumulates in recycling endosomes (RE) [13–16]. Once a critical intracellular langerin concentration is reached in the RE, a budding event facilitated by the Rab11a/myosin Vb/Rab11-FIP2 complex induces BG formation through membrane superimposition and a zippering of langerin interactions at the carbohydrate recognition domains (CRD) [4,5,7,17]. Cytoplasmic BG are proposed to process antigens for presentation via CD1a, an MHC-class I-like protein, to initiate the adaptive immune response through T cell activation [18].

Once a foreign antigen is recognized, LC undergo phenotypic and functional changes to become mature. This is characterized by an upregulation of co-stimulatory molecules such as CD80 and CD86, secretion of proinflammatory cytokines, and chemokine-mediated migration to lymph nodes where antigen-specific T cell priming occurs. LC are often the first immune cells to be in contact with pathogens that target or must bypass the mucosal epithelium such as human immunodeficiency virus (HIV) and human papillomavirus (HPV). BG structures sequester HIV and prevent its dissemination through selective degradation [10]. When exposed to HPV capsids, LC fail to mount a proper immune response. There is delayed maturation, a reduced level of MHC surface expression, and little to no costimulatory signals, which results in improper T cell priming [19]. We previously reported that this manipulation of LC by HPV is facilitated by the interaction with the annexin A2 S100A10 heterotetramer (A2t) [20]. Further investigation showed that A2t was directly involved in infectious trafficking of HPV virions [21]. In exploring the role of A2t in HPV–LC interactions, we made the interesting observation that BG structures were impacted by modulation of A2t expression, warranting further investigation into the relationship between A2t and BG formation.

### **2. Materials and Methods**

### *2.1. Cell Culture*

The CD34+ human acute myeloid leukemia cell line, MUTZ-3, was gifted by Rik J. Scheper from the VU Medical Center in Amsterdam, The Netherlands. MUTZ-3 cells were cultured at a density of <sup>2</sup> <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL in minimum essential media, Alpha 1X (MEMα) with Earle's salts, ribonucleosides, deoxyribonucleosides, and L-glutamine (Corning, NY, USA) supplemented with 20% heat-inactivated human fetal bovine serum (Omega Scientific, Tarzana, CA, USA), 10% conditioned medium from the renal carcinoma cell line 5637, and 50 μM 2-mercaptoethanol (Thermo Fisher, Carlsbad, CA, USA) at 37 ◦C with 5% CO2. To induce a Langerhans cell phenotype, MUTZ-3 cells were seeded at a density of <sup>1</sup> <sup>×</sup> 105 cells/mL and were cultured for 14 days in the medium conditions described above. On days 0, 4, and 8, the cells were treated with a cytokine regimen containing 100 ng/mL GM-CSF (Sanofi, Bridgewater, NJ, USA), 2.5 ng/mL TNFα (PeproTech, Rocky Hill, NJ, USA), and 10 ng/mL human TGFβ (Thermo Fisher). Medium was replenished on day 8 of differentiation.

To generate 5637 conditioned medium, 5637 cells were cultured in RMPI 1640, 1X with L-glutamine (Corning) supplemented with 10% heat-inactivated human fetal bovine serum (Omega Scientific), 50 μM 2-mercaptoethanol (Thermo Fisher), and 1X gentamycin (Lonza, Walkersville, MD, USA) at 37 ◦C with 5% CO2. At 80% confluency, cells were harvested, seeded at a density of 15 <sup>×</sup> 106 in a 175 cm2 tissue culture flask, and were allowed to reach confluency overnight. The medium was replaced at 24 h and collected at 72 h post seeding.

The spontaneously immortalized keratinocyte cell line, HaCaT, was cultured in Dulbecco's modification of Eagle's medium (DMEM) with 4.5 g/L glucose, L-glutamine, and sodium pyruvate (Corning, 10-013CV, NY, USA) supplemented with 10% heat-inactivated human fetal bovine serum (Omega Scientific), and 1X gentamycin (Lonza, Walkersville, MD, USA) at 37 ◦C with 5% CO2. Cells were passaged at 80% confluency.

### *2.2. Transmission Electron Microscopy*

Cells were pelleted and fixed in 2.5% glutaraldehyde. Cells were post fixed with 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, USA) in deionized water for 1 h at room temperature. The pellets were treated with 1% uranyl acetate for 1 h and, then, dehydrated through a graded addition of ethanol for 5 min each. Finally, the samples were embedded in epoxy resin of Eponate, NMA, DDSA, and DMP-30. Then, the pellets were sectioned into 70 nm ultrathin sections. Samples were imaged using a Jeol 2000 transmission electron microscope (JEOL USA, Peabody, MA, USA).

### *2.3. Immunoelectron Microscopy*

Cells were processed for immunogold labeling, as previously described [22–24]. They were high-pressure frozen in PBS containing 20% BSA (Millipore-Sigma, Burlington, MA, USA) using an EMPact2 with RTS (Leica Microsystems, Vienna, Austria). Freeze-substitution was performed in acetone containing 0.1% uranyl acetate and 2% water in a Leica AFS2 (Leica Microsystems, Vienna, Austria). Then, cells were embedded in HM20 and UV polymerized at −50 ◦C for 24 h. Next, samples were sectioned into 70 nm sections, picked up on formvar-coated copper grids, and blocked for 10 min in blocking buffer (0.5% BSA in PBS). Primary goat anti-langerin (1:100; R&D Systems, Minneapolis, MN, USA), goat anti-anxA2 (1:1000; R&D Systems), and goat anti-S100A10 (1:1000; R&D Systems) were diluted in blocking buffer. After centrifugation at 14,000 rpm for 2 min, the supernatant was used to label the blocked sections for 30 min at RT, followed by five washes for 2 min each in 0.01% PBS Tween-20. Then, a rabbit anti-goat bridging antibody was diluted at 1:50 in blocking buffer. After centrifugation at 14,000 rpm for 2 min, the supernatant was used to label the sections for 30 min at RT, followed by five washes for 2 min each in 0.01% PBS Tween-20. Finally, 10 nm protein A gold (Electron Microscopy Sciences) was diluted to 1:50 in blocking buffer and used to label sections for 30 min at RT, followed by three washes for 2 min each with PBS and two washes for 2 min each with deionized water. The antibody-labeled sections were examined at 80 kV on a Morgagni 268 (FEI, Hillsboro OR, USA).

### *2.4. shRNA-Mediated Knockdown of A2*/*A2t*

MUTZ-3 cells were transduced at a MOI of 5 with human annexin A2 shRNA or control shRNA lentiviral particles (Santa Cruz Biotechnology, Dallas, TX, USA) following the manufacturer's protocol. Stably transduced cells were selected with puromycin. Then, A2/A2t knockdown MUTZ-3 cells were differentiated to M-LC, as described above. Knockdown of annexin A2 and S100A10 was, then, confirmed via Western blot.

### *2.5. Western Blot and Protein Quantification*

Cells were lysed using RIPA buffer (Thermo Fisher) with Halt protease inhibitor (Thermo Fisher). Protein was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher). Samples were run on a 10% Bis-Tris agarose gel with MES (Thermo Fisher). Gels were transferred using the iBlot2 mini system (Thermo Fisher) and were blocked in PBS Starting Block Blocking Buffer (Thermo Fisher) prior to primary antibody staining overnight at 4 ◦C with anti-anxA2 (BD Biosciences, San Jose, CA, USA), anti-S100A10 (BD Biosciences), anti-langerin (Cell Signaling Technology, Danvers, MA, USA), anti-beta-actin (Cell Signaling Technology), or GAPDH (Cell Signaling Technology). Secondary antibodies, anti-mouse IRDye 800CW (LI-COR, Lincoln, NE, USA), and anti-rabbit (H+L) Alexa Fluor 680 (Thermo Fisher), were added for 1 h at room temperature. Membranes were imaged using the Odyssey imaging system (LI-COR) and protein images were analyzed and quantified using Image Studio Lite software (version 5.2.3, LI-COR).

### *2.6. Flow Cytometry*

Flow cytometry samples were collected using a Cytomics FC500 flow cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed using CXP software v2.2 (Beckman Coulter, Indianapolis, IN, USA). The following antibodies were purchased from BioLegend (San Diego, CA, USA): PE anti mouse/human CD207, PerCP/Cy5.5 anti-human CD1a, PE mouse IgG1, and PerCP/Cy5.5 mouse IgG1. For intracellular staining, cells were prepared according to the Affymetrix eBioscience protocol. Briefly, single cell suspensions were treated with IC Fixation Buffer, 1X Permeabilization Buffer, and the appropriate antibody, prior to being analyzed via flow cytometry.

### *2.7. Quantification of BG*

Stereology was performed as described in Griffiths 1993 and was used to quantitate BG present in sections of 20 M-LC and 20 anxA2/A2t KD M-LC. Samples were blinded, and cells were chosen at random. Undifferentiated, aberrant, apoptotic cells, cells with no dendrites, and cells damaged during preparation (large cracks) were excluded. Each cell was imaged at a magnification of 22,000× (scale = 325 pixels/μm) and the images were stitched together using Fiji software package (v20170530, National Institutes of Health, Bethesda, MD, USA) to create whole-cell images. A lattice grid (grid distance of 1 μm) containing a systematic system of test points (points of intersection of test lines) was placed over each of the whole-cell micrographs. The number of points landing over BG and BG-like profiles was counted, as was the total number of points over the entire cytoplasm profile (Figure S3). To quantify differences in BG abundance, the following parameters were studied where P = points:

Points landing on BG per points landing on cytoplasm = PBG/Pcytoplasm; Points landing on BG-like structures per points landing on cytoplasm = PBG-like/Pcytoplasm. A membrane structure was counted as BG-like if:


A membrane was counted as BG if as BG-like but with a clear membrane-membrane boundary ("zipper") anywhere within the membrane = .

### *2.8. Statistical Analysis and Software Used*

For EM images, statistical analysis was done using Stata v14.0 software (StataCorp, College Station, TX, USA) and verified by biostatistics consultation through the Southern California Clinical and Translational Science Institute (SC CTSI, Los Angeles, CA, USA). Data were plotted as box and whisker plots, and the Wilcoxon rank sum test was used to determine significance between groups. Significance was assigned to a value of *p* ≤ 0.05. For all other experiments, statistical analyses were performed using GraphPad Prism (v8, GraphPad Software, San Diego, CA, USA). Details for each individual experiment can be found in figure legends.

### **3. Results**

### *3.1. MUTZ-3-Derived LC Are an Appropriate Model to Study BG Structure*

In lieu of a commercially available cell line, LC studies often rely upon peripheral blood mononuclear cells (PBMC) isolated from human donors. However, using freshly isolated primary cells for structural studies of BG has several limitations, including donor heterogeneity. PBMC-derived LC cannot be maintained in a culture long term and are not amenable to gene manipulation. To work around these limitations, our study of BG structure utilized the immortalized MUTZ-3 cell line, which was generated from a CD34+ human acute myeloid leukemia [25]. Following a 14-day differentiation via cytokine regimen, langerin-expressing MUTZ-3-derived LC (M-LC) are generated with a consistent conversion rate of 30% to 40% as assessed by positive langerin (CD207) and CD1a expression and negative DC-SIGN expression (Figure S1A). These differentiated cells are phenotypically similar to primary human LC and have the same expression profile for langerin, CD1a, E-cadherin, HLA-DR, and other markers that are characteristic of LC [26,27]. M-LC are also functional in inducing anti-tumor T cell immunity [27], have similar transcription profiles to primary LC [28], and have been previously used to study BG sequestration of HIV [29]. Furthermore, M-LC have an abundance of BG [27], making it an ideal model system for our research question.

### *3.2. Immunogold Staining of Langerin in M-LC Gives Insight into Proper BG Structure Formation and Demonstrates a Novel BG Structure*

The gold standard to study BG morphology is using TEM to capture 2D cross-sections of LC. The samples used for this process were prepared by high-pressure freezing and freeze-substitution, which provided excellent preservation of cellular structures and preserved epitopes for immunolabeling. We used a primary antibody, followed by a bridging rabbit anti-primary antibody and protein A bound to 10 nm gold particles to visualize the distribution of langerin in M-LC. The bridging antibody allowed us to standardize the labeling reaction across primary antibodies and also amplify the signal [30]. Langerin is a highly abundant, locally concentrated protein; gold particles found within images are localized to the cell and are rarely found in extracellular spaces (background = 0.22 gold/μm2), indicating the specificity of the labeling (Figure S1B).

