**1. Introduction**

Human cysteine proteases participate in several physiological processes, such as the degradation of peptides and proteins [1], and constitute the major components of lysosomes [2]. In this group of enzymes, cathepsin L (CatL) is an endopeptidase that degrades intracellular and endocytosed proteins in the lysosome. Recent studies have suggested that this protease plays many critical roles in diverse cellular settings. Thus,

**Citation:** Linhares, D.d.C.; Faria, F.; Kodama, R.T.; Amorim, A.M.X.P.; Portaro, F.C.V.; Trevisan-Silva, D.; Ferraz, K.F.; Chudzinski-Tavassi, A.M. Novel Cysteine Protease Inhibitor Derived from the *Haementeria vizottoi* Leech: Recombinant Expression, Purification, and Characterization. *Toxins* **2021**, *13*, 857. https://doi.org/ 10.3390/toxins13120857

Received: 1 October 2021 Accepted: 11 November 2021 Published: 2 Decemeber 2021

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overexpression of CatL has been reported in several human diseases, such as liver fibrosis, Type I and II diabetes, cardiac, bone, immune, and kidney disorders [3–5]. Additionally, membrane-bound or released CatL mediates the cleavage of the S1 subunit of the coronavirus surface spike glycoprotein, participating in the invasion into human host cells via the so-called late pathway [6,7]. Although the preferred pathway for infection is via the serine protease TMPRSS2 (early pathway), CatL, together with TMPRSS2, present tempting targets for pharmacological inhibition [7].

The endogenous inhibitors named cystatins are the most effective mechanism for controlling the activity of cathepsin L [1,2]. It is interesting to note that CatL inhibitors can also be found in several organisms, such as hematophagous animals, where they play an important role in their survival. Cystatins from hematophagous animals participate in the immunological modulations [8], reducing the processing capacity and presentation of antigens by the host's antigen-presenting cells. The production of cytokines by the host's macrophages is also affected, resulting in an anti-inflammatory response [9]. Tick cystatins have been characterized as capable of inhibiting several cathepsins involved in blood digestion, embryonic development of the tick, and the immune response of the host [10,11]. Cystatins that specifically inhibit CatL have also been described, such as sialostatin L, present in the *Ixodes scapularis* tick. Sialostatin L inhibits the protective proteolytic activity of host cells at infestation sites, thus promoting tick survival [12]. In addition, it also holds anti-inflammatory and immunosuppressive activities through the inhibition of killer T cells [13].

Tick cystatins have been described as potent and specific protease inhibitors, expanding their potential to be used further as new vaccine antigens and anti-tick drugs of medical importance [10,14]. Furthermore, studies of cystatins in cell models, such as cancer [15], inflammation [16], and immunomodulation [17], demonstrate the significant potential of cystatins as new drugs or prototypes for developing new drugs for veterinary and human use.

Similar to ticks, leeches are hematophagous parasites that possess interesting compounds in their saliva capable of assisting the maintenance of blood flow for successful feeding. Thus, many anticoagulant and antiplatelet molecules have been characterized for this purpose [18–21]. However, cysteine protease inhibitors have rarely been studied in the literature, and, until now, no cystatin—native or recombinant—was characterized for leeches apart from the cystatin B gene first characterized in *Theromyzon tessulatum* leeches. It was demonstrated that the innate immune response in the leech involves a cysteine protease inhibitor not previously detected in other invertebrate models, highlighting the need for further study of the innate immunity mechanism in these animals [22].

The present work is the first of its kind to characterize the recombinant cystatin of leeches attained through the sequence of the Hviz00340 transcript from the transcriptome of *Haementeria vizottoi* [23]. Cystatin-Hv was successfully expressed in *Pichia pastoris*, and after purification, was characterized for its ability to inhibit cathepsin L. The present study of the first recombinant cystatin derived from leeches will allow a more detailed investigation of its role in feeding the parasite. In addition, the molecule itself can be further investigated in cellular and in vivo models to better understand its potential role in the search for new molecules of therapeutic interest.

#### **2. Results**

#### *2.1. Selection and Purification of Cystatin-Hv*

The cystatin-Hv cDNA sequence, coding for the predicted cysteine protease inhibitor derived from the leech *Haementeria vizottoi*, is composed of 396 nucleotides, resulting in 131 amino acids. The signal peptidase cleavage site of Cystatin-Hv predicted by SignalP and Expasy was located between the 19th and 20th amino acid residue. To express and secrete the recombinant protein in *Pichia pastoris*, the DNA fragment corresponding to the mature protein was cloned into pD912-AK. The calculated molecular mass of this predicted protein is 12,487.92 Da, and the pI is 5.23.

