Vesicle-associated Membrane Protein (VAMP) Cleavage by a New Metalloprotease from the Brazilian Scorpion Tityus serrulatus

Fletcher Paul L,Maryann D Fletcher,Keith Weninger,Trevor E Anderson,Brian M Martin

doi:10.1074/jbc.m109.028365

Abstract

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.

Highlights

Eukaryotic intracompartmental transport and secretory processes require fusion of vesicles with cellular membranes [1,2,3]
2 The abbreviations used are: sensitive factor attachment protein receptors (SNAREs), soluble N-ethylmaleimide-sensitive factor attachment protein receptor; VAMP, vesicle-associated membrane protein; TSV, T. serrulatus venom; Fx, fraction; Zymogen granules (ZG), zymogen granule(s); ZGM, zymogen granule membrane(s); WT, wild type; carbachol, carbamylcholine chloride; SELDI, surface-enhanced laser desorption ionization; tures are available for the final, postfusion ternary SNARE complex that combines one vesicle protein (v-SNARE) with target membrane proteins (t-SNAREs); our understanding of the transient, intermediate states involved in assembly, disassembly, and catalysis of membrane fusion remains incomplete
SNARE complex formation is integral to membrane fusion in vesicular trafficking and exocytosis in both neuronal and nonneuronal cells

Summary

EXPERIMENTAL PROCEDURES

Materials—Carbamylcholine chloride (carbachol) was from Sigma, and EDTA was from Calbiochem. All protein used for protease sequence analysis was recovered from reverse phase separation of Fx ␯ using a C5 or C18 column (Supelco Wide Pore, 25 cm ϫ 4.6 mm) at a flow rate of 500 ␮l/min and gradient from 10% solvent B to 70% B in 60 min. CNBr cleavage was carried out with an aliquot of ϳ20 ␮g of S-alkylated protein reduced to dryness in a small microcentrifuge tube This sample was redissolved in 100 ␮l of 70% formic acid to which was added 1 mg of CNBr and allowed to react overnight (16 h) at room temperature in the dark. All His6-tagged proteins were initially purified via nickel-nitrilotriacetic acid-agarose (Qiagen, Germantown, MD) according to the manufacturer’s instructions using native conditions for the syntaxin and SNAP25 and denaturing protocols for VAMP2 and VAMP8. The purified ternary SNARE complex was eluted in a NaCl gradient

RESULTS

Isolation and Purification of

DISCUSSION