As others have reported, we observed abundant cytoplasmic BG and robust labeling of langerin localized to the BG rods (Figure 1A). The distribution of langerin labeling, as indicated by the arrows, highlights that langerin primarily localizes to the rod and is absent from the head portions of BG. These findings support previous observations that BG rod striations are formed through langerin interactions [17,31]. Langerin was also found at the cell surface and in invaginations at the plasma membrane, likely demonstrating endocytosis of surface langerin or recycling of langerin back to the surface (Figure S1C). As langerin trafficking and accumulation in the RE is a requisite to BG formation, it is not surprising that cytoplasmic vesicles containing langerin staining were also observed (Figure S1D).

BG have been established as subdomains of the ERC and bud from the RE through interactions between accumulated langerin and the Rab11a/myosin Vb/Rab11-FIP2 complex [5,16]. BG morphology is unique as compared with the RE in TEM; while RE are typically large vesicles (>500 nm diameter) containing internal membranes, BG are relatively smaller, consisting of a translucent vesicular lobe (<200 nm diameter) connected to a striated rod. For the first time, we captured the proposed BG budding event, from an RE containing intracellular langerin stores (Figure 1B). In this cross-section, distinct langerin labeling and membrane zippering show the formation of a BG budding from the RE. This vesicle is likely an RE, due to its size, the presence of internal membranes, and labeling of accumulated langerin. From this image, it appears that BG formation begins with langerin interactions creating the CMS as the head portion is not apparent and the budding rod is still attached to the RE. These findings provide visual support of the collective evidence regarding langerin recycling, trafficking and accumulation, and its driving role in BG formation from the RE.

In addition to the typical tennis-racket shaped BG structures, we also observed BG that resembled dumbbells in these electron micrographs (Figure 1C). According to the current model, BG are disk-shaped structures with a spherical vesicle-like formation at one of the ends [3]. However, these dumbbells demonstrate that BG are composed of multiple lobules or vesicles connected to a central CMS. To further understand the three-dimensional (3D) morphology of these dumbbell-shaped BG, we imaged serial sections of M-LC (Figure S1E). The same connectivity between a BG-sheet and the RE was also visible in serial sections (Figure S1E, arrow in bottom left corner of −200 nm through +200 nm). As these structures have not been observed in primary LC, these dumbbells could be unique to the MUTZ-3 system. It is possible that these structures do exist in primary cells but have not yet been reported. It is noteworthy, however, that these dumbbell structures were seen frequently in M-LC, as represented in Figure 1C and Supplemental Figure S1E.

**Figure 1.** Birbeck granules in wild type MUTZ-derived langerhans cells (LC) (M-LC) have abundant langerin labeling localized to the cytomembrane sandwiching structure (CMS). (**A**) 10 nm gold particles (black arrows) show langerin labeling is localized to the CMS in Birbeck granules (BG); (**B**) Langerin labeling is contained within a cytoplasmic multivesicular endosome, likely a recycling endosome. Here, a BG CMS through langerin zippering from these stores is visible (red arrow); (**C**) BG structures containing multiple vesicular lobes on each end of the CMS (boxes). Images are representative of at least three biological replicates with a minimum of three grids each.

### *3.3. Neither Subunit of A2t Colocalizes to BG Structures*

A2t localization in M-LC was determined by immunolabeling using a polyclonal goat anti-annexin A2 antibody. This polyclonal antibody was raised against the full length of the anxA2 protein and allowed us to maximize the epitopes that could be recognized. AnxA2 binds membrane phospholipids, which was observed with gold labeling localized to the plasma membrane and membranes of organelles, as well as in the nucleus. We did not observe consistent localization of anxA2 labeling to BG (Figure 2A,B, Figure S2A,B). Since the anxA2 staining was not abundant, we verified the lack of A2t localizing to BG by also staining for S100A10. A polyclonal goat anti-S100A10 antibody raised against the full length of S100A10 was used to label the protein in M-LC. Similar to anxA2, S100A10 labeling was predominantly localized to non-BG structures, but some BG were labeled (Figure 2C,D and Figure S2C,D). The low levels of labeling only allowed us to speculate, based on the abundance of BG structures and the rare labeling of either anxA2 or S100A10 on them, that BG do not contain substantial amounts of A2t at steady state.

**Figure 2.** Neither subunit of the annexinA2 S100A10 heterotetramer (A2t) localize to BG in wild type M-LC. Immunoelectron microscopy with 10 nm gold particles was used to visualize the cellular distribution of anxA2 (**A**,**B**) and S100A10 (**C**,**D**). BG are boxed and gold labels are indicated by arrows. Images represent staining from tree independent biological replicates and a minimum of 3 grids.

### *3.4. Knockdown of A2t Results in Reduced Expression and Non-BG Localization of Langerin*

Because anx A2 is a key player in endosome biogenesis [32], next, we sought to investigate other ways that A2t could contribute to BG formation, such as mediating internal and surface langerin expression. Interestingly, predifferentiated MUTZ-3 do not express anxA2, however, M-LC do. M-LC lysates collected throughout the 14-day differentiation showed A2t expression beginning around day 10 (Figure 3A). AnxA2 was knocked-down in predifferentiated MUTZ-3 using a lentiviral sh RNA, causing S100A10 degradation as well (Figure 3B). We also found that overall langerin expression was decreased with anxA2 knockdown M-LC as compared with wild type (WT) M-LC (Figure 3C). Quantification of Western blot band densities are found in Supplemental Table S1.

In the steady state, langerin is regularly recycled from the cell surface through the ERC to form BG, and then trafficked back [5,14]. We sought to determine if the decreased langerin expression could be attributed to a recycling defect resulting in an altered distribution between intracellular and surface langerin. To examine this, we compared expression of surface and intracellular langerin in WT and anxA2 knockdown MUTZ-3 throughout the 14 days of M-LC differentiation via flow cytometry. Within anxA2 knockdown M-LC, surface langerin expression was significantly decreased on days 7 and 10 as compared with the WT M-LC (Figure 3D). Internal langerin expression was also significantly decreased in anxA2 knockdown M-LC on days 7, 10, and 14 (Figure 3D). These data indicate that anxA2 is involved in transport of langerin to the cell surface, as its absence significantly decreases surface langerin expression. It also suggests that langerin expression is dependent upon the expression of anxA2.

**Figure 3.** A2t knockdown in M-LC causes a decrease in langerin expression and aberrant cytoplasmic localization. (**A**) M-LC were collected throughout the 14-day differentiation for analysis of A2t expression via Western blot. The human keratinocyte cell line HaCaT were used as a positive control; (**B**) Western blot of MUTZ-3 and fully differentiated, day-14 M-LC show successful A2t knockdown in M-LC; (**C**) At day 14 of differentiation, langerin expression is decreased with A2t knockdown as compared with the WT M-LC; (**D**) Surface and internal langerin expression was assessed via flow cytometry in both wild type and A2t knockdown MUTZ-3 throughout differentiation. Data represents three biological replicates (*n* = 9) (\* *p* < 0.05, paired Student's *t*-test); (**E**) Black arrows indicate langerin labeling specific to BG and red arrows show abnormal, non-BG associated langerin clusters.

### *3.5. The Absence of A2t Results in Abnormal Cellular Distributionof Langerin and Incomplete BG Formation*

To investigate changes with intracellular langerin distribution, we compared immunolabeled anxA2 in WT and knockdown M-LC. Intracellular langerin labeling in anxA2 knockdown M-LC visibly differed from WT M-LC (Figure 3E). In striking contrast to the robust rod-shaped labeling patterns seen in WT M-LC (Figure 1), langerin was found highly concentrated in clusters contained in vesicles throughout the cytosol, similar to the occasional endosomal labeling staining seen in WT M-LC. In the few discernable BG, langerin labeling was inconsistent and only partially localized to some BG structures (Figure 3E). Aberrant distribution of langerin observed with anxA2 knockdown suggests impaired or abnormal trafficking since langerin is no longer localized to BG structures and is instead contained in clusters throughout the cytosol.

The most significant effect of anxA2 knockdown on M-LC was the near-disappearance of proper BG formation (Figure 4A,B). In addition to the decreased BG abundance, most BG found were incomplete as they were missing the central rod striations and had fewer misshapen heads but retained the unique flattened dimensions of BG rods (Figure 4C). We termed these abnormal structures as "BG-like". To quantify the effect of anxA2 knockdown on BG formation, we compared WT and anxA2 knockdown M-LC using stereology [30], stitching together whole-cell cross-sections from individual TEM micrographs taken at 22,000×. Randomly chosen cells from two biological replicates were imaged and analyzed in 20 mock-infected, 30 wild type, and 27 anxA2 knockdown M-LC. BG and BG-like structures were quantified by overlaying 1 μm spaced grid on the cell and counting the grid intersection points that fall either on BG (PBG and PBG-like) or in the cytoplasm (Pcytoplasm) (Figure S3). The ratio of BG to cytoplasm in anxA2 knockdown M-LC was significantly decreased (*p* < 0.0001) as compared with wild type M-LC (Figure 4D). BG-like occurrence in anxA2 knockdown was also significantly increased as compared with wild type M-LC (*p* = 0.0056) (Figure 4E).

**Figure 4.** BG formation is significantly reduced in the absence of A2t. Wild type M-LC (**A**) contain an abundance of cytoplasmic BG as compared with A2t knockdown M-LC (**B**). At the same magnification, the relative abundance of cytoplasmic BGin WT and A2t knockdown M-LC is compared; (**C**) BG-like structures (red arrows) were observed in anxA2 knockdown M-LC and the missing striation through the center of the CMS is visualized (red box, insert i); (**D**,**E**) Stereology was used to analyze images for BG/BG-like quantification in wild type, mock-infected, and anxA2 knockdown M-LC. A 1 μm grid was overlaid to quantify cytoplasmic volume (Pcytoplasm) and BG abundance (PBG). Each point represents a single cell. Box and whisker plots show minimum, 1st quartile, median, 3rd quartile, and maximum values for each of the groups. \*\* *p* < 0.01, \*\*\*\* *p* < 0.0001, one-way ANOVA followed by Tukey's multiple comparisons test. Data are representative of two biological replicates.

### **4. Discussion**

In this body of work, we have described an essential role for A2t in BG formation. Annexins have been shown to have diverse cellular functions and are known for their membrane remodeling capabilities [33]. A2t itself is a multifunctional protein comprised of two monomeric annexin A2 (anxA2) subunits bridged by an S100A10 dimer and has been described in cellular trafficking events such as promoting actin remodeling and facilitating endosomal transport [32,33]. Interestingly, anxA2 is found specifically within the membranes of Rab11a<sup>+</sup> RE and proper intracellular distribution of RE is dependent upon anxA2 [34,35]. Using the transferrin receptor to model the ERC, A2t depletion drastically alters the shape and distribution of recycling endosomes [35]. The literature demonstrates A2t regulation of RE morphology and cellular distribution, processes which are also utilized in LC for the formation of BG.

Given the relatively unknown processes regulating BG formation, we cannot definitively describe how A2t is involved in this process. The loss of proper BG formation in the absence of A2t shows a defect in the ability of langerin to interact to form CMS and ultimately BG. We speculate, based upon the literature and the observed BG structural abnormalities in this study, that A2t acts in conjunction with Rab11a at the RE to promote langerin interactions and the formation of BG. Despite our efforts, we were not able to discern any localization of A2t in BG or a direct functional role that A2t plays in mediating langerin trafficking and BG formation. However, since A2t often acts as a scaffolding protein, it could facilitate BG structural formation indirectly. As such, we would not necessarily expect to find it localized to BG via IEM imaging following formation in a steady state.

On the basis of the data presented in this study, the loss of BG and the formation of BG-like structures can be attributed to anxA2 deficiency. In the absence of A2t, langerin trafficking through the ERC is disrupted, leading to langerin clustering and accumulation throughout the cytosol and a significant loss of BG formation. For further elucidation of the role of anxA2 in BG formation, additional studies would be needed to investigate whether anxA2 physically interacts with langerin or Rab11a. Future mechanistic studies could also involve time course experiments studying changes in trafficking of langerin in the absence of anxA2. Overall, we have highlighted A2t as a new, previously unidentified, player in Langerhans cell BG formation and added to the foundational knowledge of BG for future studies that focus on characterizing their biogenesis, structures, and functions.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/9/4/974/s1, Figure S1: MUTZ-3 derived LC is an acceptable model to study BG morphology and langerin localization using IEM, Figure S2: Supplemental images to Figure 2 of AnxA2 and S100A10 labeling in WT M-LC, Table S1: Western blot band quantifications. Figure S3: Whole-cell composite example for quantifying BG an BG-like structures using stereology.

**Author Contributions:** Conceptualization, W.M.K., D.M.D.S., and J.G.S.; methodology, W.M.K., D.M.D.S., S.M.T., V.D.S., C.B., and J.G.S.; formal analysis, S.M.T. and C.B.; investigation, S.M.T., V.D.S., and C.B.; resources, C.B., J.G.S., K.P.L., J.R.T., D.M.D.S., and W.M.K.; writing—original draft preparation, S.M.T.; writing—review and editing, S.M.T., C.B., J.G.S., J.R.T., D.M.D.S., and W.M.K.; visualization, S.M.T. and C.B.; supervision, D.M.D.S. and W.M.K.; project administration, D.M.D. and W.M.K.; funding acquisition W.M.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by NIH grants R01 CA074397 (to W.M.K.) and F31 AI136312 (to J.R.T.). Experiments were facilitated through the Norris Comprehensive Cancer Center FACS & Immune Monitoring and Cell & Tissue Imaging core facilities that are supported by a NIH grant P30 CA014089. Additionally, gifts from Connie De Rosa, Shirley Cobb, and The Netherlands American Foundation are gratefully acknowledged.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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1. Birbeck, M.S.; Breathnach, A.S.; Everall, J.D. An Electron Microscope Study of Basal Melanocytes and High-Level Clear Cells (Langerhans Cells) in Vitiligo. *J. Invest. Dermatol.* **1961**, *37*, 51–64. [CrossRef]


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Annexin A2 Egress during Calcium-Regulated Exocytosis in Neuroendocrine Cells**

### **Marion Gabel 1, Cathy Royer 2, Tamou Thahouly 1, Valérie Calco 1, Stéphane Gasman 1, Marie-France Bader 1, Nicolas Vitale <sup>1</sup> and Sylvette Chasserot-Golaz 1,2,\***


Received: 8 July 2020; Accepted: 6 September 2020; Published: 9 September 2020

**Abstract:** Annexin A2 (AnxA2) is a calcium- and lipid-binding protein involved in neuroendocrine secretion where it participates in the formation and/or stabilization of lipid micro-domains required for structural and spatial organization of the exocytotic machinery. We have recently described that phosphorylation of AnxA2 on Tyr<sup>23</sup> is critical for exocytosis. Considering that Tyr<sup>23</sup> phosphorylation is known to promote AnxA2 externalization to the outer face of the plasma membrane in different cell types, we examined whether this phenomenon occurred in neurosecretory chromaffin cells. Using immunolabeling and biochemical approaches, we observed that nicotine stimulation triggered the egress of AnxA2 to the external leaflets of the plasma membrane in the vicinity of exocytotic sites. AnxA2 was found co-localized with tissue plasminogen activator, previously described on the surface of chromaffin cells following secretory granule release. We propose that AnxA2 might be a cell surface tissue plasminogen activator receptor for chromaffin cells, thus playing a role in autocrine or paracrine regulation of exocytosis.

**Keywords:** annexinA2 egress; exocytosis; chromaffin cells

### **1. Introduction**

Molecules such as neurotransmitters and hormones are secreted by calcium-regulated exocytosis [1,2]. In neuroendocrine cells, exocytosis implies the recruitment and subsequent fusion of secretory granules at specific sites of the plasma membrane. The scaffolding protein annexin A2 (AnxA2) is a promoter of these sites of exocytosis in cells activated for secretion. AnxA2 binds two major actors of exocytosis, actin and phospholipids. In chromaffin cells, electron tomography has revealed that actin filaments bundled by AnxA2 contribute to the formation of lipid micro-domains at the plasma membrane required for the spatial and functional organization of the exocytotic machinery [3–5]. More recently, we have found that AnxA2 needs to be phosphorylated on Tyr<sup>23</sup> to stabilize the lipid platform determining the exocytotic site, and then dephosphorylated to bundle actin filaments for stably anchoring granules, implying that the phosphorylation cycle of AnxA2 on Tyr23 is critical for neuroendocrine secretion [6].

In some cell types, one of the consequences of the AnxA2 phosphorylation is the egress of AnxA2, i.e., its passage through the plasma membrane. For instance, AnxA2 appeared on the cell surface of endothelial cells following a thermal shock [7], calcium-stimulated fibroblasts [8] or depolarized cortical neurons [9]. In neurons, cell surface AnxA2 was found to interact with tissue plasminogen activator (t-PA), which is involved in synaptic plasticity and memorization [10] as well as in neuronal death

via plasmin proteolysis activity [11]. In chromaffin cells, one study reported that AnxA2 is "secreted" unconventionally in the extracellular medium following nicotine stimulation [12]. This release of AnxA2 was correlated with catecholamine secretion and found to be independent of cell death.

Having recently shown that the AnxA2 is phosphorylated on Tyr23 during exocytosis [6], we examined whether Tyr<sup>23</sup> phosphorylation of AnxA2 could lead to its externalization in chromaffin cells. Using immunolabeling and biochemical approaches, we show here that nicotine stimulation triggers the egress of AnxA2 to the external leaflets of the plasma membrane and identify tissue plasminogen activator (t-PA) as a potential partner at the chromaffin cell surface.

### **2. Materials and Methods**

### *2.1. Antibodies and Reagents*

Rabbit polyclonal antibodies directed against AnxA2 purified from bovine aorta were a generous gift from J.C. Cavadore (Inserm U-249) [13]. Monoclonal antibodies anti-AnxA2 and anti-pTyr23-AnxA2 (85.Tyr24) were purchased respectively from BD Transduction Laboratories (Pont de Claix, France) and Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Mouse monoclonal antibodies directed against dopamine ß-hydroxylase (EC.1.14.17.1: DBH), used to specifically label secretory granules in chromaffin cells [14], were purchased from Sigma-Aldrich (St. Louis, MO, USA) and the rabbit polyclonal anti-chromogranin A (CgA) from Abcam (Amsterdam, Netherlands). Rabbit polyclonal anti-mouse tissue plasminogen activator (t-PA) antibodies were from Molecular Innovations (Novi, MI, USA). Mouse monoclonal antibodies against S100A10 were from Transduction Laboratories (Lexington, KY, USA). Sheep polyclonal antibodies against bovine phenyl ethanolamine *N*-transferase (PNMT) were purchased from Chemicon International Inc., (Temecula, CA, USA) and TRITC- or Atto-647 *N*-phalloidin from Sigma-Aldrich (St. Louis). Rabbit polyclonal anti-CD63 antibodies were from Santa Cruz Biotechnology Inc (Dallas, TX, USA)**.** Secondary antibodies coupled to Alexa Fluor® conjugates (488, 561 or 647) or gold particles were from Molecular Probes (Invitrogen, Cergy Pontoise, France) and Aurion (Wageningen, Netherlands) respectively.

The construct allowing for the simultaneous expression of the catalytic subunit of the tetanus toxin (TTx) and the GFP was generated by subcloning both the eGFP and the TTx (residues 1 to 457, a generous gift from Thomas Binz [15]) into a bidirectional expression vector. The eGFP was PCR amplified as a BglII/PstI fragment using 5 -TATAGATCTCGCCACCATGGTGAGCAAGGGCGA-3 and 5 -CGCCTGCAGTTACTTGTACAG CTCGTCCATGC-3 primers and cloned into the MCS2 of the pBI-CMV1 vector (TAKARA Bio USA, Mountain View, CA, USA). Then, the TTx was PCR amplified as an MluI/NotI fragment using 5 -TATACGCGTGCCACCATGCCGATCACCATCAAC-3 and 5 -TATGCGGCCGCTTAAGCGGT ACGGTTGTACAG-3 primers and cloned into the MCS1 of the pBI-GFP vector. The botulinum C toxin (Bot C Tx) plasmid expressing the light chain of the toxin [16] was co-transfected with pmaxGFP to identify cells expressing the toxin.

### *2.2. Chroma*ffi*n Cell Culture and Transfection*

Chromaffin cells were isolated from fresh bovine adrenal glands by perfusion with collagenase A, purified on self-generating Percoll gradients and maintained in culture as previously described [17]. To induce exocytosis, chromaffin cells were washed twice with Locke's solution (140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 11 mM glucose, 0.56 mM ascorbic acid, 0.01 mM ethylene diamine tetraacetic acid (EDTA) and 15 mM Hepes, pH 7.5), and then stimulated either with Locke's solution containing 20 μM nicotine or high K<sup>+</sup> solution (86,9 mM NaCl, 59 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 11 mM glucose, 0.56 mM ascorbic acid, 0.01 mM EDTA and 15 mM Hepes, pH 7.2).

TTx-GFP was transfected into chromaffin cells (5×106 cells) by electroporation (Amaxa Nucleofactor systems, Lonza, Levallois, France) according to the manufacturer's instructions. Electroporated cells were immediately recovered in warm culture medium and plated onto fibronectin-coated glass coverslips. Experiments were performed 48 h after transfection.

### *2.3. Cell Stimulation and Extracellular AnxA2 and t-PA Measurements*

Two days after plating in 5 cm Petri dishes, 10 <sup>×</sup> 106 chromaffin cells were washed for 5 min with Locke's solution, then 5 min with Locke's solution without Ca2<sup>+</sup> (140 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 0.25 mM ethylene glycol-tetraacetic acid (EGTA), 1 mM glucose, 125 μM ascorbate and 15 mM HEPES, pH 7.5) to discard cellular debris. Cells were then stimulated for 5 min with 1 mL of either 20 μM nicotine in Locke's solution or of high K<sup>+</sup> solution. Similarly, control cells were treated with Locke's solution. The media of stimulated cells were recovered (fraction "secreted material") and the cells were further treated for 5 min with calcium-free Locke's solution containing 20 mM EGTA (fraction "EGTA eluate"). Finally, cells were scraped into 500 μL lysis buffer (Cell Extraction Buffer, Novex®, Fisher Scientific, Illkirch-Graffenstaden, France) supplemented with protease inhibitor cocktail (P8340, Sigma-Aldrich, St. Louis). The lysates were sonicated and centrifuged and the supernatants were recovered. Secreted material and the EGTA eluate fractions were concentrated 10 times to obtain 50 μL (Spin-X UF, Corning, Wiesbaden, Germany) for AnxA2 and t-PA detection. Aliquots of supernatants were used for the lactate dehydrogenase (LDH) activity assay (QuantiChromTM LDH, D2DH-100, BioAssay Systems, Hayward, CA, USA). To isolate extracellular vesicles, the secreted material was centrifuged at 300× *g* for 10 min at 4 ◦C to discard cellular debris, then the supernatant was centrifuged at 100,000× *g* for 2 h at 4 ◦C. The pellet was suspended in 15 μL of lane marker reducing sample buffer (Thermo Scientific, Illkirch Graffenstaden, Franch). Then the samples were separated on a 4–12% SDS-PAGE gel (Novex, Thermo Scientific), blotted to nitrocellulose with the Trans-blot Turbo System (Biorad, Marnes-la Coquette, Franch) and revealed with Substrat chemiluminescent SuperSignal™ (West Femto, Thermo Scientific).

