*2.2. Inhibition Studies*

Enzymatic kinetics assays were performed with three different concentrations of Cystatin-Hv (8 nM, 16 nM, and 24 nM respectively), cathepsin L (0.4 nM) and two concentrations of Z-FR-MCA substrate (1 Km and 2 Km). Experimental data of reaction rates were linearized and treated further as proposed by Dixon [29] (Figure 6), thus allowing us to determine the inhibition constant, Ki, of 7.9 nM.

**Figure 6.** Dixon diagram presenting kinetic enzymatic assays of cathepsin L inhibition by Cystatin-Hv. Two substrate concentrations (1 Km or 2.65 μM and 2 Km or 5.3 μM) and three cystatin-Hv concentrations (8 nM, 16 nM, and 24 nM) were used. The inhibition constant was set as 7.9 nM. The graphic was made using the software GraphPad Prism 5. Slope value: 7.964 <sup>×</sup> <sup>10</sup>−<sup>5</sup> to 9.909 <sup>×</sup> <sup>10</sup>−<sup>5</sup> (2.65 <sup>μ</sup>M) and 2.496 <sup>×</sup> <sup>10</sup>−<sup>5</sup> to 4.547 <sup>×</sup> <sup>10</sup>−<sup>5</sup> (5.3 <sup>μ</sup>M) for a 95% confidence interval. UF: Units of Fluorescence.

### **3. Discussion**

The present work was carried out to assess biodiversity and contribute to developing new molecules that can generate and inspire new therapeutic possibilities. In this context, the search for molecules from animal secretions related to feeding is rather compelling since, from the evolutionary perspective, the proteins present in such secretions have been subjected to selective pressure for better efficiency to ultimately facilitate the animal's survival and perpetuation [30]. Hence, it is expected that proteins present in the saliva of hematophagous animals should have a specific action on the host or prey, and it is up to the researchers to isolate these components, identify their actions, and study how these molecules can be used for our benefit.

Cystatins present in hematophagous have the key function in inhibiting endogenous cysteine proteases of the animal and in helping the feeding process as well. The saliva of these animals contains not only inhibitors that reduce host blood clotting and premature blood clotting inside the gut but also molecules that interfere and inhibit the performance of the host's immune system [17,24] and allow the hematophagous to keep feeding for an extended period.

An important dimension in the discussion on cystatins present in the salivary complexes of leeches relates to the issue of the innate immune response. It is reported that symbiotic bacteria, antimicrobial peptides, and phagocytic immune cells play a protective role in defending from harmful agents and preventing premature degradation of the ingested blood meal, which is concentrated and maintained over a period of many weeks inside the digestive tract [31]. Although the leech defense system has been poorly investigated, studies with cystatin B have demonstrated the involvement of this cysteine protease inhibitor in the innate immunity of *Theromyzon tessulatum* leeches since an increase in cystatin B gene expression has been shown in large circulating coelomic cells after bacterial challenge [22,25]. While more studies are needed to further elucidate the function of cysteine protease inhibitors for leeches, it is likely that these molecules also work as immunoregulators, given the major implication of cathepsins in immunity [22], similarly to what has been described for ticks, a better characterized group of hematophagous.

In ticks, this group of inhibitors has been extensively explored. It was noted that in tick saliva, the majority (84%) of cystatin transcripts belong to a group that is secreted extracellularly, suggesting a predominantly immunoregulation function [32]. Cystatin OmC2, from the *Ornithodoros moubata* tick, for example, targets two lysosomal cathepsins, S and C, which perform the function of processing antigens in antigen-presenting cells, apart from affecting the maturation of dendritic cells [17]. Cystatin Iristatin, identified

from the tick *Ixodes ricinus*, inhibited the proteolytic activity of cathepsins L and C and decreased the production of several inflammation inducers (IL-1, IL-4, IL-9, IFN-γ) by different populations of T cells, among other anti-inflammatory activities [16].

Cystatins are also present in humans, where, as in other animal species, they act as inhibitors of endogenous cysteine proteases, such as cathepsins. Overexpression of these enzymes has been observed in a number of tumorous cells, such as breast, lung, brain, head, neck, and melanoma cancers, where they act on the degradation of the extracellular matrix enabling tumor growth, invasion of other tissues, and migration into the bloodstream [2,8]. In particular, cathepsin L is a lysosomal endopeptidase widely expressed and involved in the degradation of intracellular or phagocyted proteins that can also be found in a variety of extracellular media as well as in the cell nucleus [33,34]. In this way, positive regulation of the lysosomal endopeptidase cathepsin L has often been observed in a number of human cancers, and its levels of expression in tumor tissues or their presence in the environment adjacent to the tumors is considered to be largely correlated with their aggressiveness [2,34–36].

There is little information available about cystatins regarding leeches, most of which are the results of transcriptomic analyses suggesting the participation of these molecules in the immune response [25]. Functional studies with cystatins present in the leeches have not yet been reported in the literature.

The present study started with the library of transcripts of the salivary complexes of the leech *Haementeria vizottoi*, where 1204 Isotigs were obtained, and among them, 123 were identified as related to feeding [23]. After further screening, one Isotig was selected for this study, starting with the gene sequence, through the cloning and recombinant production of the protein, Cystatin-Hv, to its functional characterization.

In general, the benefits of protein production by *P. pastoris* system include appropriate folding, especially for cysteine-rich proteins (in the endoplasmic reticulum) and secretion (by Kex2 as signal peptidase) of recombinant proteins to the supernatant environment of the expression [37]. In the case of Cystatin-Hv, a protein with five cysteines, the expression occurred satisfactorily, as expected, with compatible quality acceptable to the scalability of the process. Furthermore, the use of the *P. pastoris* expression system, due to its limited production of endogenous secretory proteins, is known to favor an easy purification protein process [37]. In this sense, the isolation in two chromatography steps was sufficient to achieve a pure form of recombinant Cystatin-Hv, similar to the purification process performed by Cardoso [38], characterizing a tick cystatin that presented an inhibitory effect against the activity of a hemoglobin lytic enzyme.

The inhibition assays allowed us to confirm the activity of Cystatin-Hv, in its recombinant form, as a strong inhibitor of cathepsin L. Further, results plotted in the Dixon diagram (Figure 6), with curves intercept on the X-axis, suggest a noncompetitive mechanism of action for this inhibitor. Although cystatins are usually described as competitive inhibitors, the noncompetitive mechanism was observed for soybean [39], corn [40], and chestnut seed [41] plant cystatins, as well as for human Cystatin SA [42]. In order to improve Cystatin-Hv characterization and understanding of its mechanism, complementary assays are to be performed, also against other known cathepsins. The inhibition constant (Ki) in the order of nM (7.9 nM) is compatible with the one found in the literature for the dissociation constant of cathepsin L with human cystatins [1]. Similar Ki values were obtained related to cystatins of hematophagous animals such as the bovine ectoparasite *Rhipicephalus microplus*, whose protein identified as Rmcystatin-4 was cloned, expressed, and purified, and has demonstrated inhibitory activity against cathepsin L with a Ki of 11.1 nM [38]. The cystatin OmC2 from the tick genus *Ornithodoros* also presented similar Ki values in the range of nM against lysosomal cathepsins S and C [17].

Although the Ki value in relation to papain has not been obtained, the IC50 value of approximately 0.12 μM, considering Cystatin-Hv dominant in pool 2, indicates a greater potency of cystatin-Hv for the inhibition of cathepsin L. However, future studies should be carried out with papain and other cathepsins to assess the specificity of cystatin-Hv in relation to a particular protease.

The character of recombinant Cystatin-Hv as an inhibitor of cysteine proteases, especially human cathepsin L, opens interesting possibilities for its potential biological function as an immunoregulator and an anti-inflammatory molecule, justifying our efforts to study this protein in its recombinant form. Inhibition of CatL has also been recognized as having a significant role in the prevention of cell invasion by viruses of the coronavirus family in vitro. Given the recent emergence of the novel SARS-CoV-2, calls for more attention to inhibitors of this cysteine protease are well justified [7]. Thus, the first recombinant cystatin from leeches will allow a more detailed investigation of its role in feeding the parasite. In addition, the molecule itself can be investigated in cellular and in vivo models to understand its significance in the possible search for new molecules of therapeutic interest.
