A single gamma-carboxyglutamic acid residue in a novel cysteine-rich secretory protein without propeptide.
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.
Hansson
Karin
K
Department of Clinical Chemistry, Lund University, University Hospital, Malmö, S-20502 Malmö, Sweden. Karin_M.Hansson@med.lu.se
Thämlitz
Ann-Marie
AM
Furie
Bruce
B
Furie
Barbara C
BC
Stenflo
Johan
J
D015055:1-Carboxyglutamic Acid / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019735:Carbon-Carbon Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D020916:Conotoxins / Q000378:metabolism; D017931:DNA Primers; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012908:Snails
D020916:Conotoxins; D017931:DNA Primers; D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D015055:1-Carboxyglutamic Acid; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase