Novel γ‐carboxyglutamic acid‐containing peptides from the venom of Conus textile

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Abstract
The cone snail is the only invertebrate system in which the vitamin K‐dependent carboxylase (or γ‐carboxylase) and its product γ‐carboxyglutamic acid (Gla) have been identified. It remains the sole source of structural information of invertebrate γ‐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 γ‐carboxylation recognition site (γ‐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 Conusγ‐carboxylase and synthetic peptide substrates localized the γ‐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 µm 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.