gamma -Glutamyl carboxylation: An extracellular posttranslational modification that antedates the divergence of molluscs, arthropods, and chordates.

The posttranslational γ-carboxylation of glutamate residues in secreted proteins to γ-carboxyglutamate is carried out by the vitamin K-dependent enzyme γ-glutamyl carboxylase. γ-Carboxylation has long been thought to be a biochemical specialization of vertebrates, essential for blood clotting. Recently, a γ-carboxylase was shown to be expressed in Drosophila, although its function remains undefined in this organism. We have characterized both cDNA and genomic clones for the γ-glutamyl carboxylase from the marine mollusc, Conus, the only nonvertebrate organism for which γ-carboxyglutamate-containing proteins have been biochemically and physiologically characterized. The predicted amino acid sequence has a high degree of sequence similarity to the Drosophila and vertebrate enzymes. Although γ-carboxylases are highly conserved, the Conus and mammalian enzymes have divergent substrate specificity. There are striking parallels in the gene organization of Conus and human γ-carboxylases. Of the 10 Conus introns identified, 8 are in precisely the same position as the corresponding introns in the human enzyme. This remarkable conservation of intron/exon boundaries reveals that an intron-rich γ-carboxylase was present early in the evolution of the animal phyla; although specialized adaptations in mammals and molluscs that require this extracellular modification have been identified, the ancestral function(s) and wider biological roles of γ-carboxylation still need to be defined. The data raise the possibility that most introns in the genes of both mammals and molluscs antedate the divergence of these phyla.

[1]  C. Nicchitta,et al.  A topological study of the human γ-glutamyl carboxylase , 2000 .

[2]  C. Schneider,et al.  The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade , 1993, Molecular and cellular biology.

[3]  K. Clark,et al.  On a potential global role for vitamin K-dependent gamma-carboxylation in animal systems. Evidence for a gamma-glutamyl carboxylase in Drosophila. , 2001, The Journal of biological chemistry.

[4]  J. Stenflo Vitamin K and the biosynthesis of prothrombin. IV. Isolation of peptides containing prosthetic groups from normal prothrombin and the corresponding peptides from dicoumarol-induced prothrombin. , 1974, The Journal of biological chemistry.

[5]  T. Südhof,et al.  alpha-Latrotoxin receptor CIRL/latrophilin 1 (CL1) defines an unusual family of ubiquitous G-protein-linked receptors. G-protein coupling not required for triggering exocytosis. , 1998, The Journal of biological chemistry.

[6]  S J de Souza,et al.  Origin of genes. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[7]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[8]  D. Stafford,et al.  Identification and purification to near homogeneity of the vitamin K-dependent carboxylase. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[9]  J. Logsdon,et al.  The recent origins of spliceosomal introns revisited. , 1998, Current opinion in genetics & development.

[10]  J. Haack,et al.  Conantokin-T. A gamma-carboxyglutamate containing peptide with N-methyl-d-aspartate antagonist activity. , 1990, The Journal of biological chemistry.

[11]  K. Kurachi,et al.  Isolation and characterization of a cDNA coding for human factor IX. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[12]  P. Fernlund,et al.  Vitamin K dependent modifications of glutamic acid residues in prothrombin. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[13]  D. Stafford,et al.  Identification of a Drosophila Vitamin K-dependent γ-Glutamyl Carboxylase* , 2000, The Journal of Biological Chemistry.

[14]  M. Miyagi,et al.  Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[15]  K. High,et al.  Genomic sequence and transcription start site for the human γ-glutamyl carboxylase , 1997 .

[16]  P. Price,et al.  Primary structure of bovine matrix Gla protein, a new vitamin K-dependent bone protein. , 1985, The Journal of biological chemistry.

[17]  J E Darnell,et al.  Speculations on the early course of evolution. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[18]  S. Geromanos,et al.  The Propeptide Binding Site of the Bovine γ-Glutamyl Carboxylase* , 1997, The Journal of Biological Chemistry.

