Vesicle-associated membrane protein (VAMP) cleavage by a new metalloprotease from the Brazilian scorpion Tityus serrulatus.

We present evidence that venom from the Brazilian scorpion Tityus serrulatus and a purified fraction selectively cleave essential SNARE proteins within exocrine pancreatic tissue. Western blotting for vesicle-associated membrane protein type v-SNARE proteins (or synaptobrevins) reveals characteristic alterations to venom-treated excised pancreatic lobules in vitro. Immunocytochemistry by electron microscopy confirms both the SNARE identity as VAMP2 and the proteolysis of VAMP2 as a marked decrease in secondary antibody-conjugated colloidal gold particles that are predominantly associated with mature zymogen granules. Studies with recombinant SNARE proteins were used to determine the specific cleavage site in VAMP2 and the susceptibility of VAMP8 (endobrevin). The VAMP2 cleavage site is between the transmembrane anchor and the SNARE motif that assembles into the ternary SNARE complex. Inclusion of divalent chelating agents (EDTA) with fraction nu, an otherwise active purified component from venom, eliminates SNARE proteolysis, suggesting the active protein is a metalloprotease. The unique cleavages of VAMP2 and VAMP8 may be linked to pancreatitis that develops following scorpion envenomation as both of these v-SNARE proteins are associated with zymogen granule membranes in pancreatic acinar cells. We have isolated antarease, a metalloprotease from fraction nu that cleaves VAMP2, and report its amino acid sequence.

Crystal structure of peptide-bound neprilysin reveals key binding interactions.

Neprilysin (NEP) is a promiscuous zinc metalloprotease with broad substrate specificity and cleaves a remarkable diversity of substrates through endopeptidase action. Two of these - amyloid-ß and natriuretic peptides - implicate the enzyme in both Alzheimer's disease and cardiovascular disease, respectively. Here, we report the creation of a catalytically inactive NEP (E584D) to determine the first peptide-bound crystal structure at 2.6 Å resolution. The structure reveals key interactions involved in substrate binding which we have identified to be conserved in other known zinc metalloproteases. In addition, the structure provides evidence for a potential exosite within the central cavity that may play a critical role in substrate positioning. Together, these results contribute to our understanding of the molecular function of NEP.

The C-terminal region of human plasma fetuin-B is dispensable for the raised-elephant-trunk mechanism of inhibition of astacin metallopeptidases.

Human fetuin-B plays a key physiological role in human fertility through its inhibitory action on ovastacin, a member of the astacin family of metallopeptidases. The inhibitor consists of tandem cystatin-like domains (CY1 and CY2), which are connected by a linker containing a "CPDCP-trunk" and followed by a C-terminal region (CTR) void of regular secondary structure. Here, we solved the crystal structure of the complex of the inhibitor with archetypal astacin from crayfish, which is a useful model of human ovastacin. Two hairpins from CY2, the linker, and the tip of the "legumain-binding loop" of CY1 inhibit crayfish astacin following the "raised-elephant-trunk mechanism" recently reported for mouse fetuin-B. This inhibition is exerted by blocking active-site cleft sub-sites upstream and downstream of the catalytic zinc ion, but not those flanking the scissile bond. However, contrary to the mouse complex, which was obtained with fetuin-B nicked at a single site but otherwise intact, most of the CTR was proteolytically removed during crystallization of the human complex. Moreover, the two complexes present in the crystallographic asymmetric unit diverged in the relative arrangement of CY1 and CY2, while the two complexes found for the mouse complex crystal structure were equivalent. Biochemical studies in vitro confirmed the differential cleavage susceptibility of human and mouse fetuin-B in front of crayfish astacin and revealed that the cleaved human inhibitor blocks crayfish astacin and human meprin α and ß only slightly less potently than the intact variant. Therefore, the CTR of animal fetuin-B orthologs may have a function in maintaining a particular relative orientation of CY1 and CY2 that nonetheless is dispensable for peptidase inhibition.

Structural and functional characterization of a P-III metalloproteinase, leucurolysin-B, from Bothrops leucurus venom.

Leucurolysin-B (leuc-B) is an hemorrhagic metalloproteinase found in the venom of Bothrops leucurus (white-tailed-jararaca) snake. By means of liquid chromatography consisting of gel filtration on Sephracryl S-200, S-300 and ion-exchange on DEAE Sepharose, leuc-B was purified to homogeneity. The proteinase has an apparent molecular mass of 55kDa as revealed by the reduced SDS-PAGE, and represents approximately 1.2% of the total protein in B. leucurus venom. The partial amino acid sequence of leuc-B was determined by automated Edman sequencing of peptides derived from digests of the S-reduced and alkylated protein with trypsin. Leuc-B exhibits the characteristic motif of metalloproteinases, HEXXHXXGXXH and a methionine-containing turn of similar conformation ("Met-turn"), which forms a hydrophobic basis for the zinc ions and the three histidine residues involved as ligands. Leuc-B has been characterized as a P-III metalloproteinase and possesses a multidomain structure including a metalloproteinase, a disintegrin-like (ECD sequence instead of the typical RGD motif) and a cysteine-rich C-terminal domain. Leuc-B contains three potential sites of N-glycosylation. The enzyme only cleaves the Ala14-Leu15 peptide bond of the oxidized insulin B-chain and preferentially hydrolyzes the Aalpha-chain of fibrinogen and the alpha-chain of fibrin. Its proteolytic activity was completely inhibited by metal chelating agents but not by other typical proteinase inhibitors. In addition, its enzymatic activity was stimulated by the divalent cations Ca2+ and Mg2+ but inhibited by Zn2+ and Cu2+. The catalytic activity of leuc-B on extracellular matrix proteins could readily lead to loss of capillary integrity resulting in hemorrhage occurring at those sites (MHD=30ng in rabbit), with alterations in platelet function. In summary, here we report the isolation and the structure-function relationship of a P-III snake venom metalloproteinase.

Structure and function analysis of protein HD73_0859 produced by Bacillus thuringiensis.

HD73_0859 is a secretory protein from Bacillus thuringiensis (Bt). In this study, its structural feature was analyzed and its function was predicted. The results revealed that HD73_0859 is a peptidase consists of a signal peptide, three well-separated SH3_3 domains and one Peptidase_M23 domain. The peptidase belongs to the M23/37-metallopeptidase family, and might have enzymatic activity to cell wall peptidoglycan and some elastins. This paper improves the understanding of both the protein HD73_0859 and the M23/37-metallopeptidases family, and lays a foundation for further experimental testing and theoretical study.