Cloning of the bone Gla protein gene from the teleost fish Sparus aurata. Evidence for overall conservation in gene organization and bone-specific expression from fish to man

Bone Gla protein (BGP, Osteocalcin) is a bone-specific vitamin K-dependent protein which has been intensively studied in mammals. Although BGP is the most abundant non-collagenous protein of bone, its mode of action at the molecular level remains unclear. From an evolutionary point of view, the appearance of BGP seems to parallel the appearance of hydroxyapatite-containing bone structures since it has never been found in elasmobranchs, whose skeleton is composed of calcified cartilage. Accordingly, recent work indicates that, in mammalian bone, BGP is required for adequate maturation of the hydroxyapatite crystal. Taken together, these data suggest that teleost fishes, presumably the first vertebrates to develop a BGP-containing skeleton, may be a useful model to further investigate BGP function. In addition, fish offer several advantages over mammalian models, due to a large progeny, external embryonic development and transparency of larvae. In the present work, the BGP cDNA and gene were cloned from a teleost fish, Sparus aurata, and its tissue distribution, pattern of developmental expression and evolutionary pathways analyzed. The molecular organization of the Sparus BGP (spBGP) gene is similar to mammalian BGP genes, and its expression throughout development follows the onset of calcification. The spBGP gene encodes a pre-propeptide of 97 amino acid residues, expressed only in bone and showing extensive homology to its mammalian homologs. Phylogenetic analysis of the available BGP sequences supports the hypothesis that all BGPs have a single origin and share a common ancestor with a related vitamin K-dependent protein (Matrix Gla protein).     

Discovery of bone gamma-carboxyglutamic acid protein in mineralized scales. The abundance and structure of Lepomis macrochirus bone gamma-carboxyglutamic acid protein.

The mineralized scale of the freshwater sunfish Lepomis macrochirus (bluegill) contains a Gla protein. The protein was identified in extracts of scale by a new colorimetric assay for Gla-containing proteins. The protein was purified by gel filtration chromatography followed by reversed phase high performance liquid chromatography (HPLC). Several tests establish the identity of scale Gla protein and bone Gla protein (BGP). First, the proteins exhibit identical mobilities on electrophoresis and by reversed phase HPLC. Second, they have identical amino-terminal amino acid sequences. Finally, identical peptides are generated by proteolytic digestion. The 45-residue amino acid sequence of the bone Gla protein from L. macrochirus has a high sequence homology with swordfish, as well as homology to mammalian bone Gla protein. The BGP of bluegill shares with swordfish BGP a truncated NH2 terminus and an extended COOH terminus. These features may be unique to fish, as they have not been observed in terrestrial vertebrates. The bluegill BGP is the first vitamin K-dependent protein to contain a non-gamma-carboxylated residue to the NH2-terminal side of all of its Gla residues. In all other vitamin K-dependent proteins, Gla always appears to the NH2-terminal side of the first Glu. The implications of this result are discussed. The bluegill rib bone is curiously enriched in BGP, as are other mineralized tissues of this species. One hypothesis is that this may be due to the acellular nature of the bone in this species. The abundance of BGP in the bones of this fish may provide clues to the unknown function of this bone protein.     

Primary structure of bovine matrix Gla protein, a new vitamin K-dependent bone protein

The complete amino acid sequence of bovine bone matrix Gla protein (MGP) was determined by automatic sequence analysis of the intact protein and of peptides isolated from tryptic and BNPS-skatole digests. This 79-residue, vitamin K-dependent protein contains a single disulfide bond and 4.8 gamma-carboxyglutamate (Gla) residues, one each at positions 37, 41, 48, and 52, and 0.8 Gla and 0.2 Glu at position 2. There is sufficient sequence homology between MGP and bone Gla protein (BGP) to indicate that these two bovine bone proteins arose by gene duplication and subsequent divergent evolution. Although MGP has a very low solubility in water compared to BGP, there is no hydrophobic domain in MGP which could account for its insolubility, and the overall fraction of hydrophobic residues is 32% for MGP compared to 43% for BGP. MGP is the first vitamin K-dependent protein to be discovered which has several non-gamma-carboxylated residues to the NH2-terminal side of its Gla residues. The presence of NH2-terminal Glu residues between the putative targeting domain for the gamma-carboxylase in the MGP leader sequence and the mid-molecule Gla residues suggests that the gamma-carboxylase may have additional, as yet unrecognized, specificity requirements which determine the susceptibility of Glu residues for gamma-carboxylation.     

