**3. Galectin 13**

#### *3.1. The PP13 Protein and its mRNA*

Galectin 13 (Gal-13), also known as LGALS13 and placental protein 13 (PP13), is the most studied galectin of the anthropoid primates. As one of a six cluster primate genes, it is located on chromosome 19q13 [32], and is one of 56 known placental proteins. It was first isolated from human term placenta in 1983 and characterized by Bohn et al. [37]. Normal term placenta has approximately 2.5 mg of PP13, and, according to Bohn, PP13 represents ~7% of the total placental proteins. PP13 shows structural and functional homologies to the ß-galactoside-binding lectins [1], with high homology to the other members of the cluster in their CRD [31,32,38,39]. Although so far no specific individual receptor for PP13 (in the classical sense) has been identified, affinity chromatography and mass spectroscopy determined high affinity binding of PP13 to annexin IIa, a member of Ca2+ and phospholipid binding proteins of the extracellular matrix, and to beta/gamma actin in the cytoplasm [10,40]. PP13 has high affinity to sugar residues, especially to N-acetyl glucose amine, fucose, and N-acetyl galactose amine [10]. It also binds sugar residues of the B and AB antigen of the ABO blood groups [11], a binding that regulates the availability of free PP13 in the blood of pregnan<sup>t</sup> women. This binding has

been found to influence the risk assessment and preeclampsia prediction of PP13 [41], as will be further detailed below.

PP13 is expressed from a very early stage of pregnancy, and can be detected in the maternal blood already at week five of gestation [42], or 3 weeks after embryo return in IVF (Meiri, unpublished results). Immunohistochemistry and RNA hybridization studies have pointed to its predominant localization in the placental syncytiotrophoblast layer, placental blood vessels, and specific sites within the placental bed [31,42,43]. Early studies by Than et al. indicated its presence in the syncytiotrophoblast [10]. PP13 is detected in the cytoplasm and mainly along the apical plasma membrane of the syncytiotrophoblast [42,43]. It can also be detected in their nuclei, at least during very early gestation [43]. In cases of oxidative stress, strong staining for PP13 appears in the increasingly appearing syncytiotrophoblast microparticles (STBM, or necrotic bodies) [42,44]. A process of aponecrosis is accompanied by placental shedding of STBM during preeclampsia [42,45].

#### *3.2. Insights on the Gene and Protein Structures*

The LGALS13 gene encodes for PP13, and is comprised of a long promoter region at the 5 prime end followed by four exons: E1 (60 bp), E2 (72 bp), E3 (211 bp), and E4 (251 bp) spaced by introns (Figure 1). Intronic regions vary between 499 bp and 1834 bp in length. Exon 4 and part of exon 3 of the LGALS13 gene exclusively code for the entire amino acids included in the CRD domain [6,10,38,46].

**Figure 1.** Schematic diagram of the LGALS13 gene and its mRNA variants. Top—The exons and introns are marked by boxes and lines, respectively. Lower panels represent the mRNA and the protein coding region. The wild type Gal-13 (PP13) consists of four full exons. The truncated Gal-13 variant delT221 is missing part of exon 3 and the full exon 4, while the Dex-2 variant is missing exon 2. The two variants—the truncated delT221 variant and the spliced variant Dex-2 are both naturally occurring variants along with the promoter polymorphic variant of -98 A/C.

The open reading frame of PP13 encodes for 139 amino acids [10,46]. The calculated molecular weight of the monomer is ~ 16.12 kDa. In-vitro studies have shown that its expression is up-regulated by the binding of the TFAP2A transcription factor [32]. Other studies pointed to the link between PP13 expression and human chorionic gonadotropin (hCG) [47] that drives the fusion of villous cytotrophoblasts with the overlying syncytiotrophoblast [48]. Indeed, fusion of differentiating trophoblasts to form the syncytiotrophoblast is accompanied by increased PP13 expression. Fusion also increases PP13 expression in the trophoblast-derived BeWo cell line [47,49].

We engineered several recombinant PP13 variants. Initially, a Histidine-tag (His-PP13) variant was constructed, produced in E. coli, purified, and characterized [10,38]. The resultant His-PP13 fails to dimerize via disulfide bonds since the His-tag prohibits one of its cysteine SH residues from forming a dimer. The molecular conformation of such a monomeric state of PP13 prohibits the formation of the naturally occurring homodimer, and this variant tends to form a long chain of *head-to-tail* linked oligomers, which are characterized by low stability in solutions. Treatment of the His-PP13 variant with the reducing agen<sup>t</sup> dithiothreitol (DTT) keeps the protein in a monomeric form, prohibiting the formation of long chain oligomers. This monomeric form exhibits long stability in solution, and in the presence of DTT lyophilized His-tag PP13 has an estimated shelf-life of 12 years or longer [50]. The second recombinant PP13 variant lacks the histidine tag (rPP13) and is expressed in E. coli [38,51]. The resultant protein was isolated from the inclusion bodies as a monomer that spontaneously homo-dimerizes to form a 32 kDa protein that is very stable in aqueous solutions. Further aggregation to trimers and tetramers is marginal [46]).

### *3.3. PP13 Secretion from the Placenta*

Lacking a signal sequence for transmembrane transport [6], it was estimated that the release of PP13 is accomplished in a manner typical to other galectins, namely via the liberation of extracellular vesicles [12,52,53] (Figure 2). A release of un-packed protein via co-transfer with carrier proteins or endosomes was also suggested to be a calcium dependent mechanism [54,55]. In fact, it has been shown that the PP13 release from immortalized placental cells (BeWo cells) is significantly augmented with the use of a calcium ionophore [44]. Like other galectins, PP13 can re-enter cells by endocytosis via recycling of endocytic vesicles [56].

**Figure 2.** PP13 release from placental syncytiotrophoblast. Extracellular vesicles are cell-derived membrane particles, including exosomes (30–200 nm), microvesicles (100–1000 nm), and apoptotic bodies (>1000 nm). They are released from the placental syncytiotrophoblast layer. During normal turnover, the syncytiotrophoblast releases late-apoptotic syncytial knots (1–5 μm) as large corpuscular structure into the maternal blood. At the same time, microvesicles and exosomes are released and can pass through capillary blood vessels. PP13 cargo of microvesicles and exosomes appears on both types of these extracellular vesicles, on the surface and inside the vesicles. These vesicles may interact with various cell types (red and white blood cells or endothelial cells) and convey di fferent messages to the maternal body.

Sammar et al. [52] discovered a novel pathway for PP13 secretion that may be most relevant to the protein level in maternal blood. PP13 liberation is executed through the release of extracellular vesicles (EVs), mainly microvesicles and exosomes, carrying PP13 on the surface of EVs and/or inside them [52]. The microvesicles and exosomes that carry the PP13 cargo communicate with maternal organs to influence their response, both during normal and complicated pregnancies. Evidence has been obtained for the potential interaction of PP13 in such extracellular vesicles with red and white blood cells, as well as the endothelium (Figure 2).
