Novel γ-carboxyglutamic acid-containing peptides from the venom of Conus textile
2006-04-25,
Czerwiec, Eva,
Kalume, Dario E.,
Roepstorff, Peter,
Hambe, Bjorn,
Furie, Bruce,
Furie, Barbara C.,
Stenflo, Johan
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)1 have been identified. It remains the sole source of structural
information of invertebrate γ-carboxylase subtrates. Four novel γ-
carboxyglutamic acid (Gla)1 containing peptides were purified from the
venom of Conus textile and characterized by biochemical methods and mass
spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B
and Gla(3)-TxVI each have 6 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 Conus γ-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 10-fold.