Gla-rich Protein (GRP), A New Vitamin K-dependent Protein Identified from Sturgeon Cartilage and Highly Conserved in Vertebrates

Abstract

We report the isolation of a novel vitamin K-dependent protein from the calcified cartilage of Adriatic sturgeon (Acipenser nacarii). This 10.2-kDa secreted protein contains 16 γ-carboxyglutamic acid (Gla) residues in its 74-residue sequence, the highest Gla percent of any known protein, and we have therefore termed it Gla-rich protein (GRP). GRP has a high charge density (36 negative + 16 positive = 20 net negative) yet is insoluble at neutral pH. GRP has orthologs in all taxonomic groups of vertebrates, and a paralog (GRP2) in bony fish; no GRP homolog was found in invertebrates. There is no significant sequence homology between GRP and the Gla-containing region of any presently known vitamin K-dependent protein. Forty-seven GRP sequences were obtained by a combination of cDNA cloning and comparative genomics: all 47 have a propeptide that contains a γ-carboxylase recognition site and a mature protein with 14 highly conserved Glu residues, each of them being γ-carboxylated in sturgeon. The protein sequence of GRP is also highly conserved, with 78% identity between sturgeon and human GRP. Analysis of the corresponding gene structures suggests a highly constrained organization, particularly for exon 4, which encodes the core Gla domain. GRP mRNA is found in virtually all rat and sturgeon tissues examined, with the highest expression in cartilage. Cells expressing GRP include chondrocytes, chondroblasts, osteoblasts, and osteocytes. Because of its potential to bind calcium through Gla residues, we suggest that GRP may regulate calcium in the extracellular environment. We report the isolation of a novel vitamin K-dependent protein from the calcified cartilage of Adriatic sturgeon (Acipenser nacarii). This 10.2-kDa secreted protein contains 16 γ-carboxyglutamic acid (Gla) residues in its 74-residue sequence, the highest Gla percent of any known protein, and we have therefore termed it Gla-rich protein (GRP). GRP has a high charge density (36 negative + 16 positive = 20 net negative) yet is insoluble at neutral pH. GRP has orthologs in all taxonomic groups of vertebrates, and a paralog (GRP2) in bony fish; no GRP homolog was found in invertebrates. There is no significant sequence homology between GRP and the Gla-containing region of any presently known vitamin K-dependent protein. Forty-seven GRP sequences were obtained by a combination of cDNA cloning and comparative genomics: all 47 have a propeptide that contains a γ-carboxylase recognition site and a mature protein with 14 highly conserved Glu residues, each of them being γ-carboxylated in sturgeon. The protein sequence of GRP is also highly conserved, with 78% identity between sturgeon and human GRP. Analysis of the corresponding gene structures suggests a highly constrained organization, particularly for exon 4, which encodes the core Gla domain. GRP mRNA is found in virtually all rat and sturgeon tissues examined, with the highest expression in cartilage. Cells expressing GRP include chondrocytes, chondroblasts, osteoblasts, and osteocytes. Because of its potential to bind calcium through Gla residues, we suggest that GRP may regulate calcium in the extracellular environment. Vitamin K-dependent proteins are characterized by the presence of several γ-carboxyglutamic acid residues (Gla) 3The abbreviations used are: Glaγ-carboxyglutamic acidGRPGla-rich proteinMGPmatrix Gla proteinOCosteocalcinHPRT1hypoxanthine phosphoribosyltransferase 1ICimmature chondrocytesMCmature chondrocytesHChypertrophic chondrocytesPCproliferating chondrocytesFCfibrocartilageRP-HPLCreverse phase-high performance liquid chromatographyRACErapid amplification of cDNA endsaaamino acidGAPDHglyceraldehyde-3-phosphate dehydrogenaseqPCRquantitative PCR resulting from vitamin K-dependent post-translation modifications of specific glutamates (Glu) by the γ-glutamyl carboxylase (1Vermeer C. Biochem. J. 1990; 266: 625-636Crossref PubMed Scopus (283) Google Scholar, 2Cranenburg E.C. Schurgers L.J. Vermeer C. Thromb. Haemostasis. 2007; 98: 120-125Crossref PubMed Scopus (187) Google Scholar). The identification in 1974 of the new amino acid Gla in prothrombin (3Stenflo J. Fernlund P. Egan W. Roepstorff P. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 2730-2733Crossref PubMed Scopus (618) Google Scholar) opened the way for subsequent identifications of other Gla containing proteins in blood, all involved in coagulation (factors VII, IX, and X, and proteins C, S, and Z) and containing from 9 to 13 Gla residues (4Suttie J.W. Annu. Rev. Biochem. 1985; 54: 459-477Crossref PubMed Scopus (374) Google Scholar). Lack of adequate γ-carboxylation was found to prevent Gla-mediated calcium binding and thus inhibit clot formation, highlighting the importance of Gla for normal coagulation process (5Zhang L. Castellino F.J. J. Biol. Chem. 1993; 268: 12040-12045Abstract Full Text PDF PubMed Google Scholar). Finding high levels of Gla in non-hepatic tissues such as bone led to the discovery of a different group of Gla-containing proteins not involved in hemostasis, e.g. osteocalcin (OC), characterized in 1976 (6Hauschka P.V. Lian J.B. Gallop P.M. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3925-3929Crossref PubMed Scopus (644) Google Scholar, 7Price P.A. Otsuka A.A. Poser J.W. Kristaponis J. Raman N. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 1447-1451Crossref PubMed Scopus (686) Google Scholar), and matrix Gla protein (MGP), first described in 1983 (8Price P.A. Urist M.R. Otawara Y. Biochem. Biophys. Res. Commun. 1983; 117: 765-771Crossref PubMed Scopus (285) Google Scholar). Both proteins contain 3 to 5 Gla residues, appear to play a role in tissue mineralization, and form a distinct group within the VKD protein family (9Laizé V. Martel P. Viegas C.S. B. Price P.A. Cancela M.L. J. Biol. Chem. 2005; 280: 26659-26668Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Later, additional VKD proteins were identified and found to be involved in diverse biological functions such as growth control and apoptosis (growth arrest-specific protein 6, Gas6) (10Manfioletti G. Brancolini C. Avanzi G. Schneider C. Mol. Cell. Biol. 1993; 13: 4976-4985Crossref PubMed Scopus (532) Google Scholar), and signal transduction (proline-rich Gla proteins 1, 2, 3, and 4, PRGP1-4) (11Kulman J.D. Harris J.E. Haldeman B.A. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9058-9062Crossref PubMed Scopus (94) Google Scholar, 12Kulman J.D. Harris J.E. Xie L. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1370-1375Crossref PubMed Scopus (91) Google Scholar). Although VKD proteins were initially identified in vertebrates, the presence of a γ-glutamyl carboxylase in Drosophila melanogaster and the identification of several of its substrates in marine snails from the genus Conus (13Walker C.S. Shetty R.P. Clark K. Kazuko S.G. Letsou A. Olivera B.M. Bandyopadhyay P.K. J. Biol. Chem. 2001; 276: 7769-7774Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 14Brown M.A. Hambe B. Furie B. Furie B.C. Stenflo J. Stenberg L.M. Toxicon. 2002; 40: 447-453Crossref PubMed Scopus (6) Google Scholar, 15Hansson K. Thamlitz A.M. Furie B. Furie B.C. Stenflo J. Biochemistry. 2006; 45: 12828-12839Crossref PubMed Scopus (19) Google Scholar), together with the ubiquity of γ-glutamyl carboxylase expression in mammalian tissues (16de Boer-van den Berg M.A. Verstijnen C.P. Vermeer C. J. Investig. Dermatol. 1986; 87: 377-380Abstract Full Text PDF PubMed Scopus (13) Google Scholar), suggests a wider prevalence of γ-carboxylation, with a broad range of biological roles. We hereby report the purification and characterization of a new Gla-containing protein from the calcified cartilage of sturgeon, an ancient bony fish with a cartilaginous endoskeleton and in which bone is found essentially in the ganoid plaques forming the exogenous skeleton. An in silico approach, coupled with a cloning strategy, identified orthologs of this protein in all vertebrate groups, including humans. Its unprecedented high number of Gla residues and high evolutionary conservation suggests a fundamental function. γ-carboxyglutamic acid Gla-rich protein matrix Gla protein osteocalcin hypoxanthine phosphoribosyltransferase 1 mature phase-high performance liquid amplification of cDNA amino acid PCR of were by and used at in a with a biological and a were a with from sturgeon were also obtained from of were obtained from from the of GRP and a proteins were from of a of acid for at as described J.B. Viegas C.S. Price P.A. Cancela M.L. J. Res. PubMed Scopus Google Scholar). The proteins were from the insoluble matrix by through and at 3, with of to all The was in and 5 to GRP. of this was and of the resulting protein were by The protein was by with acid acid as described Vermeer C. Biochem. PubMed Scopus Google Scholar). The was by phase-high performance liquid to GRP. protein was in and were a in acid in and at a of 1 The was for at and proteins were from the with a to acid in of 1 were and at from purification containing GRP were in of and a in of were and at were and to acid and as described P.A. Otsuka A.A. Poser J.W. Kristaponis J. Raman N. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 1447-1451Crossref PubMed Scopus (686) Google Scholar, P.A. Urist M.R. Otawara Y. Biochem. Biophys. Res. Commun. 