Characterization and expression profiling of serine protease inhibitors in the yellow mealworm Tenebrio molitor

Authors: LI, GUANG-YA - Southwest Forestry University; YANG, LI - Southwest Forestry University; XIAO, KAI-RAN - Southwest Forestry University; SONG, QI-SHENG - University Of Missouri; Stanley, David; WEI, SHU-JUN - Southwest Forestry University; ZHU, JIA-YING - Southwest Forestry University

Submitted to: Archives of Insect Biochemistry and Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/3/2022
Publication Date: 6/24/2022
Citation: Li, G., Yang, L., Xiao, K., Song, Q., Stanley, D.W., Wei, S., Zhu, J. 2022. Characterization and expression profiling of serine protease inhibitors in the yellow mealworm Tenebrio molitor. Archives of Insect Biochemistry and Physiology. 111(3). Article e2194. https://doi.org/10.1002/arch.21948.
DOI: https://doi.org/10.1002/arch.21948

Interpretive Summary: Serine protease inhibitors (SPIs) are among the most intensively studied protease inhibitors. They are widely distributed in all living organisms (Bode & Huber, 1992). SPIs can be called trypsin inhibitors, chymotrypsin inhibitors, elastase inhibitors, and subtilisin inhibitors due to their inhibitory focus on particular proteases (Zhao et al., 2012). Also, according to their modes of action, SPIs can be divided into four distinct types, serpins, canonical inhibitors (standard mechanism), noncanonical inhibitors, and a-macroglobulins (A2Ms) (Kanost, 1999; Krowarsch et al., 2003; Yang et al., 2017). Serpins make up a structurally conserved protein superfamily, typically about 45'kDa (Meekins et al., 2017). They generally contain a core structure with an exposed reactive center loop (RCL) of 25-30 amino acids situated in the C-terminus, responsible for defining their primary peptidase specificity (Silverman et al., 2001). The serpin RCL serves as a guide for serine proteases to occupy their active site, followed by an inhibitory reaction to permanently inactivate them. During the inhibitory reaction, the RCL is cleaved at the scissile bond between residues P1 and P1', leading to structural changes inside the serpin in a suicide inhibitory mechanism (Reichhart, 2005). Although most serpins function as SPIs, some inhibit other kinds of proteases, such as caspases and cathepsins (Schick et al., 1998). A few serpins have no inhibitory function but act in other biological roles, including tumor suppressors, hormone transporters, and molecular chaperones (Ito & Nagata, 2019; Lin et al., 2017; Zou et al., 1994). Similar to serpins, canonical inhibitors interact with their target proteases in a substrate-like manner (Krowarsch et al., 2003). The difference between the two is that canonical inhibitors are kept in reversible tight-binding interactions (Yang et al., 2017). As the largest group of protease inhibitors, canonical inhibitors include proteins from 14 to about 200 amino acid residues, and hold canonical conformations of the binding loops that are complementary to the active site of the enzyme (Laskowski & Kato, 1980). Based on sequence homology, position of active center, and disulfide bond, they are assorted into structurally diverse families such as Kazal, trypsin inhibitor-like, Pacifastin, and Kunitz SPI (Krowarsch et al., 2003). Compared with canonical inhibitors and serpins, noncanonical inhibitors such as thrombin or factor Xa interact through their N-terminal segment. They are only found in blood-sucking organisms (ticks and leeches, Hirudo nipponica Whitman) and inhibit proteases involved in clot formation (Silverman et al., 2001). A2M, a key member of the thioester superfamily, is the only enzyme inhibitor known to operate by physical entrapment of the target protein, exhibiting a unique “trapping” mechanism (Armstrong & Quigley, 1999). When A2M interacts with protein, it forms a reversible complex by wrapping the target protein (Husted et al., 2002; Rattanachai et al, 2004). As large glycoprotein, it is unique in its ability to inactivate a wide array of proteinases such as serine protease, papain, aspartate protease, and metalloproteinase without directly occupying the proteinase active site (Rehman et al., 2013). Since the first insect serpin gene was sequenced from a fat body cDNA library of Manduca sexta (Kanost et al., 1989), a large number of SPI genes have been identified in many insect species. In particular, completion of insect genome projects has led to identifying the SPI gene repertoire by exhaustive bioinformatics-based screening, coupled with manual refinements in the species listed in Table 1. Among these studies, most of them focused on identifying serpin genes encoded in the genomes of Table 1 species other than Bombyx mori, Plutella xylostella, and Pteromalus puparum. Canonical SPI genes were ident

Technical Abstract: Serine protease inhibitors (SPIs) act in diverse biological processes in insects such as immunity, development, and digestion by preventing the unwanted proteolysis. So far, the repertoire of genes encoding SPIs has been identified from few insect species. In this study, 62 SPI genes were identified from the genome of the yellow mealworm, Tenebrio molitor. According to their modes of action, they were classified into three families, serpin (26), canonical SPI (31), and a-macroglobulins (A2M) (5). These SPIs feature eight domains including serpin, Kazal, TIL, Kunitz, WAP, Antistasin, pacifastin, and A2M. In total, 39 SPIs contain a single SPI domain, while the others encode at least two inhibitor units. Based on the amino acids in the cleaved reactive sites, the abilities of these SPIs to inhibit trypsin, chymotrypsin, or elastase-like enzymes are predicted. The expression profiling based on the RNA-seq data showed that these genes displayed stage-specific expression patterns during development, suggesting to us their significance in development. Some of the SPI genes were exclusively expressed in particular tissues such as hemocyte, fat body, gut, ovary, and testis, which may be involved in biological processes specific to the indicated tissues. These findings provide necessary information for further investigation of insect SPIs.