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 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.

FIGURE 1.

Isolation (A) and purification (B and C) of Adriatic sturgeon GRP. A, SDS-PAGE analysis of crude precipitate after acid extraction of branchial arches. The gel was stained with Coommassie Brilliant Blue (CBB) and 4-diazobenzenesulfonic acid (DBS). Arrow indicates the position of the GRP band for which the N-terminal sequence was determined. B, RP-HPLC separation of proteins in crude precipitate (see “experimental Procedures” for details). Fractions of 1 ml were collected and absorbance was determined at 220 nm. Arrow indicates the GRP elution peak. C, purification of GRP by Sephadex G-75 chromatography (see “Experimental Procedures” for details). Fractions of 0.5 ml were collected and absorbance was determined at 220 nm. The excluded and included volumes correspond to fractions 16 and 48 respectively. Four μg of RP-HPLC fractions 49-52 and 2 μg of Sephadex G-75 fractions 16 and 28-38 were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue (inset in panel C).

FIGURE 2.

GRP tissue distribution (A), sites of gene expression (B), and corresponding quantification of GRP expression levels per cell type(C) in Adriatic sturgeon. A, levels of GRP gene expression measured by real-time qPCR in adult tissues related to levels in heart (Ht). Lv, liver; Kd, kidney; Ms, muscle; Gn, gonads; Br, brain; GP, ganoid plate; AK, anterior kidney; Sl, spleen; Sp, spine; Ct, cleithrum; HP, head plate; Op, operculum; Sk, skull; Md, mandibula; BA, branchial arches; AV, anterior vertebra; PV, posterior vertebra. B, sites of gene expression determined by in situ hybridization in vertebra (panels 1 and 2) and mandibula (panel 3). Cb, chordoblast layer of the notochord; Cd, chordocytes; NtSh, notochord sheath; Ct, cartilage; IC, immature chondrocytes; MC, mature chondrocytes. Hybridization with sense probe is presented in panel 4. Panels 1, 2, 3, and 4, magnification ×20. C, the percentage of IM, MC, and chordoblasts (Cd) expressing GRP was determined by dividing the number of cells of the indicated type expressing GRP by the total number of cells of that type detected in 3 consecutive vertebra sections. p was calculated using the unilateral Student's t test.

FIGURE 3.

Alignment of GRP pre-propeptide of 7 species representative of vertebrates showing the conservation of residues at particular positions and highlighting functionally important domains and motifs. Stars indicate identical residues. Numbering is according to the first residue of the mature protein. Highly conserved residues in important motifs or in Gla domain are highlighted in gray (see supplemental Fig. S4). Arrows indicate cleavage position between the signal peptide and propeptide. RXXR, furin-like proteolytic cleavage site.

FIGURE 4.

Phylogenetic analysis of the mature GRP peptides. Neighbor-joining tree constructed with MEGA3 demonstrating the taxon-specific clustering of mature GRP peptides and illustrating the bony fish-specific gene duplication event. Internal numbers are supporting values for the corresponding branch points (supporting values below 60% were omitted). ^*, fish species where only one GRP isoform was found, either GRP1 or GRP2.

FIGURE 5.

GRP, MGP, and OC gene expression in rat cartilage and bone, determined by in situ hybridization in sections of rib (A) and tail (B), and corresponding quantification of GRP expression levels per cell type, performed through morphometric analysis (C). A, co-localization of GRP, MGP, and Oc in consecutive sections of rib. CC, columnar chondrocytes; HyC, hyaline cartilage; HZ, hypertrophic zone; TB, trabecular bone. White arrow and black arrowhead represents osteoblasts and osteocytes, respectively. Red asterisk is located within the hyaline cartilage central zone, which contain HC. B, co-localization of GRP, MGP, and Oc in consecutive sections of tail, as described for panel A. Hybridization with GRP sense probe is presented in the SP panel (×20). Magnification for panels A1-6 and panels B1 and B2, ×10. Magnification for panels A 1′, 2′, 3′, 4′, 5′, 6′, and panel B 1′, 2′, and 3, × 20. C, the percentage of immature and proliferating chondrocytes (IM), mature, columnar, and hypertrophic chondrocytes (MC), and osteocytes (Oc) expressing GRP was determined by dividing the number of cells of the indicated type expressing GRP by the total number of cells of that type detected in 4 consecutive sections of rib (IC and MC) and tail (Oc).

FIGURE 6.

Structural organization of the Gla domain of human VKD proteins at protein (A) and gene (B) levels. VKD protein superfamily is composed of 4 families, i.e. the Gla-rich protein family, the bone-associated Gla protein family (containing osteocalcin and matrix Gla protein), the coagulation factor family (containing factors 7, 9, and 10, prothrombin, proteins C, Z, and S, and gas6), and the proline-rich Gla protein family (PRGP1, -2, -3, and -4). A, SP, signal peptide; PP, propeptide; MM, mature peptide; GGCX, γ-glutamyl carboxylase recognition site; AXXF, conserved motif and proteolytic cleavage site in MGP; RXXR, proteolytic cleavage site, absent in MGP and PRGP4; P, serine phosphorylation; C, conserved cysteine residues; E, epidermal growth factor-like domain; PS, peptidase S1 domain; K, kringle domain; LG, laminin-G like domain; TM, transmembrane-like domain; PY, potential WW domain-binding motifs PPXY. Dashed line indicates disulfide bond. B, gene structure of human VKD proteins (limited to coding sequences and Gla domain; complete gene structure is presented in supplemental Fig. S8). Boxes represent coding exons; black boxes represent exons coding for the Gla domain; triangles indicate the phase of intron insertion; dash line represents the continuation of the gene. a indicates extra coding exon in coagulation factor 7 and protein Z, and b indicates fused exon in PRGP1 and -3 (see supplemental Fig. S9).