This protein presents domain and typical cystatin active sites, such as the highly conserved first hairpin loop QVVAG and the second hairpin loop PW [24], associated with cystatins type C (Figure 1). The highest identity is found with a hypothetical protein of the leech *Helobdella robusta* (47% identity, access number: XP\_009012188.1), presenting low identity with the preliminary characterized cystatin B of the leech *Theromyzon tessulatum* (21% identity, access number: AAN28679) [25], the Sialostasin of the tick *Ixodes scapularis* (19% identity, access number: Q8MVB6) [26], the Iristasin of the tick *Ixodes ricinus* (14% identity, access number: 5O46\_A) [16], the OmC2 of the tick *Ornithodoros moubata* (21% identity, access number: 3L0R\_B) [27], and with the cystatin 2a of the tick *Rhipicephalus (Boophilus) microplus* (21% identity, access number: AGW80657.1) [28].

**Figure 1.** Multiple alignments of cystatin-Hv with other cystatins from leeches and ticks. Regions highlighted in red show the first hairpin loop and the second hairpin loop. The black arrow indicates the signal peptide cleavage site. The conservation of the sequences is shown in yellow bars, and the score values 9 and 10 (or asterisk) indicate total conservation of the aligned sequences; the consensus sequence is shown in black bars (Clustal Omega).

> For the expression of the recombinant Cystatin-Hv, the selected expression vector carries a secretion signal derived from *S. cerevisiae* (SS alpha-factor), located upstream of the insert, which fused to the recombinant protein, promotes its secretion out of the cell. The production of Cystatin-Hv using *P. pastoris* (X33) was performed as described, with four 100 mL replicates, beginning the expression step with OD600nm around 5, going up to OD600nm 69 after 44 h of assay (average values), as shown in Figure 2.

> Culture supernatant after 44 h of expression was recovered, concentrated, dialyzed, submitted to ion-exchange chromatography, and pooled fractions were analyzed on SDS-PAGE and used for inhibition assays against papain (Figure 3). Inhibitory activity was detected relative to pool 2 (eluted within 10% to 15% of NaCl 1 M buffer), evidenced by the decrease in fluorescence emission (the result of proteolysis) when compared to other pooled fractions and positive control (reaction without pooled fractions). Inhibition of papain occurred in a dose–response manner, as shown in Figure 4.

**Figure 2.** Growth curve of *P. pastoris* (X-33) pD912-AK: Cystatin-Hv during the assay of recombinant protein expression (**a**) and Coomassie-stained SDS-PAGE (15%) of proteins recovered from culture supernatant collected during the experiment (**b**). Vertical arrows: points of methanol feeding and supernatant collection; lane M: low molecular weight protein markers "Precision Plus Protein™ Dual Color Standards" (Bio-Rad); lane BMMY: fresh culture medium; lanes 0 h to 44 h: proteins recovered at different incubation times; horizontal arrow: expected sized protein band.

**Figure 3.** Chromatogram showing the elution profile of culture supernatant proteins from MonoQ resin, using 1 M NaCl as elution buffer, whose fractions were pooled 1–6 for analysis (**a**) SDS-PAGE of pooled fractions (**b**) and inhibition assay of papain activity (papain 10.7 nM, zFR-MCA substrate 5 μM) in the presence of protein pools (250 ng) (**c**). (**b**) Lane M: low molecular weight protein markers "SDS standards low range" (Bio-Rad); O: culture supernatant; lanes 1 to 6: proteins recovered from pools; vertical arrow indicates protein band compatible with Cystatin-Hv. (**c**) Here C+ indicates the positive control (papain and substrate), and C− indicates negative control (substrate).

**Figure 4.** Inhibition assay of Cystatin-Hv (1 μg, 5 μg, 10 μg e 15 μg of pool 2 from ion-exchange chromatography) against papain (21.4 nmol/L or 50 ng) using 5 μM of Z-FR-MCA substrate. An estimate of the half maximal inhibitory concentration (IC50) is given, considering the dominance of Cystatin-Hv in pool 2.

Once the inhibitory activity was detected, pooled fractions (referred to as pool 2) were further purified by size-exclusion chromatography, leading to a single band named Cystatin-Hv (Figure 5). Mass spectrometry analysis (LC-MS/MS) was performed to confirm the accuracy of the molecular mass and allowed the identification of Cystatin-Hv with eight unique peptides, covering 89% of the mature protein sequence (Supplementary Figure S1).

**Figure 5.** Size exclusion chromatography of pool 2 (from previous ion-exchange chromatography), highlighting protein bands into fractions B10 and B11 (purified Cystatin-Hv), on SDS-PAGE, related to inhibitory activity against papain and cathepsin L (**a**), SDS-PAGE analysis of purified Cystatin-Hv under reducing (1) and nonreducing (2) conditions (**b**). Molecular markers used were "Precision Plus Protein™ Dual Color Standards" (Bio-Rad) (**a**) and "SDS standards low range" (Bio-Rad) (**b**).