### *2.4. Immunofluorescence and Confocal Microscopy*

For immunocytochemistry, chromaffin cells, grown on fibronectin-coated glass coverslips, were fixed and labeled as described previously [14]. The transient accessibility of DBH to the plasma membrane of chromaffin cells was tested by incubating cells for 5 min in Locke's solution containing 20 μM nicotine and anti-DBH antibodies diluted to 1:100. F-actin was stained with TRITC-phalloidin (0.5 μg/mL) for 15 min in the dark at room temperature. Labeled cells were visualized using a Leica SP5II confocal microscope. Nonspecific fluorescence was assessed by incubating cells with the secondary fluorescent-conjugated antibodies. To compare the labeling of cells from different conditions within the same experiment, images were acquired at the equatorial plane of the nucleus with the same parameters of the lasers and photomultipliers. The amount of AnxA2 or t-PA labeling associated with the plasma membrane was measured with ICY software [18] and expressed as the average fluorescence intensity normalized to the labeling surface, and divided by the total area of each cell. This allowed a quantitative cell-to-cell comparison of the fluorescence detected in cells.

### *2.5. Plasma Membrane Sheet Preparation and Transmission Electron Microscopy Observation*

Cytoplasmic face-up membrane sheets were prepared and processed as previously described [19]. Briefly, carbon-coated Formvar films on nickel electron grids were inverted onto unstimulated or nicotine-stimulated chromaffin cells incubated with antibodies. To prepare membrane sheets, pressure was applied to the grids for 20 s, then the grids were lifted so that the fragments of the upper cell surface adhered to the grid. These membrane portions were fixed in 2% paraformaldehyde for 10 min at 4 ◦C. After blocking in PBS with 1% BSA and 1% acetylated BSA, the immune labeling was performed and revealed with gold particle-conjugated secondary antibodies. These membrane portions were fixed in 2.5% glutaraldehyde in PBS, postfixed with 0.5% OsO4, dehydrated in a graded ethanol series, treated with hexamethyldisilazane (Sigma-Aldrich, St. Louis), air-dried and observed using a Hitachi 7500 transmission electron microscope.

### *2.6. Statistical Analysis*

As specified in figure legends, groups of data are presented as mean (±SEM) or median and were analyzed using a Mann-Whitney test. Asterisks in each box and whisker plot indicate statistical significance.

### **3. Results**

### *3.1. AnxA2 Crosses the Plasma Membrane in Stimulated Chroma*ffi*n Cells in a Ca2*<sup>+</sup>*-Dependent Manner and Accumulates on the Extracellular Membrane Leaflet*

Bovine chromaffin cells represent a good model to study regulated exocytosis [20]. They express various nicotinic receptors and accordingly exocytosis is triggered by nicotinic agonists [21,22]. To examine whether AnxA2 could be found on the cell surface of stimulated chromaffin cells, living chromaffin cells maintained under resting conditions or stimulated with nicotine were incubated in the presence of anti-AnxA2 antibodies to specifically label AnxA2 potentially present on the cell surface. Cells were then fixed and labeled with phalloidin-TRITC to reveal the actin cytoskeleton. Resting cells characterized by a typical F-actin ring at the cell periphery displayed only faint cell surface AnxA2 labeling (Figure 1a). In contrast, while F-actin labeling decreased in stimulated cells in line with the actin depolymerization occurring during exocytosis, the labeling of AnxA2 on the cell surface increased (Figure 1a). In some experiments, cells stimulated with nicotine in the presence of the AnxA2 antibodies were further washed with a calcium-free Locke's solution containing 20 mM EGTA to collect proteins bound in a calcium-dependent manner to the cell surface (S + EGTA). Washing cells with EGTA led to a diminution of the cell surface AnxA2 labeling. Semi-quantitative analysis of the confocal images (Figure 1b) indicated that cell stimulation increased by approximately three times the amount of AnxA2 detected on the surface of chromaffin cells and confirmed the calcium sensitivity of AnxA2 binding to the plasma membrane.

**Figure 1.** Annexin A2 (AnxA2) is present on the outer face of the plasma membrane of stimulated chromaffin cells. (**a**) AnxA2 labeling at the surface of chromaffin cells in the resting condition (Resting), stimulated with 20 μM nicotine without (Stimulated) or after washing with calcium-free Locke's solution

containing 20 mM EGTA (Stimulated + EGTA). Anti-AnxA2 antibodies were revealed with Alexa Fluor®488-conjugated anti-rabbit antibodies and F-actin with TRITC-phalloidin. Confocal images were recorded in the same optical section by a dual exposure procedure with the same parameters of lasers and photomultipliers. Scale bar: 10 μM. (**b**) Semi-quantitative analysis of cell surface AnxA2 labeling in chromaffin cells in resting condition (R) or stimulated with 20 μM nicotine (S) without (Control) or after EGTA wash (EGTA). AnxA2 labeling is expressed in arbitrary units. Statistical significance for medians was determined using a Mann-Whitney test. Dotted lines indicate the mean and asterisks statistical significance (\*\*\* = *p* < 0.001, \*\* = *p* < 0.01). Three experiments were done on independent cell cultures and pooled (*n* = 32 and 63 control cells, 28 and 63 EGTA-treated cells for resting and stimulated conditions, respectively). (**c**) Lysate, secreted material and EGTA eluate from chromaffin cells in the resting condition (R), or stimulated with 20 μM nicotine (S) were analyzed by western blot and revealed with anti-CgA and anti-AnxA2 antibodies. Data correspond to a typical experiment representative of three independent experiments. (**d**) The 100,000 g pellet of secreted materials from chromaffin cells in the resting condition (R), or stimulated with high K<sup>+</sup> solution (K+) or 20 μM nicotine (Nic) were analyzed by western blot and revealed with anti-CD63 and anti-AnxA2 antibodies. Samples analyzed were obtained from two independent experiments.

Next, a biochemical approach was performed using fractions collected from nicotine-stimulated chromaffin cells. As illustrated in Figure 1c, AnxA2 was not found in the secreted material containing chromogranin A (CgA), but it could be detected in the calcium-free EGTA solution (EGTA eluates) used to wash cells after stimulation and in the cell lysates. In line with the absence of AnxA2 in secretory granules, these data suggest that AnxA2 is not secreted via the conventional exocytotic pathway but it is found bound in a calcium-dependent manner on the extracellular face of the plasma membrane following cell stimulation. Moreover, we did not detect AnxA2 in 100,000 g pellets of secreted materials (Figure 1d) containing the specific marker of extracellular vesicle CD63 [23]. Thus, AnxA2 did not seem to be associated with the extracellular vesicles released after cell stimulation.

### *3.2. Tyr <sup>23</sup> Phosphorylated AnxA2 Tetramer Binds the External Face of the Plasma Membrane*

In chromaffin cells, we have previously showed that AnxA2 can be found in monomeric and tetrameric forms, associated with two S100A10 molecules [19]. We examined whether cell surface AnxA2 was phosphorylated on Tyr23 and whether S100A10 was present on the cell surface upon cell stimulation. Live chromaffin cells were stimulated with nicotine in the presence of anti-pTyr23 AnxA2 or anti-S100A10 antibodies. Both antibodies labeled the surface of stimulated cells, suggesting that cell surface AnxA2 is Tyr<sup>23</sup> phosphorylated and associated with S100A10. Thus, AnxA2 tetramer could be present on the external face of the plasma membrane (Figure 2a,b). Bovine chromaffin cells are constituted of two populations: the adrenergic cells secreting adrenaline and noradrenergic cells secreting noradrenaline [24], and we have previously showed that S100A10 is selectively expressed in adrenergic cells [14]. Thus, we performed a staining experiment for cell surface AnxA2 together with S100A10 or phenylethanolamine *N*-methyltransferase (PNMT), selectively expressed in adrenergic cells [24]. Figure 2c shows a S100A10-positive cell close to two S100A10-negative cells and all cells display a similar staining of cell surface AnxA2. Accordingly, cell surface AnxA2 labeling was observed on PNMT-positive and -negative cells (Figure 2d). Altogether, these data suggest that AnxA2 translocated through the plasma membrane of stimulated chromaffin cells even in the absence of S100A10.

### *3.3. The Egress of AnxA2 is Linked to Exocytosis*

To probe the idea that AnxA2 egress is related to exocytosis, we labeled in parallel cell surface AnxA2 and the exocytotic sites using anti-DBH antibodies [14]. In live cells stimulated for exocytosis, the granule-associated DBH becomes transiently accessible to the antibody only at sites of exocytosis, leading to the appearance of fluorescent patches at the cell surface [14].

**Figure 2.** Tyr23-phosphorylated AnxA2 and S100A10 are associated with the surface of stimulated chromaffin cells. (**a**) The p-Tyr23AnxA2 labeling at the surface of stimulated chromaffin cells. Anti-p-Tyr23AnxA2 antibodies were revealed with Alexa Fluor®488-conjugated anti-mouse antibodies and F-actin with TRITC-phalloidin to visualize the cell shape. (**b**) S100A10 labeling at the surface of stimulated chromaffin cells. Anti-S100A10 antibodies were revealed with Alexa Fluor®488-conjugated anti-mouse antibodies and F-actin with TRITC-phalloidin. (**c**) Confocal micrograph of the triple labeling of cell surface AnxA2, of intracellular S100A10 and of F-actin labeled with Atto-647 *N*-phalloidin in stimulated chromaffin cells. (**d**) Confocal micrographs of the triple labeling of cell surface AnxA2, of intracellular PNMT and of F-actin labeled with Atto-647 *N*-phalloidin in stimulated chromaffin cells. For (**a**–**d**), confocal images were recorded in the same optical section by a dual exposure procedure. Scale bar: 10 μM.

As illustrated in Figure 3a, resting chromaffin cells exhibited only a few DBH patches, confirming the low levels of baseline exocytotic activity in the absence of secretagogue, and displayed only a weak cell surface AnxA2 staining. Stimulation for 5 min with nicotine triggered the appearance of a patchy pattern of DBH surface staining and concomitantly increased cell surface AnxA2 labeling (Figure 3a). We also observed the co-localization between DBH and AnxA2 at the cell surface (Figure 3a, merge). Semi-quantitative analysis indicated that 65.5 ± 4.12% (±SEM, *n* = 27) of the cell surface AnxA2 labeling colocalized with DBH labeling. This indicated that the AnxA2 egress primarily takes place in the vicinity of the exocytotic sites. Next, we examined the time course of AnxA2 appearance on the cell surface of nicotine-stimulated chromaffin cells using a semi-quantitative analysis (Figure 3b). Egress of AnxA2 was observed after 30 s of stimulation and it peaked after 60 s. Thus, the kinetics of AnxA2 egress correlated well with the rapid kinetics of AnxA2 Tyr<sup>23</sup> phosphorylation during exocytosis, but preceded the maximal exocytotic response usually observed after 180 s of stimulation in our experimental conditions [6]. To confirm the link between the AnxA2 egress and exocytosis, we examined whether the formation of the Soluble NSF Attachment Proteins REceptor (SNARE) complexes was necessary for the externalization of AnxA2. A bicistronic plasmid encoding for tetanus toxin (TTx) and GFP was expressed in chromaffin cells. TTx is known to specifically cleave VAMP2, a protein required for SNARE complex formation and exocytosis [25]. As illustrated in Figure 3c, cell surface AnxA2 labeling was reduced in stimulated cells expressing TTx as compared to electroporated control cells not expressing TTx. Semi-quantitative analysis confirmed that expression of TTx reduced AnxA2 egress in stimulated cells (Figure 3d). Thus, VAMP2 cleavage and the consequent inhibition of the SNARE complex formation reduced AnxA2 externalization and its appearance on the cell surface. Similar results were obtained in cells expressing botulinum toxin C, which cleaves SNAP-25 and syntaxin-1 (Figure 3c,d). These findings indicate that the egress of AnxA2 seems to depend on the formation of SNARE complexes and therefore on the completion of exocytosis in chromaffin cells.