1983; 117: 765-771Crossref PubMed Scopus (285) Google Scholar). acid was was with to (10Manfioletti G. Brancolini C. Avanzi G. Schneider C. Mol. Cell. Biol. 1993; 13: 4976-4985Crossref PubMed Scopus (532) Google Scholar). Both were a acid at the of were an with a and the by the were a and by the were by at and was from sturgeon tissues bony and cartilaginous and and rat and as described by and P. N. Biochem. PubMed Scopus Google Scholar). was by and was by at was from tissue to a described Cancela M.L. 2001; PubMed Scopus Google Scholar). cDNA the sequence of the sturgeon protein, a was and used in combination with to by PCR the of sturgeon, and GRP from The of were by PCR specific and cDNA to the from 1 of a of from and calcified The rat GRP cDNA was by from of specific to sequences from sequence and The rat and were by from of and specific and to and sequences from the sequence and of all PCR used in this are in PCR were and and were the to the GRP gene was as and and first cDNA sequence and also sequences as was used to the exon 1 to 3 with and exon 3 with and exon to 5 with and and 3 were obtained with and with the of by of was with and at with specific and PCR was with an to sturgeon to sturgeon and to sturgeon hypoxanthine phosphoribosyltransferase 1 in were as of cDNA of at and of PCR were to an at for and of amplification is at and at was at the of each in the and of each were to for of gene expression were the comparative and gene expression levels of expression in was to 1 and used as for expression in other was in and a was of all used in this are in were in at with in for 1 in and in of sturgeon GRP cDNA from to and a of rat GRP cDNA from to the in were with and with to an with and with to a of rat cDNA from to and a of rat cDNA from to in were with and and with to were with the to the were with as described J.B. Viegas C.S. Price P.A. Cancela M.L. J. Res. PubMed Scopus Google Scholar, Cancela M.L. 2001; PubMed Scopus Google Scholar). were with in containing of 20 for and at in a were and the signal with the and as described J.B. Viegas C.S. Price P.A. Cancela M.L. J. Res. PubMed Scopus Google Scholar, Cancela M.L. 2001; PubMed Scopus Google Scholar). for GRP mRNA were with of to from each chondrocytes, mature and were from each of 3 of sturgeon and used to the of expressing within the rat the were groups to chondrocytes, which and and mature chondrocytes, which and of of each group were from each of of rat and used to the of expressing GRP as of of each in the The of rat expressing GRP was obtained from of the and and as as were for sequences to sturgeon sequences were first and of each were the from 9 to highly and were from the sequence gene and were comparative and of GRP mature and were sequence C. Res. 2006; PubMed Scopus Google Scholar) with to were in a to were Res. 1990; PubMed Scopus Google Scholar). The sequence are as the of each is to its This the conserved residues as signal were identified in protein sequences the J. S. G. 1997; Scopus Google Scholar). was from of GRP mature S. K. PubMed Scopus Google Scholar). The matrix was and the of was as of a of was and in all The was the which are an of are and of Adriatic proteins from the calcified cartilage of Adriatic sturgeon were acid from of as described J.B. Viegas C.S. Price P.A. Cancela M.L. J. Res. PubMed Scopus Google Scholar), and the resulting insoluble by in protein with an of and a positive signal with the acid was to a and protein sequence the first 20 were identified as were for residues at and to sequences in at identified sequences with of sturgeon GRP. all were the discovery of a novel protein. The presence of a positive with obtained for 3 within the sequence, identified as Glu residues cDNA additional the that this was a new Gla-containing protein, an and thus it was Gla-rich protein (GRP). sturgeon the insoluble obtained was in and to a were and by This was found to be with proteins first and was by a The resulting a of the of were by the of as a from the high GRP and of cDNA of sturgeon GRP was obtained through a combination of and PCR The cDNA and an of a of residues and and of and The was Analysis of the amino acid sequence a signal of as by a propeptide of containing a γ-glutamyl carboxylase recognition site with of other VKD an and a and a mature protein of with an of and The presence of 16 Glu residues in the mature protein and the of Gla residues by amino acid in acid and of the protein that all Glu residues are γ-carboxylated in sturgeon GRP its to The of the protein the sequence from the cDNA and the first 20 residues of the mature protein were to by protein sequence that the cDNA encodes the GRP protein. The sturgeon GRP gene was through a combination of gene and PCR is in 5 and all of 1 as by L. PubMed Scopus Google Scholar). The mature protein is by 3, 4, and all containing Gla residues, of them a of are in exon 4, forming the core of the protein. of GRP gene expression were in a of sturgeon tissues calcified and by and as gene GRP gene expression was in all tissues the highest levels were in cartilaginous tissues and with the highest from the high of protein in high levels of gene expression were also in this were obtained a different was used to GRP gene expression not of GRP gene expression were at by in of from a of sturgeon that GRP mRNA was in mature and of and and and in the of the in that cartilaginous tissues significant levels of GRP in sturgeon. The found in the cartilaginous matrix of sturgeon are identified as mature chondrocytes, and are by a of identified as and