Isolation and Characterization of a Cone Snail Protease with Homology to CRISP Proteins of the Pathogenesis-related Protein Superfamily

Abstract

The pathogenesis-related (PR) protein superfamily is widely distributed in the animal, plant, and fungal kingdoms and is implicated in human brain tumor growth and plant pathogenesis. The precise biological activity of PR proteins, however, has remained elusive. Here we report the characterization, cloning and structural homology modeling of Tex31 from the venom duct of Conus textile. Tex31 was isolated to >95% purity by activityguided fractionation using a para-nitroanilide substrate based on the putative cleavage site residues found in the propeptide precursor of conotoxin TxVIA. Tex31 requires four residues including a leucine N-terminal of the cleavage site for efficient substrate processing. The sequence of Tex31 was determined using two degenerate PCR primers designed from N-terminal and tryptic digest Edman sequences. A BLAST search revealed that Tex31 was a member of the PR protein superfamily and most closely related to the CRISP family of mammalian proteins that have a cysteine-rich C-terminal tail. A homology model constructed from two PR proteins revealed that the likely catalytic residues in Tex31 fall within a structurally conserved domain found in PR proteins. Thus, it is possible that other PR proteins may also be substrate-specific proteases.Of the genomes that have been completely sequenced, as many as 2% of the gene products encode known proteases, many of which regulate physiological processes such as blood coagulation, fibrinolysis, the complement system, and the processing of protein hormone precursors by specific convertases. Prohormones and neuropeptides (3-40 amino acids), important coordinators of cellular function in the endocrine and nervous systems, are often synthesized as propeptides. The propeptide is subsequently processed by substrate-specific proteases to yield the mature bioactive form (1). As many of the substratespecific proteases remain unidentified, predicting new bioactive peptides from cDNA sequences is presently difficult, if not impossible (2, 3). Linderström-Lang coined the term "limited proteolysis" to describe enzymes with restricted specificity (4). Enzyme activity can be restricted by substrate structure, enzyme processing state, endogenous protease inhibitor levels or a combination of these factors (5). Peptides derived from prohormones are typically flanked by a pair of dibasic residues (Lys-Arg, Arg-Arg, Lys-Lys, or Arg-Lys) and are cleaved by proteases found in secretory vesicles (1). However, many precursor peptides contain multiple sets of basic residues, suggesting that highly substrate specific or differentially expressed proteases can determine processing outcomes.The venom of cone snails (predatory marine molluscs of the genus Conus) has yielded a rich source of novel neuroactive peptides ("conotoxins") (6). However, the enzymes responsible for cleaving the conotoxins from their propeptide precursors have not been isolated. Here we report the purification, cloning, and initial characterization of Tex31, a protease from venom Conus textile. Interestingly,...

Cysteine‐rich secretory proteins are not exclusively expressed in the male reproductive tract

“…suggest that CAP domains may have enzymatic (Milne et al, 2003), immunological (Moyle et al, 1994), or cellular adhesion functions (Maeda et al, 1999). A more detailed understanding of the expression profile of CRISPs provides insights into their potential biological functions.…”

“…All members of this family share a common CAP domain (also known as a sperm-coating protein, SCP, domain or a PR-1 domain), which is characterized by the presence of two signature motifs that are involved in the formation of a cleft-like structure forming a putative active site containing (in the CRISPs) three intra-molecular disulphide bonds (Henriksen et al, 2001;Shikamoto et al, 2005). CAPs are typically secreted and found in an extraordinary range of species in both bacteria and eukaryotes including, for example, yeast, fungi, plants, cone snails, drosophila, lampreys, snakes, mice, and humans (Pfitzner and Goodman, 1987;Schuren et al, 1993;Miosga et al, 1995;Murphy et al, 1995;Schreiber et al, 1997;Yamazaki et al, 2002b;Milne et al, 2003;Ito et al, 2007)). The presence of such an evolutionarily diverse, yet conserved, structure is suggestive of a common function, although the identification of this function remains a challenge.…”

“…The presence of such an evolutionarily diverse, yet conserved, structure is suggestive of a common function, although the identification of this function remains a challenge. Data from a range of species, however, imply roles for CAPs as antifungal agents in plants (Niderman et al, 1995); venoms of wasps and as a significant allergen in humans (King et al, 1978;Lu et al, 1993); as a pro-tease in cone snails (Milne et al, 2003); as a protease inhibitor in human glioblastoma cells (Yamakawa et al, 1998); as an anti-coagulant protein in lampreys (Ito et al, 2007); and, as outlined in more detail below, in mediating cell-cell binding in mammals.…”

Probing the equatorial groove of the hookworm protein and vaccine candidate antigen, Na-ASP-2

“…In particular, protease activity has been suggested as a shared feature of PR-1 proteins based on structure-function inference from the cone snail protease Tex31 (Milne et al, 2003), but could not be demonstrated to date. We were intrigued by the structural similarity of the tandem histidine motif of Group 1 ASPs with enzymatic sites found in phosphodiesterases such as the Arabidopsis CPDase (Hofmann et al, 2000b), and thus investigated the possibility that Na-ASP-2 might possess phosphohydrolase activity.…”

“…Proteolytic activity has been speculated for this protein based on enzymatic activity observed for cone snail protease Tex31, a member of the CRISP family of pathogenesis-related proteins (Milne et al, 2003). Proteolytic activity was not detected for Na-ASP-1 or ASP-2, and from the crystal structure of the protein it is obvious that the proposed residues for this activity are not in spatial vicinity.…”