**Figure 3.** AnxA2 egress was correlated with exocytosis. (**a**) Dual labeling of cell surface AnxA2 and exocytotic sites. Cells were stimulated with nicotine 20 μM in the presence of anti-DBH and anti-AnxA2 antibodies. Cells were then fixed and incubated with secondary antibodies coupled to Alexa Fluor®561 and Alexa Fluor®488, respectively. Confocal images were recorded in the same optical section and with the same parameters of lasers and photomultipliers. Scale bar: 10 μM. (**b**) Time course of AnxA2 egress after cell stimulation. Chromaffin cells were stimulated with 20 μM nicotine during different times in the presence of AnxA2 antibodies. The cell surface AnxA2 labeling is expressed in arbitrary units. Statistical significance for medians was determined using a Mann-Whitney test. Dotted lines indicate the mean and asterisks statistical significance (\*\*\* = *p* < 0.001, \*\* = *p* < 0.01, \* = *p* < 0.05). Two experiments were done on independent cell cultures and pooled. Number of cells analyzed were 19 (0 s), 25 (15 s), 29 (30 s), 23 (60 s), 21 (180 s). (**c**) Effect of tetanus and botulinum C toxins on the AnxA2 egress in chromaffin cells. Electroporated cells expressing TTx/GFP, BotC Tx/GFP or no toxin (Control, C) were stimulated with high K<sup>+</sup> solution in the presence of anti-AnxA2 antibodies, fixed and then incubated with secondary antibodies coupled to Alexa Fluor®561. Confocal images were recorded in the same optical section. Scale bar: 10 μM. (**d**) Semi-quantitative analysis of cell surface AnxA2 labeling in stimulated cells is expressed in arbitrary units (Control *n* = 79 cells, TTx/GFP *n* = 69 cells, Control *n* = 39 cells, BotC Tx/GFP *n* = 39 cells). Statistical significance for medians was determined using a Mann-Whitney test. Dotted lines indicate the mean and asterisks statistical significance (\*\*\* = *p* < 0.001, \*\* = *p* < 0.01). Three experiments were done on independent cell cultures and pooled.

### *3.4. Cell Surface Membrane-Associated t-PA is Present Close to Exocytotic Sites*

Cell surface AnxA2 has been described as a co-receptor of t-PA [26]. Cell surface AnxA2 tetramer was shown to capture circulating plasminogen and t-PA to promote plasmin generation that participates in fibrinolysis [26]. Moreover, previous studies reported the presence of t-PA in secretory granules and its release upon chromaffin cell stimulation [27], and t-PA was found to bind to the cell surface by interacting with an unknown receptor [28]. We confirmed the presence of t-PA at the surface of chromaffin cells (Figure 4). Double labeling of living cells stimulated with nicotine in the presence of anti-t-PA and anti-DBH antibodies revealed the appearance of a patchy pattern of DBH surface staining and a concomitant increase in t-PA labeling (Figure 4a). We also observed the co-localization between t-PA and DBH at the cell surface (Figure 3a, merge). Semi-quantitative analysis indicated that 68 ± 2.7% (±SEM, *n* = 33) of the cell surface t-PA labeling colocalized with DBH labeling.

**Figure 4.** Tissue plasminogen activator (t-PA) is present on the outer leaflet of the plasma membrane of stimulated chromaffin cells. (**a**) Dual labeling of cell surface t-PA and exocytotic sites. Cells were stimulated with nicotine 20 μM in the presence of anti-t-PA and anti-DBH antibodies. Cells were then fixed and incubated with secondary antibodies coupled to Alexa Fluor®488 and Alexa Fluor®551,

respectively. Confocal images were recorded in the same optical section and with the same parameters of lasers and photomultipliers. Scale bar: 10 μM. (**b**) The t-PA labeling on the surface of chromaffin cells in the resting condition (R), stimulated with 20 μM nicotine without (S) or after EGTA wash (S + EGTA). Anti-t-PA antibodies were revealed with Alexa Fluor®488-conjugated anti-rabbit antibodies and F-actin with TRITC-phalloidin to visualize the cell shape. Confocal images were recorded in the same optical section by a dual exposure procedure. Scale bar: 10 μM. (**c**) Semi-quantitative analysis of t-PA labeling on the cell surface of chromaffin cells in the resting condition (R), stimulated with 20 μM nicotine (S) without (Control) or after EGTA wash (S + EGTA). The t-PA labeling is expressed in arbitrary units. Statistical significance for medians was determined using a Mann-Whitney test. Dotted lines indicate the mean and asterisks statistical significance (\*\*\* = *p* < 0.001, \*\* = *p* < 0.01). Three experiments were done on independent cell cultures and pooled (*n* = 36 and 65 control cells, 40 and 75 EGTA-treated cells for resting and stimulated conditions, respectively). (**d**) Lysate, secreted material and EGTA eluate from chromaffin cells in the resting condition (R) or stimulated with nicotine 20 μM (S) were analyzed by western blot and revealed with anti-t-PA and anti-AnxA2 antibodies. Data correspond to a typical experiment representative of three independent experiments.

We further characterized the binding of t-PA at the cell surface of stimulated chromaffin cells using a similar approach to that used to visualize cell surface AnxA2. Live cells were maintained at rest or stimulated with nicotine in the presence of anti-t-PA antibodies prior to fixation. In resting cells, t-PA labeling was barely visible, whereas in stimulated cells, a clear increase in cell surface labeling was observed (Figure 4b). Washing the nicotine-stimulated cells with calcium-free EGTA containing Locke's solution prior to incubation with the anti-t-PA antibodies resulted in a clear reduction in t-PA labeling (Figure 4b). Semi-quantitative analysis (Figure 4c) confirmed that cell stimulation increased by approximately 2.5-fold the t-PA staining on the surface of chromaffin cells and validated the calcium sensitivity of t-PA binding to the plasma membrane. Figure 4d illustrates a western blot analysis of the fractions collected from cells stimulated with nicotine or high K<sup>+</sup> solution and subsequently washed for 5 min with EGTA solution. In the cell lysates and secreted material, t-PA was found, in line with the presence of t-PA in secretory granules. It was also detected in the EGTA eluates, indicating that part of the t-PA released by exocytosis remained bound to the cell surface in a calcium-dependent manner. CgA, a major component stored in chromaffin granules and released by exocytosis, also bound to the cell surface following secretion (Figure 1c).

### *3.5. AnxA2 and t-PA are Side by Side at Cell Surface of Stimulated Chroma*ffi*n Cells*

To further explore the spatial relationship between t-PA, AnxA2 and the sites of exocytosis, experiments were designed to analyze at the ultrastructural level the outer face of the chromaffin cell plasma membrane. The localization of cell surface AnxA2 or t-PA and granule membranes transiently inserted into the plasma membrane after exocytosis was examined on plasma membrane sheets from chromaffin cells stimulated with 20 μM nicotine in the presence of anti-DBH antibodies to reveal exocytotic granule membranes [29] and anti-AnxA2 or anti-t-PA antibodies (Figure 5). Cells were fixed and labeled with anti-mouse antibodies revealed with 10 nm gold particles and anti-rabbit antibodies revealed with 15 nm gold particles to label DBH/anti-DBH complexes and AnxA2/anti-AnxA2 or t-PA/anti-t-PA, respectively. Ultrastructural images obtained by transmission electron microscopy revealed the appearance of gold-labeled DBH clusters on the cell surface (Figure 5a), corresponding to the insertion of the granular membrane in the plasma membrane of nicotine-stimulated cells [29]. Cell surface AnxA2, as well as t-PA (Figure 5a,b), tended to localize at the periphery of the DBH-labeled areas. Double-labeling experiments with anti-t-PA and anti-AnxA2 antibodies using a combination of 10 and 15 nm gold particles indicated AnxA2 and t-PA formed mixed clusters at the cell surface (Figure 5c,d). Both types of beads were found in close proximity, suggesting a possible interaction of t-PA with AnxA2 at the surface of stimulated chromaffin cells, in line with the idea that AnxA2 could be a t-PA receptor at the surface of chromaffin cells.

**Figure 5.** Membrane topography of AnxA2, t-PA and exocytotic sites after immunogold labeling of the outer face of the plasma membrane sheets prepared from stimulated chromaffin cells. (**a**) Plasma membrane sheets were prepared from bovine chromaffin cells stimulated by nicotine for 5 min. To label DBH, AnxA2 and t-PA exposed at the surface of cells undergoing exocytosis, anti-DBH, anti-AnxA2 and anti-t-PA antibodies were added during stimulation. Membrane sheets were labeled with anti-mouse antibodies coupled to 10 nm gold particles to detect DBH antibodies revealing exocytotic sites (red circle) and rabbit antibodies coupled to 15 nm gold particles to label AnxA2 or t-PA (green circle). (**b**) The histogram represents the relative distribution of 15 nm gold particles as a function of their distance from the granule membrane once inserted in the plasma membrane (blue line). The distance was measured and the number of particles was counted manually with Photoshop. Three experiments were done on independent cell cultures (**c**). Double staining experiment for t-PA (10 nm gold particles) and AnxA2 (15 nm gold particles) were performed with the same protocol. Scale bar: 100 nm. (**d**) The histogram represents the relative distribution of 10 nm gold particles (t-PA) as a function of their distance from 15 nm gold particles (AnxA2). The distance was measured and the number of particles was counted manually with Photoshop. Two experiments were done on two independent cell cultures.

### **4. Discussion**

During neuroendocrine secretion, AnxA2 participates together with the actin cytoskeleton in the formation of lipid micro-domains required for the docking and fusion of secretory granules with the plasma membrane [5,13,19]. We have recently observed that AnxA2 needs to be phosphorylated on Tyr23 to form these lipid platforms supporting exocytosis [6]. Since Tyr23 phosphorylation of AnxA2 has been reported to induce its translocation from the inner to the outer side of the plasma membrane [7], we examined here whether AnxA2 might be externalized in neurosecretory chromaffin cells. In agreement with these findings, the present report favors a model in which AnxA2 is not conventionally secreted in the extracellular medium, but rather translocates through the plasma membrane and remains attached to the surface of stimulated chromaffin cells. Here, p-Tyr23-AnxA2 was found associated with the outer face of the plasma membrane in a calcium-dependent interaction. Hence, our observations are in agreement with previous results obtained in endothelial cells [7] and keratinocytes [30], but also in neurosecretory GABAergic neurons [9]. AnxA2 egress is closely linked

to the process of exocytosis since the externalization of AnxA2 required the formation of the SNARE complexes and occurred near the sites of secretory granule fusion. In addition, at the cell surface, AnxA2 was found to co-localize with t-PA in a calcium-dependent manner. We propose that cell surface AnxA2 could function as a receptor for secreted t-PA.

### *4.1. By Which Mechanism is AnxA2 Translocated to the Cell Surface in Chroma*ffi*n Cells?*

Despite the description of many extracellular functions for AnxA2, the mechanism underlying its translocation across the plasma membrane to the cell surface remains unclear. There are a number of soluble proteins lacking signal peptides which are secreted in the extracellular medium through a process called "unconventional secretion" [31]. Two major pathways for this secretion involve either the direct translocation across the plasma membrane or the secretion via extracellular vesicles. For instance, in NIH 3T3 fibroblasts, the cell surface appearance of AnxA2 has been linked to the fusion of multi-vesicular bodies with the plasma membrane, whereas in intestinal epithelial cells, it depends on the fusion of secretory vesicles with the plasma membrane, and in stimulated macrophages or ultraviolet-irradiated keratinocytes it requires caspase-1 activation [32]. In chromaffin cells, the implication of secretory granules and extracellular vesicles is unlikely since AnxA2 was neither found in the soluble secreted material nor associated with the extracellular vesicles released after cell stimulation, but detected on the outer face of the plasma membrane where it bound in a calcium-dependent manner. Furthermore, we did not find AnxA2 in a 100,000 g pellet of the secreted material, suggesting that AnxA2 is not associated with the extracellular vesicles released after cell stimulation. Consequently, the most likely mechanism for AnxA2 membrane translocation involves direct insertion into the lipid bilayer, allowing the passage of AnxA2 through the plasma membrane.

The insertion of annexins into model membranes has been demonstrated in vitro for several members of this large family of proteins (AnxA1, A2, A4, A5 and A6) [33]. Within cells, AnxA2 was also shown to translocate across membranes [34]. AnxA2 membrane translocation requires calciumdependent binding to negatively charged phospholipids such as phosphatidylserine (PS) and a lipid flipping activity. Accordingly, the phospholipid scramblase TMEM16F was reported to contribute to AnxA2 membrane crossing [35]. A similar mechanism has been described for the unconventional secretion of fibroblast growth factors (FGFs) [36], which also requires calcium and is linked to PS egress [37]. It is tempting to draw parallels with AnxA2 in chromaffin cells, since secretagogue-evoked stimulation of chromaffin cells triggers the appearance of PS at the cell surface, presumably at the periphery of the granule fusion sites [29,38]. This PS egress depends on the lipid scramblase PLSCR-1 [39]. The contribution of PLSCR-1 in AnxA2 egress needs now to be tested in chromaffin cells. Finally, an alternative mechanism has been proposed in enterocytes, which involves the extrusion of AnxA2 during hemifusion [40].