[19]  John M. Logsdon,et al.  The recent origins of introns. , 1991 .

[20]  G. S. Begley,et al.  A Conserved Motif within the Vitamin K-dependent Carboxylase Gene Is Widely Distributed across Animal Phyla* , 2000, The Journal of Biological Chemistry.

[21]  C Vermeer,et al.  Gamma-carboxyglutamate-containing proteins and the vitamin K-dependent carboxylase. , 1990, The Biochemical journal.

[22]  J. Howard,et al.  The mode of action of vitamin K. Identification of gamma-carboxyglutamic acid as a component of prothrombin. , 1974, The Journal of biological chemistry.

[23]  Kathryn E. Crosier,et al.  New insights into the control of cell growth; The role of the Axl family , 1997, Pathology.

[24]  B. Olivera,et al.  Venomous cone snails: molecular phylogeny and the generation of toxin diversity. , 2001, Toxicon : official journal of the International Society on Toxinology.

[25]  G. Heijne Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. , 1992, Journal of molecular biology.

[26]  T. Cavalier-smith,et al.  Intron phylogeny: a new hypothesis. , 1991, Trends in genetics : TIG.

[27]  T. Stanley,et al.  Identification of a vitamin K‐dependent carboxylase in the venom duct of a Conus snail , 1997, FEBS letters.

[28]  B. Olivera,et al.  The T-superfamily of Conotoxins* , 1999, The Journal of Biological Chemistry.

[29]  P. Fossier,et al.  A conotoxin from Conus textile with unusual posttranslational modifications reduces presynaptic Ca2+ influx. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[30]  J. Harris,et al.  Primary structure and tissue distribution of two novel proline-rich gamma-carboxyglutamic acid proteins. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[31]  L. M. Canfield Vitamin K-dependent oxygenase/carboxylase; differential inactivation by sulfhydryl reagents. , 1987, Biochemical and biophysical research communications.

[32]  S. Woodward,et al.  Diversity of Conus neuropeptides. , 1990, Science.

[33]  S. Ham,et al.  Vitamin K and energy transduction: a base strength amplification mechanism. , 1995, Science.

[34]  Gregg B. Fields,et al.  Peptides for the New Millennium , 2002, American Peptide Symposia.

[35]  B. Eisenstein,et al.  The polymerase chain reaction. A new method of using molecular genetics for medical diagnosis. , 1990, The New England journal of medicine.

[36]  B. Olivera,et al.  Conantokin-G Precursor and Its Role in γ-Carboxylation by a Vitamin K-dependent Carboxylase from a ConusSnail* , 1998, The Journal of Biological Chemistry.

[37]  L. Helgeland The submicrosomal site for the conversion of prothrombin precursor to biologically active prothrombin in rat liver. , 1977, Biochimica et biophysica acta.

[38]  B. Olivera,et al.  The spasmodic peptide defines a new conotoxin superfamily. , 2000, Biochemistry.

[39]  D. Stafford,et al.  Cloning and expression of the cDNA for human gamma-glutamyl carboxylase. , 1991, Science.

[40]  D. Roth,et al.  Cloning, structural organization, and transcriptional activity of the rat vitamin K-dependent gamma-glutamyl carboxylase gene. , 1998, Biochemical and biophysical research communications.

[41]  C. Walsh,et al.  Localization of the factor IX propeptide binding site on recombinant vitamin K dependent carboxylase using benzoylphenylalanine photoaffinity peptide inactivators. , 1995, Biochemistry.

[42]  J. McIntosh,et al.  Gamma-carboxyglutamate in a neuroactive toxin. , 1984, The Journal of biological chemistry.

[43]  W. Gilbert,et al.  The exon theory of genes. , 1987, Cold Spring Harbor symposia on quantitative biology.

[44]  D. Stafford,et al.  A Missense Mutation in γ-Glutamyl Carboxylase Gene Causes Combined Deficiency of All Vitamin K-Dependent Blood Coagulation Factors , 1998 .