A Single γ-Carboxyglutamic Acid Residue in a Novel Cysteine-Rich Secretory Protein without Propeptide^,

Abstract

Gamma-glutamyl carboxylase catalyzes the modification of specific glutamyl residues to gamma-carboxyglutamyl (Gla) residues in precursor proteins that possess the appropriate gamma-carboxylation recognition signal within the propeptide region. We describe the immunopurification and first biochemical characterization of an invertebrate high molecular weight Gla-containing protein with homologues in mammals. The protein, named GlaCrisp, was isolated from the venom of the marine cone snail Conus marmoreus. GlaCrisp gave intense signals in Western blot experiments employing the Gla-specific antibody M3B, and the presence of Gla was chemically confirmed by amino acid analysis after alkaline hydrolysis. Characterization of a full-length cDNA clone encoding GlaCrisp deduced a precursor containing an N-terminal signal peptide but, unlike other Gla-containing proteins, no apparent propeptide. The predicted mature protein of 265 amino acid residues showed considerable sequence similarity to the widely distributed cysteine-rich secretory protein family and closest similarity (65% identity) to the recently described substrate-specific protease Tex31. In addition, two cDNA clones encoding the precursors of two isoforms of GlaCrisp were identified. The predicted precursor isoforms differed at three amino acid positions (-6, 9, and 25). Analysis by Edman degradation and nanoelectrospray ionization mass spectrometry, before and after methyl esterfication, identified a Gla residue at amino acid position 9 in GlaCrisp. This is the first example of a Gla-containing protein without an obvious gamma-carboxylation recognition site. The results define a new class of Gla proteins and support the notion that gamma-carboxylation of glutamyl residues is phylogenetically older than blood coagulation and the vertebrate lineage.

Activated protein C cofactor function of protein S: a novel role for a γ-carboxyglutamic acid residue

“…Most hypothesized roles of Gla residues in the many proteins involve coordination of calcium. 41,43,44 In contrast, Gla36 in protein S is not predicted to coordinate calcium 19 (Figure 5B), neither is it involved in phospholipid binding of protein S. In a model of Gla-TSR-EGF1 domains of protein S, 19 Gla36 lies outside of the -loop and is predicted to point away from the phospholipid membrane ( Figure 5B). Collectively, this suggests that the ␥-carboxylation of Glu36 is essential for protein S function and is involved in the interactions through which protein S enhances APC function.…”

“…16 However, Gla residues have also been identified in peptides from invertebrates, 41,42 which are thought to have evolved before blood coagulation. Most hypothesized roles of Gla residues in the many proteins involve coordination of calcium.…”

Structures of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels: implications for movement of the C-terminal cysteine-rich domain

“…With the discovery of testis-specific protein 1 (Tpx-1, CRISP-2; Hardy et al, 1988;Kasahara et al, 1989) and specific granule protein of 28 kDa (SGP28, CRISP-3; Haendler et al, 1993;Kjeldsen et al, 1996), four CRISPs (CRISP-1-CRISP-4) have now been identified in mammalian tissues. Furthermore, it has also been shown that CRISPs are widely distributed in non-mammalian species, including Xenopus (Olson et al, 2001), Caenorhabditis elegans (Ookuma et al, 2003) and the venoms of lizards (Nobile et al, 1994(Nobile et al, , 1996Morrissette et al, 1995), cone snails (Milne et al, 2003;Hansson et al, 2006) and snakes (Yamazaki & Morita, 2004, 2007. Although clear evidence for either function or target molecules is lacking for most of the CRISPs, including endogenous CRISPs, recent evidence has emerged suggesting that some exogenous CRISPs derived from venomous snakes may function as ion-channel blockers (Nobile et al, 1994(Nobile et al, , 1996Brown et al, 1999Brown et al, , 2003Yamazaki, Brown et al, 2002;Yamazaki, Koike et al, 2002;Wang et al, 2005).…”

Fatal hemorrhage in mice lacking γ-glutamyl carboxylase

“…22,23 Given the generalized expression of the ␥-carboxylase in most or all tissues, it is likely that a number of other Gla-containing proteins with specialized functions remain to be identified, including additional members of a recently characterized class of proteins lacking a conventional ␥-carboxylase recognition site. 24 The ␥-carboxylase requires reduced vitamin K as an obligate cofactor. The oral anticoagulant warfarin functions by inhibiting vitamin K epoxide reductase (VKOR) and thereby blocking the regeneration of active reduced vitamin K. 2 The VKOR gene has recently been cloned and shown to encode a small transmembrane protein localized to the ER.…”