### *4.2. What Might be the Role of Cell Surface AnxA2?*

The most well-documented role of cell surface AnxA2 is as co-receptor of t-PA and plasminogen [26]. Plasminogen and t-PA were previously described on the surface of chromaffin cells but their receptors remained unidentified [41]. Of note, t-PA was also detected in the secretory granules of a subpopulation of chromaffin cells [42]. In the present report, t-PA and AnxA2 were both found on the surface of chromaffin cells. Both t-PA and cell surface AnxA2 were eluted by EGTA, indicating that both proteins bind to the membrane in a calcium-dependent manner. Although the direct interaction of t-PA with chromaffin cell surface AnxA2 remains to be demonstrated, AnxA2 is able to interact with t-PA and its primary substrate, plasminogen [43]. Thus, by recruiting released t-PA and circulating plasminogen, cell surface AnxA2 might well serve as an extracellular proteolytic center that locally generates plasmin. Plasmin is known to cleave released CgA into a variety of biologically active peptides, some of which may significantly inhibit the nicotinic stimulation of catecholamine release from PC12 cells and primary bovine adrenal chromaffin cells [28]. For instance, the fragment CgA360-373 is selectively generated by plasmin and the corresponding synthetic peptide markedly inhibited nicotine-induced catecholamine

release [44]. Plasmin generated at the cell surface could also activate signals, leading to protein kinase C-mediated phosphorylation of intracellular AnxA2, thereby dissociating the AnxA2 complex and preventing further catecholamine release [32]. In neuronal tissues, plasminogen activators and cell surface AnxA2 have both been detected at high levels and implicated in processes like neuronal plasticity and synaptic remodeling within the hippocampus and cerebellum during memory [10].

Additionally, AnxA2, via its ability to sequester and laterally organize PS [45], could promote the formation or stabilization of PS-rich domains in the external leaflet of the plasma membrane. These AnxA2-mediated clusters of PS at the cell surface may represent a concentrated signal for endocytosis as has been shown for AnxA5 and phagocytosis [46]. Finally, we cannot exclude that the AnxA2 tetramer could potentially interact with neighboring phospholipid membranes and therefore serve as a bridge between two adjacent cells [47].

To summarize, we propose that, upon cell stimulation, AnxA2 is recruited to the plasma membrane to form a tetramer with S100A10. Once phosphorylated at the plasma membrane, AnxA2 phosphorylated on Tyr<sup>23</sup> stabilizes lipid micro-domains required to recruit/organize the priming/docking machinery for exocytosis. Although AnxA2 Tyr23 dephosphorylation is required to promote the formation of actin bundles that strongly anchor secretory granules to the exocytotic sites, a fraction of Tyr23-phosphorylated AnxA2 appears to cross the plasma membrane near exocytotic sites. At the cell surface, AnxA2 as a co-receptor of t-PA could thus participate in various autocrine and/or paracrine activities.

**Author Contributions:** Conceptualization, S.C.-G.; Data curation, M.G., C.R. and S.C.-G.; Formal analysis, S.C.-G.; Funding acquisition, S.G. and N.V.; Resources, M.G., C.R., T.T., V.C. and S.C.-G.; Supervision, S.G. and N.V.; Visualization, M.G. and S.C.-G.; Writing—original draft, M.G. and S.C.-G.; Writing—review and editing, M.G., M.-F.B., S.G., N.V. and S.C.-G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Fondation pour la Recherche Médicale (DEI20151234424) and the ANR (ANR-19-CE44-0019) to N.V.

**Acknowledgments:** We are grateful to Thomas Binz for generously providing us with tetanus toxin plasmid. We thank the municipal slaughterhouse of Haguenau (France) for providing bovine adrenal glands.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Membrane Binding Promotes Annexin A2 Oligomerization**

### **Anna Lívia Linard Matos 1,**†**, Sergej Kudruk 1,**†**, Johanna Moratz 2, Milena Heflik 1, David Grill 1, Bart Jan Ravoo <sup>2</sup> and Volker Gerke 1,\***


Received: 6 April 2020; Accepted: 6 May 2020; Published: 8 May 2020

**Abstract:** Annexin A2 (AnxA2) is a cytosolic Ca2<sup>+</sup> regulated membrane binding protein that can induce lipid domain formation and plays a role in exocytosis and endocytosis. To better understand the mode of annexin-membrane interaction, we analyzed membrane-bound AnxA2 assemblies by employing a novel 3-armed chemical crosslinker and specific AnxA2 mutant proteins. Our data show that AnxA2 forms crosslinkable oligomers upon binding to membranes containing negatively charged phospholipids. AnxA2 mutants with amino acid substitutions in residues predicted to be involved in lateral protein–protein interaction show compromised oligomer formation, albeit still being capable of binding to negatively charged membranes in the presence of Ca2<sup>+</sup>. These results suggest that lateral protein–protein interactions are involved in the formation of AnxA2 clusters on a biological membrane.

**Keywords:** annexin A2; microdomain; cross-linker; quartz crystal microbalance with dissipation monitoring (QCM-D)

### **1. Introduction**

Biological membranes can segregate into microdomains of defined lipid and protein composition that serve diverse but yet very specific tasks. One category of these microdomains, often referred to as lipid rafts, is enriched in cholesterol as well as sphingomyelin species present in the extracellular leaflet and in certain cases phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] present in the cytoplasmic leaflet. Rafts are particularly well studied in the plasma membrane of eukaryotic cells, where they serve as assembly and transmission platforms in outside-in as well as inside-out signaling and also regulate membrane trafficking events to and from the plasma membrane (for review see [1–3]). To function in these processes, rafts have to be highly dynamic, both with respect to lipid and protein composition as well as size, thus they assemble into larger structures and also disassemble again on a rapid time scale. Dynamic assembly/disassembly is driven by intrinsic properties of the raft lipids and proteins and is affected, among other things, by cholesterol content and degree of fatty acid saturation in the incorporated phospho- and sphingolipids. Importantly, raft dynamics are also controlled by membrane-associated proteins that function by binding to raft lipids or proteins and affect their properties and distribution. These associated proteins are often regulated in their membrane association and thus raft-controlling properties enable cells to rapidly respond to certain stimuli by altered membrane microdomain assembly. One important regulatory event is a change in intracellular Ca2<sup>+</sup> concentration and a number of raft-associated proteins whose membrane interaction is regulated

by Ca2<sup>+</sup> (for review see [4]). Annexins are a family of such Ca2<sup>+</sup> regulated proteins that bind to acidic phospholipids in the cytoplasmic leaflets of cellular membranes in a peripheral and reversible manner (for review see [5–7]).

Annexin A2 (AnxA2) is a member of the annexin family that has been shown to associate with raft-like microdomains in certain cells and certain physiological scenarios (for review see [8]). As other annexins, it directly binds to headgroups of negatively charged phospholipids and requires the presence of these lipids in membranes (and raft domains) for high-affinity association. One such lipid is PI(4,5)P2, and AnxA2 has been shown to interact with this phosphoinositide in a specific manner [9,10]. In addition to binding to PI(4,5)P2 and other acidic phospholipids, in particular phosphatidylserine (PS), in a Ca2<sup>+</sup> regulated manner, the protein can also form two-dimensional assemblies on PI(4,5)P2 or PS containing model membranes and can cluster these lipids into domains [11–14]. Most likely, this lipid segregating property of AnxA2 is responsible for a function of the protein in regulating membrane-cytoskeleton contacts, cell polarity, and exocytotic granule docking and fusion [15–19]. However, the molecular basis underlying the phospholipid segregating properties of AnxA2, in particular the role of potential protein-protein interactions in this process, is not known.

Structurally, AnxA2 has a fold similar to all other annexins. It comprises a conserved core domain that is built of four repeats, each with five α helices, and a unique N-terminal domain mediating interactions with protein ligands. The core forms a slightly bent structure, where type-II Ca2<sup>+</sup> binding sites as well as the membrane binding site are located on the convex side. A high resolution crystal structure of AnxA2 has been obtained revealing anti-parallel dimers of AnxA2 in the crystallized unit [20] and amino acid side chains residing at this dimer interface could provide lateral contacts in two-dimensional AnxA2 assemblies.

Here we have investigated the oligomeric state of membrane-bound AnxA2 to assess whether protein–protein interactions could represent the molecular mechanism underlying AnxA2-driven PI(4,5)P2 and PS segregation. Therefore, we developed a novel chemical crosslinker that revealed the existence of AnxA2 oligomers. These oligomers only form in the presence of Ca2<sup>+</sup> and require membrane binding, thus could represent the molecular structures driving a segregation of AnxA2-bound phospholipids. Moreover, mutating residues present at the dimer interface identified positions that reside in close proximity in membrane-bound AnxA2 oligomers and could participate in oligomer formation.

### **2. Materials and Methods**

### *2.1. Lipids, Chemicals*

Cholesterol (Chol), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-snglycero-3-phoshocholine (POPC), 1-palmitoyl-2-oleoylsn-glycero-3-phosho-L-serine (sodium salt) (POPS), and 1,2-dioleoyl-sn-glycero-3 [phosphoinositol-4,5-bisphosphate](triammonium salt) (PI(4,5)P2) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Lipids and cholesterol were dissolved in chloroform/methanol (1:1, *v*/*v*), except for PI(4,5)P2 that was dissolved in chloroform/methanol/water (20:9:1 *v*/*v*). Other chemicals were purchased from Applichem (Darmstadt, Germany), Merck KGaA (Darmstadt, Germany), Carl Roth GmbH (Karlsruhe, Germany) and Sigma-Aldrich (Munich, Germany). Water was purified and deionized with a cartridge system from Millipore (18.2 MΩ). For all the SDS-PAGE and Western blots the marker PageRuler Plus Prestained Protein Ladder from Thermo Scientific was used (Waltham, MA, USA).

### *2.2. Crosslinker Synthesis*

Biotinyl *N*-Tris((2-(2.5-dioxopyrrolidin-1-yl propionate triethyleneglycolamido) ethoxy) methyl) methylamide, herein referred to as Biotin3xNHSX-Linker, was synthesized as described in detail in the Supplementary Information (SI). Briefly, spacer synthesis started with 2- (2- (2-chloroethoxy) ethoxy) ethanol, which was converted into the ester (**1**) in a MICHAEL reaction with *t*-butyl

acrylate and sodium in tetrahydrofuran (THF) (Figure 1). The chloride (**1**) was then mixed with sodium azide (NaN3) and the azide obtained was reduced to the amine-terminated spacer with triphenylphosphine. In the convergent procedure, Tris (hydroxymethyl) aminomethane (THAM) was converted with *t*-butyl acrylate and sodium hydroxide (NaOH) in THF to the triple-functionalized amine (**3**), which was then protected with benzyl chloroformate (**4**). This protective group shows stable behavior in the case of tri-fluoro acetic acid (TFA) initiated acidic hydrolysis. This was followed by selective TFA deprotection of the ester-protected hydroxyl group with subsequent peptide coupling of the molecule (**4**) and the spacer (**2**). In the next step, the *N*-Cbz protective group was removed under reductive conditions using hydrogen and palladium on activated carbon (Pd/C). The amine (**5**) obtained was further processed under peptide coupling conditions with D(+)biotin, diisopropylethylamine (DIPEA), and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) to yield the biotin-functionalized product (**7**). The activated crosslinker (Biotin3xNHSX-linker) was obtained after deprotection of the ester units and direct functionalization of the carboxylic acids with N-hydroxysuccinimide (NHS) and N, N -dicyclohexylcarbodiimide (DCC) in THF.

