title: Novel gamma-carboxyglutamic acid-containing peptides from the venom of Conus textile.
abstract: The cone snail is the only invertebrate system in which the vitamin K-dependent carboxylase (or gamma-carboxylase) and its product gamma-carboxyglutamic acid (Gla) have been identified. It remains the sole source of structural information of invertebrate gamma-carboxylase substrates. Four novel Gla-containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have six Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-l-bromotryptophan. Gla(2)-TxVI/A and Gla(2)-TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids -46 to -27) were nearly identical and highly hydrophobic. The predicted propeptide region (-20 to -1) that contains the gamma-carboxylation recognition site (gamma-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conusgamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the Km was reduced from 1.8 mm for Gla (1)-TxVI lacking a propeptide to 24 microm when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km value tenfold.
authors.affiliation: Marine Biological Laboratory, Woods Hole, MA, USA. czerwiec@mbl.edu
authors.forename: Eva
authors.initials: E
authors.lastname: Czerwiec
authors.forename: Dario E
authors.initials: DE
authors.lastname: Kalume
authors.forename: Peter
authors.initials: P
authors.lastname: Roepstorff
authors.forename: Björn
authors.initials: B
authors.lastname: Hambe
authors.forename: Bruce
authors.initials: B
authors.lastname: Furie
authors.forename: Barbara C
authors.initials: BC
authors.lastname: Furie
authors.forename: Johan
authors.initials: J
authors.lastname: Stenflo
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000032:analysis / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D020916:Conotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D052078:Conus Snail / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D020916:Conotoxins; D018076:DNA, Complementary; D010455:Peptides; D011498:Protein Precursors; D015055:1-Carboxyglutamic Acid; D003545:Cysteine