title: Cloning and characterisation of novel cystatins from elapid snake venom glands.
abstract: Snake venoms contain a complex mixture of polypeptides that modulate prey homeostatic mechanisms through highly specific and targeted interactions. In this study we have identified and characterised cystatin-like cysteine-protease inhibitors from elapid snake venoms for the first time. Novel cystatin sequences were cloned from 12 of 13 elapid snake venom glands and the protein was detected, albeit at very low levels, in a total of 22 venoms. One highly conserved isoform, which displayed close sequence identity with family 2 cystatins, was detected in each elapid snake. Crude Austrelaps superbus (Australian lowland copperhead) snake venom inhibited papain, and a recombinant form of A. superbus cystatin inhibited cathepsin L ≅ papain > cathepsin B, with no inhibition observed for calpain or legumain. While snake venom cystatins have truncated N-termini, sequence alignment and structural modelling suggested that the evolutionarily conserved Gly-11 of family 2 cystatins, essential for cysteine protease inhibition, is conserved in snake venom cystatins as Gly-3. This was confirmed by mutagenesis at the Gly-3 site, which increased the dissociation constant for papain by 10(4)-fold. These data demonstrate that elapid snake venom cystatins are novel members of the type 2 family. The widespread, low level expression of type 2 cystatins in snake venom, as well as the presence of only one highly conserved isoform in each species, imply essential housekeeping or regulatory roles for these proteins.
authors.affiliation: The Queensland Institute of Medical Research, Herston, Brisbane, QLD, Australia.
authors.forename: Renée
authors.initials: R
authors.lastname: Richards
authors.forename: Liam
authors.initials: L
authors.lastname: St Pierre
authors.forename: Manuela
authors.initials: M
authors.lastname: Trabi
authors.forename: Lambro A
authors.initials: LA
authors.lastname: Johnson
authors.forename: John
authors.initials: J
authors.lastname: de Jersey
authors.forename: Paul P
authors.initials: PP
authors.lastname: Masci
authors.forename: Martin F
authors.initials: MF
authors.lastname: Lavin
mesh_terms: D017836:Agkistrodon / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001315:Australia; D001483:Base Sequence; D003001:Cloning, Molecular; D015891:Cystatins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D015853:Cysteine Proteinase Inhibitors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D004546:Elapid Venoms / Q000737:chemistry* / Q000235:genetics; D017815:Elapidae / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D055333:Salivary Cystatins / Q000737:chemistry* / Q000302:isolation & purification; D012469:Salivary Glands / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D004546:Elapid Venoms; D020033:Protein Isoforms; D011994:Recombinant Proteins; D055333:Salivary Cystatins