**Figure 1.** Synthesis of the Biotin3xNHSX-Linker: (**I.**) *t*-butylacrylate, sodium, THF, 16h, 13%, (**II.**) 1. NaN3, DMF, 70 ◦C, 2d, 2. PPh3, H2O, THF, 4d, quant., (**III.**) *t*-butylacrylate, NaOH, DMSO, 15 ◦C and then warm up to rt, 20h, 23%, (**IV.**) benzyl chloroformate, Na2CO3 (aq.), DCM, 4d, 94%, (**V.**) 1. TFA, DCM, 2h, 2. (**2)**, DIPEA, PyBoP, DMF, 24 h, 69%, (**VI.**) H2, Pd/C, MeOH, 3d, 88%, (**VII.**) D(+)Biotin, DIPEA, PyBoP, DMF, 18h, 38%, (**VII.**) 1. TFA, DCM, Toluol, 2h, 2. *N*-hydroxysuccinimide, DCC, THF, 3d, quant. See SI and Figures S1 and S2 for details.

### *2.3. Liposome Preparation and Co-Sedimentation*

Lipids dissolved in chloroform/methanol were mixed at the desired molar ratio and composition. Chloroform was evaporated under a stream of nitrogen and traces of solvent were removed in vacuum for 4 h. Lipid films were stored at 4 ◦C until use. Liposomes were formed by hydration of the lipid film in a PBS −/− buffer. Small unilamellar vesicles (SUVs, 50 nm) or large unilamellar vesicles (LUVs, 100 or 200 nm) were obtained by extrusion through polycarbonate membranes (Avanti Polar Lipids

Inc.). SUVs were employed in quartz crystal microbalance with dissipation monitoring (QCM-D) experiments to facilitate vesicle rupture that occurs following vesicle coalescence on the sensor surface and results in the formation of a stable bilayer [21]. LUVs were used in liposome co-sedimentation and crosslinking experiments to ensure efficient co-pelleting and prevent rupture more often observed with high curvature vesicles.

Co-sedimentation experiments employed liposomes composed of POPC:Chol:POPS (60:20:20) with a defined size of 200 nm at a final concentration of 1 mg/mL. Liposomes were incubated for 1h at 4 ◦C with the desired AnxA2 derivative at a liposome/protein ratio of 10/1 (μL/μg) in PBS −/− buffer containing 1 mM CaCl2. Ultracentrifugation (UC) was performed to pellet the liposomes (96600 *g*, 4 ◦C for 20 min), the supernatant was collected and the pellet resuspended in 500 μL of PBS with 1 mM CaCl2, followed by 20 min incubation at 4 ◦C. After a second UC, the supernatant was collected and the pellet was resuspended in 500 μL of PBS with 5 mM EGTA and incubated for 30 min at 4 ◦C. A third UC yielded a supernatant (EGTA eluate) and a pellet that was resuspended in 500 μL of PBS with 5 mM EGTA. All fractions were analyzed via SDS-PAGE and immunoblotting with AnxA2-specific antibodies [22].

### *2.4. QCM-D Measurements*

Quartz Crystal Microbalance with Dissipation (QCM-D) analysis was performed as described before [21,23] using a Q-Sense E4 QCM-D (Q-Sense, Gothenburg, Sweden) equipped with four temperature controlled flow cells in a parallel configuration connected to a peristaltic pump (Ismatec IPC, Glattbrugg, Switzerland), at a flow rate of 80.4 μL/min. A bilayer was established by fusion of SUVs composed of POPC/DOPC/Chol/POPS/PI(4,5)P2 (37:20:20:20:3). Binding measurements were performed at 20 ◦C in HBS buffer supplemented with 250 μM Ca2<sup>+</sup> and AnxA2 constructs at 50 nM. 250 μM Ca2<sup>+</sup> and a relatively complex lipid mixture were chosen in the QCM-D experiments to directly compare the results to our previous data obtained by QCM-D analysis of AnxA2 and other AnxA2 mutants [21,24]. Frequency and dissipation shifts of the 7th overtone resonance frequency of the quartz sensor (QSX 303, 50 nm SiO2, 4.95 MHz) were recorded. OriginPro v. 9.1 (OriginLab Corp.) was used for data analysis.

### *2.5. Crosslinking of AnxA2*

LUVs (100 nm) composed of POPC:DOPC:Chol:POPS (40:20:20:20) were used at 3.33 μg/μL in HBS pH 7,4. Control #1 contained only the AnxA2 derivative (60 μg) in 1 mM CaCl2, i.e., a reaction in the absence of membranes, while control #2 contained a mixture of AnxA2 (60 μg) with 100 μg of LUVs, 5 mM EGTA, and 0.3 mM Biotin3xNHSX-Linker, i.e., a reaction in the absence of Ca2<sup>+</sup>. The actual Ca2+/crosslink sample consisted of LUVs (100 μg), AnxA2 (60 μg), 1 mM CaCl2, and 0.3 mM Biotin3xNHSX-Linker. A Ca2<sup>+</sup> concentration of 1 mM was used in these experiments to ensure efficient phospholipid binding of the protein. All components were mixed with exception of the Biotin3xNHSX-Linker, which was added after 30 min, and incubation was then continued for another 30 min while shaking. The reaction was stopped with 5× PAGE sample buffer without β-mercaptoethanol. For a better separation in the gel, 3 μL of a 100 mM EGTA solution was added to each sample. Samples were kept for 15 min at RT before analysis by 10% SDS-PAGE. Gels were stained with Coomassie Brilliant Blue. Quantification of crosslinked oligomer bands was achieved by gating the area in the stained gel lane above the position of AnxA2 dimers in all samples and relating its intensity to that of the monomer band in the respective sample. In this quantification, the actual dimer band was excluded because AnxA2 species migrating at the dimer position in SDS-PAGE, which do not reflect the physical state of the protein in solution and most likely form during SDS sample preparation, have been observed before [22]. They would mask an association not caused by the crosslink. Image Studio Lite (LI-COR Corporate Offices, NE, USA) and Graphpad Prism 4 (GraphPad Software, San Diego, CA, USA) were used for quantification.

### *2.6. Mutagenesis*

The human AnxA2 cDNA carrying a substitution at amino acid 66 (glutamate-for-alanine) to establish a monoclonal antibody epitope was cloned into the pSE420 expression vector as described [11] to yield pSE420-AnxA2A66E.

AnxA2 6x (pSE420-AnxA2A66E\_6x) was generated by mutating 6 amino acids in the template pSE420-AnxA2A66E using site-directed mutagenesis as described [23]. Mutations were introduced at amino acid positions: 81 (K to A), 189 (E to K), 196 (R to S), 206 (K to A), 212 (K to S) and 219 (E to K). The following primers were employed in the mutagenesis reactions: K81A\_For 5 -CCAGAG AAGGACCAAAGCGGAACTTGCATCAGCAC-3 and K81A\_Rev 5 -GTGCTGATGCAAGTTCCGC TTTGGTCCTTCTCTGG-3 . E189K\_For 5 -GGCTCTGTCATTGATTATAAACTGATTGACCAAGA TGCTC-3 E189K\_Rev 5 -GAGCATCTTGGTCAATCAGTTTATAATCAATGACAGAGCC-3 , K206A \_For 5 -CGCTGGAGTGAAGAGGGCAGGAACTGATGTTCCC-3 K206A\_Rev 5 -GGGAACATCA GTTCCTGCCCTCTTCACTCCAGCG-3 , R196S\_For 5 -CTGATTGACCAAGATGCTAGTGATCTC TATGACGCTGGAG-3 R196S\_Rev 5 -CTCCAGCGTCATAGAGATCACTAGCATCTTGGTCAAT CAG-3 , K212S\_For 5 -CAGGAACTGATGTTCCCTCGTGGATCAGCATCATG-3 K212S\_Rev 5 -CAT GATGCTGATCCACGAGGGAACATCAGTTCCTG-3 , E219K\_For 5 -ATCAGCATCATGACCAA GCGGAGCGTGCCC-3 E219K\_Rev 5 -GGGCACGCTCCGCTTGGTCATGATGCTGAT-3 , (Biomers, Ulm, Germany):

AnxA2 10x (pSE420-AnxA2A66E\_10x) was generated using pSE420-AnxA2A66E\_6x as template by introducing 4 additional amino acid substitutions at amino acid positions: 36 (R to S), 53 (V to A), 54 (T to A) and 328 (K to A). The following primers were used: R36A\_For 5 -CCTATACTAACTTTGA TGCTGAGAGCGATGCTTTGAACATTG-3 R36A\_Rev 5 -GTTTCAATGTTCAAAGCATCGCTCTC AGCATCAAAG-3 , V53A\_T54A\_For 5 -CAAAGGTGTGGATGAGGCCGCCATTGTCAACATTTTG-3 V53A\_T54A\_Rev 5 -CAAAATGTTGACAATGGCGGCCTCATCCACACCTTTG-3 , K328A\_For: 5 -TA AGGGCGACTACCAGGCAGCGCTGCTGTACCTG-3 K328A\_Rev 5 -AGGTACAGCAGCGCT GCCTGGTAGTCGCCCTTAG-3 (Biomers, Ulm, Germany).

### *2.7. Protein Expression and Purification*

For protein expression, E. coli cells transformed with the respective pSE420 plasmid were grown at 37 ◦C in LB medium supplemented with ampicillin to an optical density of 0.6 at 600 nm (OD600). Protein expression was then induced by addition of isopropyl β-D-1-thiogalactopyranoside to a final concentration of 1 mM. After expression for 4 h, cells were harvested by centrifugation at 5000× *g* for 10 min at 4 ◦C. Protein purification was performed by diethylamioethyl- and carboxymethyl-cellulose ion exchange chromatography and proteins were alkylated specifically at Cys-8 by 2-iodoacetamide treatment to prevent disulfide mediated protein crosslink as previously described [23].

### **3. Results**

### *3.1. Protein-Protein Interaction in Membrane-Bound AnxA2*

AnxA2 has been shown by atomic force microscopy to form two-dimensional assemblies on model membranes containing negatively charged phospholipids [13]. To analyze whether these assemblies are characterized by homotypic protein–protein interactions, we performed chemical crosslinking studies of membrane-bound versus soluble AnxA2. Therefore, we first developed a novel crosslinker, herein referred to as Biotin3xNHSX-linker, that due to its trifunctional nature should efficiently link proximal amino groups in proteins (Figure 1). Biotin3xNHSX-linker also contained a biotin group enabling streptavidin-mediated detection and enrichment.

Biotin3xNHSX-linker was then used to study the nature of AnxA2 assemblies on model membranes. Purified AnxA2 (Figure S3) was treated with Biotin3xNHSX-linker either in the absence of membranes or following Ca2+-dependent binding to liposomes containing the negatively charged AnxA2-binding lipid phosphatidylserine (PS). Figure 2 shows the results of these crosslinking experiments. While a

very small amount of higher molecular mass species was observed in the control reactions, i.e., AnxA2 samples in the absence of membranes or Ca2<sup>+</sup>, significant crosslink products indicative of oligomeric AnxA2 assemblies were generated when AnxA2 bound to PS-containing liposomes was subjected to the crosslinking reaction. Thus, our crosslink approach involving the Biotin3xNHSX-Linker indicates that membrane binding triggers the formation of AnxA2 oligomers, in which the proteins engage in lateral contacts spatially close enough to allow an effective covalent linkage by the trifunctional crosslinker.

**Figure 2.** SDS-PAGE of crosslinking reactions involving alkylated AnxA2 wild-type (WT). Lane 1: Control #1 (AnxA2 WT + Ca2+); lane 2: Control #2 (AnxA2 WT + LUVs + EGTA + Biotin3xNHSX-Linker); lane 3: Ca2+/membrane sample (AnxA2 WT + LUVs + Ca2<sup>+</sup> + Biotin3xNHSX-Linker). Brackets on the right indicate the positions of AnxA2 monomers, dimers, and oligomers. Dimer formation most likely occurs during sample preparation, whereas the oligomers likely present AnxA2 assemblies that form following membrane interaction and are then stabilized by the crosslinker. A representative result of *n* = 5 independently performed experiments is shown.

