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 gamma-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 gamma-carboxylase recognition site and a mature protein with 14 highly conserved Glu residues, each of them being gamma-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.
Highlights
In the present work we report the purification and molecular characterization of a new member of the VKD protein family containing an unprecedented high density of Gla residues (22%) and named Gla-rich protein (GRP)
The presence of 16 Gla residues, assigned through comparison between amino acid composition of the pure protein, and the number of Glu residues deduced from its cDNA, identifies GRP as the protein with the highest ratio of Gla residues/size identified to date, and emphasizes its uniqueness
The putative ␥-carboxylase recognition site (GGCX) found in the GRP propeptide contains three highly conserved residues (e.g. Phe Ϫ17, Ala Ϫ11, and Leu Ϫ7) (Fig. 3 and supplemental Fig. S4) at positions shown by site-directed mutagenesis and kinetic studies to be important for carboxylase affinity (29 –31)
Summary
Gla, ␥-carboxyglutamic acid; GRP, Gla-rich protein; MGP, matrix Gla protein; OC, osteocalcin; HPRT1, hypoxanthine phosphoribosyltransferase 1; IC, immature chondrocytes; MC, mature chondrocytes; HC, hypertrophic chondrocytes; PC, proliferating chondrocytes; FC, fibrocartilage; RP-HPLC, reverse phase-high performance liquid chromatography; RACE, rapid amplification of cDNA ends; aa, amino acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; qPCR, quantitative PCR.
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