are in with not for the different of that of and mature and that a of GRP. coupled with the that cartilaginous tissues have the highest expression of GRP in that are the GRP in sturgeon. GRP and and were for sequences sturgeon GRP as and at identified proteins were to be orthologs to GRP sequence to the sturgeon sequence, and this the protein. all were and sequences of sequences were and identified as GRP orthologs were from and were a of and PCR sequences different of 3 and and including bony and was found for the presence of any GRP sequences in invertebrates. no GRP sequences were identified in other to of from any GRP homolog was identified in its presence in the which and The identification of an in the together with its to cartilaginous suggests that GRP may have with cartilage fish comparative a GRP was identified with the of bony fish the bony fish for which is and distinct by different were identified and and We that to a GRP in all fish is to sequence The presence of a in bony fish is no The of in is in has for other A. Y. Res. 13: PubMed Scopus Google Scholar, V. Viegas C.S. B. Price P.A. Cancela M.L. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) and is with a that has in the fish from and which bony fish V. Viegas C.S. B. Price P.A. Cancela M.L. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Res. 2005; PubMed Scopus Google Scholar). the other and to PCR a gene in sturgeon, we the the that the the of and from the to fish J. Res. 2005; PubMed Scopus Google Scholar). a in exon 3 resulting in a mature protein with a to and an of exon were identified in the and GRP the of GRP a of 47 mature proteins were at the conserved proteins were and through a with the of vertebrates, GRP sequences were taxonomic groups bony and fish sequences in groups the presence of GRP the 47 mature as and 9 as the bony GRP sequences and containing the mature were as the highly conserved residues residues a of from which are within the mature protein and include Glu conserved were also a signal of a propeptide of containing a γ-carboxylase recognition site the VKD proteins to the γ-glutamyl an in it as a site of and a site used to the propeptide at the and a mature protein from to 3 and Although the of the protein to including signal and the of the was found to be conserved residues between sturgeon and the of the protein to including the γ-carboxylase recognition involved in the of the protein, and the mature a high of sequence identity residues between sturgeon and in the of for protein and thus conserved vertebrate 14 conserved Glu residues are of γ-carboxylation in sturgeon. for any additional conserved of and was to in were in the and for of GRP in The of the gene was from of of each and were the mRNA to at cDNA and gene are known for a in a by comparative sequence the gene with 5 of exon exon 5 exon 3 exon exon and The of was also conserved in all the of exon 4, which encodes the protein region with Gla residues was in all in the this gene was identified and with the conserved exon and Both fish a gene in number of and of the sequence was in of and suggest a constrained for GRP in for the region the core Gla the conservation of this it the that the of GRP be and its Gla of GRP in the high of conservation between fish and mammalian GRP at protein and gene we of rat GRP gene expression were with identified in sturgeon. of rat and and and and were by in GRP mRNA expression was in cartilage from all of in mature and from rat 1, 4, and 1 and and and and the cartilage from GRP mRNA with mRNA in and in the of the it was also in of the of which not for 4, and were found to be positive for GRP mRNA negative for mRNA and GRP mRNA was also in the of between of GRP and that expression was to 5 and and 2, and 2, and and GRP mRNA was in all cartilaginous GRP mRNA was also in bone from rat and being in and 1 and and 1 and and and of with osteocalcin a known for osteoblasts, that the of GRP expression in was with the expression of and and and 3, and and expression in was 3 and and GRP was highly 1 and and and was in and to the number and of expressing GRP and the in The of and mature and that GRP is not was not to for it was not to the within each to its a between and GRP signal was and a of in osteoblasts, the presence of GRP in that GRP is not to in sturgeon fish with an bony and is also in bone of and therefore suggests a role for the protein the we report the purification and characterization of a new of the VKD protein family containing an unprecedented high density of Gla residues and thus GRP. This protein, identified in the process of purification of Gla-containing proteins from the cartilaginous of the sturgeon A. is a secreted protein with a of Gla The presence of 16 Gla residues, through between amino acid of the protein Gla and the number of Glu residues from its GRP as the protein with the highest of Gla identified to and its The high of conservation orthologs of GRP in different vertebrate from within this and in as as the presence of highly conserved specific to all VKD proteins propeptide containing a γ-glutamyl carboxylase recognition an a and a Gla-containing mature suggests that this is a new γ-carboxylated protein. The γ-carboxylase recognition site found in the GRP propeptide contains highly conserved residues and 3 and at by and to be for carboxylase Furie B. Furie B.C. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The conservation of 3 residues and the high of γ-carboxylation in the sturgeon protein suggests that the of carboxylase for GRP may be with that for VKD the at is not conserved and its of that is in mammalian is in and the of sturgeon and this in a in carboxylase binding and in of γ-carboxylation, to be conserved identified in as a site (9Laizé V. Martel P. Viegas C.S. B. Price P.A. Cancela M.L. J. Biol. Chem. 2005; 280: 26659-26668Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), was identified in all GRP The is by a site at the of the was by sequence of the sturgeon protein. The of this protein in the high number of Gla residues found in its mature This to have conserved of with an of conservation in number and of corresponding Glu residues within the protein 3, and and as as in the γ-carboxylase binding thus that this protein be γ-carboxylated in all including found in other VKD GRP also significant in protein and gene and particularly in the of its Gla domain. in all known VKD the Gla is in the mature protein, by an growth a a and a (11Kulman J.D. Harris J.E. Haldeman B.A. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9058-9062Crossref PubMed Scopus (94) Google Scholar). the Gla of GRP the mature protein. the of identified a secreted protein cartilage protein, which to be to GRP A. J. L. C. N. A. A. Biol. PubMed Scopus Google Scholar, C. U. N. J. A. P. U. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The presence of and of cartilage protein in the extracellular matrix C. U. N. J. A. P. U. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that may by a in the extracellular and is in with to the of γ-carboxylation in protein, to of γ-carboxylation the protein is in The of the protein, by may have to this and Gla residues are to Glu Furie B. Furie B.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The and of VKD proteins has to be by the in the in used Biochemistry. PubMed Scopus Google Scholar) by in any of the of this several protein are involved in the of VKD the γ-glutamyl carboxylase A. A. Furie B. Furie B.C. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar), the vitamin a protein that vitamin a vitamin and a propeptide A. A. Furie B. Furie B.C. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). with Gla-containing coagulation proteins that is the of all the involved in this process W. Biochemistry. 2006; 45: PubMed Scopus Google Scholar). in sturgeon, GRP mRNA with cartilaginous being in as as and mature cartilage has for its cartilage A. J. L. C. N. A. A. Biol. PubMed Scopus Google Scholar, C. U. N. J. A. P. U. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), with a in and and an in rat not that in Although we a GRP expression in rat to and chondrocytes, a in with the by C. U. N. J. A. P. U. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), we also found a positive signal for GRP mRNA in in and osteocytes. This the that this protein is cartilage specific in as by are by in silico and expression of the human GRP in and the of its cartilage and the of the protein cartilage by The binding of Gla residues within VKD proteins have with binding of calcium calcium through in the binding of coagulation to Furie B.C. B. Furie B. J. Biol. Chem. Full Text PDF PubMed Google Scholar), through binding to the in extracellular matrix G. J. Biol. PubMed Scopus Google Scholar). This was for and VKD proteins also known to be with of and The of GRP of the sturgeon extracellular matrix that its Gla is of binding to calcium in rat tissues and for we that mammalian GRP is of binding to highly in cartilage through a calcium with the Gla the described of the Gla in coagulation a of in and is for and of known to be involved in cartilage to the in were identified and found to be with A. 2007; PubMed Scopus (19) Google Scholar). The identification of a protein with an unprecedented high Gla extracellular matrix binding and a expression in cartilage also in other tissues including may to the for of the potential of this protein for calcium binding it a to play a role as calcium Viegas C.S. B. Cancela M.L. U. S. Scholar), and it be highly to its in cartilage and as as in in tissues with vitamin such as are used as and known to including bone and bone as as and a of that has not of the other known Gla proteins E.C. Schurgers L.J. Vermeer C. Thromb. Haemostasis. 2007; 98: 120-125Crossref PubMed Scopus (187) Google Scholar). is thus that of be by of GRP not GRP its new the in bone and cartilage and We P. for in the of in S. for in protein and A. J. and J. for of the biological used in this with