### *3.2. Annexin A2 Oligomers on Model Membranes are Stabilized by Lateral Protein-Protein Interactions*

To address the nature of the homotypic AnxA2 interaction, which occurs following membrane binding and can be stabilized by Biotin3xNHSX-linker, we generated two AnxA2 derivatives, in which residues predicted to participate in lateral protein-protein interactions in the crystal structure of an anti-parallel AnxA2 dimer [20] (see also pdb entry of the crystal structure of this AnxA2 dimer at 1XJL) were mutated to side chains of opposite charge or to alanine or serine (Figure 3). Importantly, the residues mutated are not part of the known type-II or type-III Ca2<sup>+</sup>-binding sites of AnxA2 [25] and so far have not been identified as sites of posttranslational modification. Moreover, the residues selected are characterized by polar or charged side chains and thus could engage in salt bridges and/or other ionic interactions that would favor oligomer formation. Provided that the two-dimensional AnxA2 assemblies on membranes involve these residues located on the lateral surface of the folded AnxA2 molecule, the mutants, herein named AnxA2 6x and AnxA2 10x, should show a compromised oligomer formation and thus Biotin3xNHSX-linker mediated crosslink. Moreover, as the mutations do not involve residues of the Ca2+/membrane binding sites, AnxA2 6x and AnxA2 10x are expected to retain the capability to bind to membranes containing acidic phospholipids.

**Figure 3.** AnxA2 crystal structure highlighting mutations introduced in the AnxA2 6x and 10x constructs. AnxA2 6x top [81 (Lys to Ala), 189 (Glu to Lys), 196 (Arg to Ser), 206 (Lys to Ala), 212 (Lys to Ser) and 219 (Glu to Lys)] and AnxA2 10x bottom [36 (Arg to Ser), 53 (Val to Ala), 54 (Thr to Ala) and 328 (Lys to Ala)]. Illustrations were created using the AnxA2 crystal structure (PDB code: 1XJL).

AnxA2 6x and 10x were purified following the protocol developed for the wild-type protein (Figure S1). Importantly, this also involved alkylation of the exposed cysteine-8 as disulfide bridge formation involving this cysteine residue is observed under oxidative conditions [23]. The mutants were first characterized with respect to their ability to bind to membranes containing acidic phospholipids in a Ca2<sup>+</sup>-dependent manner by employing liposome co-pelleting and solid-supported membrane-binding assays. Figure 4 shows that AnxA2 6x and 10x effectively bind to PS-containing liposomes in the presence of Ca2<sup>+</sup>. As observed for the wild-type protein, this binding is fully reversible, i.e., the bound protein is released when the liposome-protein mixtures are treated with the Ca2<sup>+</sup> chelator EGTA. Analysis of protein binding to solid supported membrane bilayers was carried out using a quartz crystal microbalance with dissipation (QCM-D). QCM-D is a well-established tool to evaluate and quantify protein–lipid interactions [21]. By applying SUVs, a bilayer can be formed on a sensor chip connected to a quartz microbalance and the ability of proteins to interact with this lipid bilayer can be measured via decrease in the resonance frequency of the quartz crystal that occurs as a result of mass adsorption. Importantly, due to the unique Ca2<sup>+</sup>-dependent and fully reversible interaction of AnxA2 with membranes, AnxA2 bound to the solid-supported membrane can be readily released from the bilayer by Ca2<sup>+</sup> chelation with EGTA [24]. QCM-D recordings performed with the different AnxA2 derivatives revealed that AnxA2 wild-type (WT) and the AnxA2 6x and 10x mutants show similar binding kinetics and resonance frequency shifts (ΔΔF) of 19 Hz for AnxA2 WT, 18.5 Hz for

AnxA2 6x, and 16.7 Hz for AnxA2 10x (Figure 4). Moreover, in each case the binding is fully reversible upon addition of EGTA and the dissipation increase is rather minimal indicating that a relatively rigid protein layer is formed on the solid-supported bilayer.

**Figure 4.** Membrane binding of AnxA2 constructs. Left, Liposome co-pelleting assay analyzed by SDS-PAGE of the different fractions. AnxA2 wild-type (WT), AnxA2 6x, or AnxA2 10x were mixed with PS-containing liposomes in the presence of 1 mM Ca2<sup>+</sup>. Liposomes were pelleted and the supernatant, i.e., non-bound material, was collected (lane 1). Liposomes were then washed in Ca2<sup>+</sup>-containing buffer, yielding a second supernatant (Ca2<sup>+</sup> wash, lane 2). Subsequently, the pelleted liposomes were washed with EGTA-containing buffer resulting in release of the Ca2<sup>+</sup>-dependently bound material (EGTA eluate, lane 3). The final liposome pellet containing non-released protein is shown in lane 4. The gel shows a representative result of *n* = 5 independently performed experiments. Right, QCM-D measurements, frequency (as deviation from resonance frequency, ΔF) is shown in the upper recordings and dissipation (ΔD) in the lower. Following formation of a solid-supported bilayer (at a ΔF of around −30 Hz in these settings), AnxA2 WT was added in the presence of Ca2+, resulting in a drop in resonance frequency to approximately −49 Hz. Addition of EGTA removed all bound protein with resonance frequency returning to its initial bilayer value (−30 Hz). Recording was continued with subsequent additions (in Ca2<sup>+</sup> containing buffer) and release (in EGTA containing buffer) of AnxA2 WT (to show reversibility of the reaction), AnxA2 6x, and AnxA2 10x. The QCM-D recordings were performed at least three times each for the different, independently purified AnxA2 derivatives.

Next, the lateral side chain mutants, AnxA2 6x and AnxA2 10x, were characterized with respect to their ability to form crosslinkable oligomers following membrane binding. Mutant proteins were bound to PS-containing liposomes and proteins residing in close proximity were covalently linked employing the Biotin3xNHSX-linker. Figure 5 shows that the capability of forming crosslinked high molecular mass products was significantly compromised in both mutants when compared to the wild-type protein (Figure 2). A quantification of the oligomeric products revealed that higher molecular mass products representing crosslinked AnxA2 oligomers are reduced to approximately 48% and 33% for AnxA2 6x and AnxA2 10x, respectively, when compared to the wild-type protein. Thus, side chains identified in AnxA2 crystals as potential protein–protein contact sites appear to reside in close proximity in membrane-bound AnxA2, suggesting that lateral protein–protein interactions accompany the membrane association of AnxA2

**Figure 5.** Chemical crosslinking of alkylated AnxA2 mutant proteins. Left, SDS-PAGE of crosslinking reactions involving AnxA2 6x and 10x. Each AnxA2 derivative was subjected to chemical crosslinking in the presence or absence of Ca2<sup>+</sup> and LUVs. Lanes 1: Controls #1 (AnxA2 + Ca2+); lanes 2: Controls #2 (AnxA2 + LUVs + EGTA + Biotin3xNHSX-Linker); lanes 3: Ca2+/membrane sample (AnxA2 + LUVs + Ca2<sup>+</sup> + Biotin3xNHSX-Linker). Right, quantification of crosslinked oligomer bands for each AnxA2 derivative (calculated in relation to the respective monomer band, see Materials and Methods). Given is the relative percentage of these oligomer bands compared to those obtained for the wild-type protein analyzed in a parallel reaction. Three independent crosslinking reactions were analyzed for each protein species and the standard error of means is indicated.

### **4. Discussion**

In addition to binding to membranes containing acidic phospholipids in the presence of elevated Ca2<sup>+</sup> concentrations, at least some annexins can also form two-dimensional clusters or assemblies on model membranes. Such assemblies have been extensively studied in the case of AnxA5, which forms highly ordered 2D crystals on the membrane [26–28]. In the case of AnxA5, the building blocks in these crystalline arrays are trimers, and residues mediating and stabilizing trimer formation have been mapped [29]. In contrast to AnxA5, AnxA2 does not form such ordered crystalline arrays on model membranes, but rather appears to associate into more irregular two-dimensional assemblies of amorphous shapes [13]. Interestingly, these assemblies have been shown to segregate the AnxA2-binding phospholipids [PS, PI(4,5)P2] into domains underneath the bound proteins, and this activity has been suggested to underlie the function of AnxA2 in Ca2+-regulated exocytosis [11,12,14, 16,30]. Thus, understanding the molecular basis of the AnxA2-mediated lipid segregation is of high relevance for understanding the cellular properties of the protein.

Here, we have used a chemical cross-linking approach employing a novel trifunctional compound and combined this with the characterization of AnxA2 mutant derivatives to address the nature of the 2D assemblies formed by this annexin on model membranes. Our analysis revealed that these assemblies are characterized by close protein proximity most likely involving protein–protein interactions that can be stabilized by chemical crosslinking. Moreover, we identified amino acid residues residing on the lateral surface of the folded protein that are either directly involved in the crosslink or mediate interactions between bound AnxA2 moieties that are lost in the 6x and 10x mutants. Thus, it appears likely that the assemblies containing AnxA2 and its interacting lipids [PS, PI(4,5)P2] are at least partly stabilized by lateral protein–protein interactions.

Our analysis benefitted from the introduction of a novel trifunctional crosslinker. Typically, chemical crosslinkers are capable of connecting spatially proximate amino acids of proteins. When selecting the crosslinker, the chemical selectivity and activity of the functional groups towards the amino acids to be linked should be taken into account. In addition, the "length/size" of the crosslinker is crucial, since it defines the maximum spatial distance of the linked amino acids. The Biotin3xNHSX-linker synthesized here is a homo-trifunctionalized NHS ester crosslinker, whose N-hydroxy-succinimide group can react with primary amines or alcohol groups, thus yielding high crosslink efficiency that allowed the detection of AnxA2 protein proximity in the membrane-bound form.

Although AnxA2 has been shown to cluster the lipids bound by the protein, the composition of the membrane patch underneath bound AnxA2 is not known. Most likely it also contains cholesterol as it has been shown that membrane cholesterol renders the AnxA2 binding cooperative, suggesting a cooperative nature of the assembly formation. As the protein assemblies formed by AnxA2 on the membrane are accompanied by close protein apposition, it appears plausible that the cooperation is at least in part mediated by a conformational change in the membrane-bound AnxA2, which renders it capable of undergoing protein–protein interactions. Future experiments involving streptavidin-mediated enrichment of membrane patches bound to Biotin3xNHSX-linker-crosslinked AnxA2 should shed some light on the lipid composition in the associated membrane. The Ca2+-regulated and reversible manner underlying the cluster formation of AnxA2 on cholesterol and PS/PI(4,5)P2 containing membranes is most likely highly relevant for its cellular function as it would support the dynamic formation of lipid platforms that could function as sites for exocytosis, and are also involved in epithelial cell polarization shown to depend on AnxA2 [15].

In summary, we introduce a novel water-soluble trifunctional NHS crosslinker, which was suitable to covalently link AnxA2 protein moieties bound to model membranes containing negatively charged phospholipids. Moreover, we could identify amino acid side chains residing on the lateral surface of folded AnxA2 that are required for efficient crosslink, and thus likely support lateral protein–protein interactions of membrane-bound AnxA2, possibly by providing salt bridges and/or other ionic interactions.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/9/5/1169/s1. Supplementary Methods including detailed protocol of the chemical synthesis of Biotin3xNHSX-Linker. Figure S1: 1H-NMR and 13C-NMR spectrum of Biotin3xNHSX-Linker, 400 MHz, CDCl3. Figure S2: HSQC spectrum of Biotin3xNHSX-Linker. Figure S3: SDS-PAGE and Western Blot of AnxA2 WT purification steps.

**Author Contributions:** Conceptualization, B.J.R., V.G.; resources, A.L.L.M., S.K., J.M., M.H., D.G.; data curation, A.L.L.M., S.K., J.M., M.H., D.G., B.J.R., V.G.; writing—original draft preparation, V.G., A.L.L.M., S.K.; writing—review and editing, B.J.R., V.G., A.L.L.M., S.K.; visualization, A.L.L.M., S.K., J.M., M.H., D.G.; supervision, B.J.R., V.G..; project administration, B.J.R., V.G.; funding acquisition, B.J.R., V.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Deutsche Forschungsgemeinschaft (grants SFB 858/B19, EXC 1003, SFB 1348/B04, Ge514/6-3).

**Acknowledgments:** We would like to thank Ursula Rescher for helpful discussions and Abigail Cornwell and Michael Hülskamp for protein purification.

**Conflicts of Interest:** The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

### **References**


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