1,25-Dihydroxyvitamin D3 stimulates the synthesis of matrix gamma-carboxyglutamic acid protein by osteosarcoma cells. Mutually exclusive expression of vitamin K-dependent bone proteins by clonal osteoblastic cell lines

Several clonal rat osteosarcoma cell lines were tested for the ability to express and secrete matrix Gla protein (MGP), a small vitamin K-dependent protein found in bone and cartilage. Two independently derived cell lines, UMR 106-01 and ROS 25/1, expressed MGP mRNA and secreted MGP antigen identical in size with that found in bone. No MGP message could be detected in ROS 17/2 and 2/3 cells, cell lines previously shown to synthesize the other known vitamin K-dependent bone protein, bone Gla protein (BGP), and no BGP mRNA could be detected in the cell lines which synthesize MGP. Since UMR 106-01 and ROS 17/2 are presently the best characterized clonal osteoblastic cell lines, the discovery of the mutually exclusive expression of MGP and BGP by these cell lines indicates that osteosarcoma cells can be fixed in different phenotypic states and that MGP and BGP should be useful markers for the analysis of phenotypic expression in bone. Treatment of UMR 106-01 cells with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) dramatically increased MGP mRNA within 4 h and, by 24 h, increased MGP secretion 15-fold. This is only the second example of a bone matrix protein whose synthesis is dramatically increased by vitamin D, the first being the 6-fold stimulation of BGP synthesis by 1,25(OH)2D3 in ROS 17/2 cells. The discovery that MGP and BGP are similarily regulated by 1,25(OH)2D3 was unexpected since the two proteins differ markedly in structure, physical properties, and tissue distribution. Since the synthesis of MGP is rapidly and dramatically increased by 1,25(OH)2D3, it is probable that MGP plays a role in the normal bone response to the hormone. MGP may also be the vitamin K-dependent protein whose abnormal synthesis in the Warfarin-treated animal modifies the bone response to 1,25(OH)2D3.     

Lung, heart, and kidney express high levels of mRNA for the vitamin K-dependent matrix Gla protein. Implications for the possible functions of matrix Gla protein and for the tissue distribution of the gamma-carboxylase

We have used cDNA probes for two small vitamin K-dependent bone matrix proteins, bone Gla protein (BGP) and matrix Gla protein (MGP), to evaluate the possibility that either of these proteins might be synthesized by the various soft tissues previously shown to have gamma-carboxylase activity. BGP mRNA was found in bone but not in any of the soft tissues tested, a result which reinforces the view that plasma BGP is a specific marker for bone metabolism. In contrast, MGP mRNA was found in all rat tissues examined. Lung and heart have 10-fold higher levels of MGP mRNA than bone, and kidney has a 5-fold higher level. Despite the high levels of MGP mRNA in heart and kidney, these tissues contain 40-500-fold lower concentrations of MGP protein than bone. Immunofluorescence was used to identify cells that contain MGP in kidney, lung, heart, and spleen. In each tissue, MGP was found in discrete tissue-specific cell types. In most of the soft tissues tested, MGP is the first well characterized substrate for the vitamin K-dependent carboxylase found to be synthesized. The exceptionally broad tissue distribution for MGP synthesis demonstrates that the function of MGP is not specific to connective tissues, and the low levels of MGP antigen in soft tissues with high MGP mRNA levels indicate that MGP is unlikely to act solely by virtue of its accumulation in an extracellular matrix.     

Matrix Gla protein, a new gamma-carboxyglutamic acid-containing protein which is associated with the organic matrix of bone

A new protein has been isolated from CaCl2/urea extracts of demineralized bovine bone matrix. This protein has five to six residues of the vitamin K-dependent amino acid, gamma-carboxyglutamic acid (Gla), and we have accordingly designated it matrix Gla protein. Matrix Gla protein is a 15,000 dalton protein whose amino acid composition includes a single disulfide bond. The absence of 4-hydroxyproline in matrix Gla protein demonstrates that it is not a precursor to bone Gla protein, 5,800 dalton protein which has a residue of 4-hydroxyproline at position 9 in its sequence. Matrix Gla protein also does not cross-react with antibodies raised against bone Gla protein.     

Periostin, a member of a novel family of vitamin K-dependent proteins, is expressed by mesenchymal stromal cells

The modification of glutamic acid residues to gamma-carboxyglutamic acid (Gla) is a post-translational modification catalyzed by the vitamin K-dependent enzyme gamma-glutamylcarboxylase. Despite ubiquitous expression of the gamma-carboxylation machinery in mammalian tissues, only 12 Gla-containing proteins have so far been identified in humans. Because bone tissue is the second most abundant source of Gla-containing proteins after the liver, we sought to identify Gla proteins secreted by bone marrow-derived mesenchymal stromal cells (MSCs). We used a proteomics approach to screen the secretome of MSCs with a combination of two-dimensional gel electrophoresis and tandem mass spectrometry. The most abundant Gla-containing protein secreted by MSCs was identified as periostin, a previously unrecognized gamma-carboxylated protein. In silico amino acid sequence analysis of periostin demonstrated the presence of four consensus gamma-carboxylase recognition sites embedded within fasciclin-like protein domains. The carboxylation of periostin was confirmed by immunoprecipitation and purification of the recombinant protein. Carboxylation of periostin could be inhibited by warfarin in MSCs, demonstrating its dependence on the presence of vitamin K. We were able to demonstrate localization of carboxylated periostin to bone nodules formed by MSCs in vitro, suggesting a role in extracellular matrix mineralization. Our data also show that another fasciclin I-like protein, betaig-h3, contains Gla. In conclusion, periostin is a member of a novel vitamin K-dependent gamma-carboxylated protein family characterized by the presence of fasciclin domains. Furthermore, carboxylated periostin is produced by bone-derived cells of mesenchymal lineage and is abundantly found in mineralized bone nodules in vitro.     

Gla-rich protein (GRP), a new vitamin K-dependent protein identified from sturgeon cartilage and highly conserved in vertebrates

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.     

The identification of matrix Gla protein in cartilage

The vitamin K-dependent bone protein matrix gamma-carboxyglutamic acid (Gla) protein (MGP) has been identified by radioimmunoassay in the guanidine extract of rat cartilage. MGP was present in all cartilages tested at levels comparable to the MGP level in bone. Western blot analysis indicated that the molecular weight of cartilage MGP is the same as bone MGP, and Northern blot analysis revealed that MGP mRNA from cartilage is the same size as the MGP mRNA from bone. The structurally related vitamin K-dependent protein bone Gla protein could not be detected in cartilage by radioimmunoassay or by Northern blot analysis. The discovery that MGP is synthesized by growth plate cartilage could provide an explanation for the excessive growth plate mineralization disorder seen in rats treated with the vitamin K antagonist warfarin and the punctate mineralization of the growth plate seen in infants whose mothers received warfarin in the first trimester of pregnancy (the fetal warfarin syndrome). Both disorders appear to be caused by the inactivation of a vitamin K-dependent mineralization inhibitor in cartilage, an inhibitor which we suggest is MGP.     

Developmental appearance of matrix GLA protein during calcification in the rat

A marked dissociation has been observed between the timed accumulation in calcified tissues of two related vitamin K-dependent proteins, bone Gla protein (BGP) and the recently discovered matrix Gla protein (MGP). In long bone diaphyses, total levels of MGP were essentially equivalent in newborn, juvenile, and adult rats. In agreement with previous studies, BGP levels were only 5% of adult levels in newborn rat bones and increased to 90% of adult levels by 19 days of age. Similar results were obtained from the analysis of the longitudinal distribution of MGP and BGP in 14-day-old rat tibia, a bone in which new mineral is added rapidly at both growth plates. Again, MGP was essentially at the same level in the regions nearest the growth plates as in the midshaft while BGP levels were 10-fold lower in the regions nearest the growth plates. These differences in the timed accumulation of MGP and BGP in calcifying tissues indicate that MGP could function earlier in bone formation than does BGP. To further characterize the MGP antigen in bone, extracts from newborn and adult rat bones were chromatographed by gel filtration over Sephacryl S-200. All of the antigen extracted by formic acid and most of the antigen subsequently extracted by guanidine HCI emerged at the position expected for the 79-residue MGP. There was a significant difference in the fraction of total MGP which was extracted by guanidine HCI in newborn (50%) and adult (20%) bone. The radioimmunoassay for rat MGP which was developed for these studies employs rabbit antibody directed against calf MGP and rat MGP for standards and radioiodinated tracer. This assay has a sensitivity of 0.1 ng and does not detect rat or calf BGP.     

The propeptides of the vitamin K-dependent proteins possess different affinities for the vitamin K-dependent carboxylase.

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the modification of specific glutamates in a number of proteins required for blood coagulation and associated with bone and calcium homeostasis. All known vitamin K-dependent proteins possess a conserved eighteen-amino acid propeptide sequence that is the primary binding site for the carboxylase. We compared the relative affinities of synthetic propeptides of nine human vitamin K-dependent proteins by determining the inhibition constants (Ki) toward a factor IX propeptide/gamma-carboxyglutamic acid domain substrate. The Ki values for six of the propeptides (factor X, matrix Gla protein, factor VII, factor IX, PRGP1, and protein S) were between 2-35 nM, with the factor X propeptide having the tightest affinity. In contrast, the inhibition constants for the propeptides of prothrombin and protein C are approximately 100-fold weaker than the factor X propeptide. The propeptide of bone Gla protein demonstrates severely impaired carboxylase binding with an inhibition constant of at least 200,000-fold weaker than the factor X propeptide. This study demonstrates that the affinities of the propeptides of the vitamin K-dependent proteins vary over a considerable range; this may have important physiological consequences in the levels of vitamin K-dependent proteins and the biochemical mechanism by which these substrates are modified by the carboxylase.     

Osteocalcin and matrix GLA protein in developing teleost teeth: identification of sites of mRNA and protein accumulation at single cell resolution

In this study, the tissue distribution and accumulation of osteocalcin or bone Gla protein (BGP) and matrix Gla protein (MGP) were determined during tooth development in a teleost fish, Argyrosomus regius. In this species, the presence of BGP and MGP mRNA in teeth was revealed by in situ hybridization. mRNA for BGP was detected in the odontoblasts as well as in its cytoplasmic processes emerging through dentinal tubules, while mRNA for MGP was expressed in the enamel portion within the apical portion of the elongated cell bodies of enameloblasts, adjacent to the root of the teeth as well as in cells within the pulpal space. Immunolocalization of BGP and MGP demonstrated that these proteins accumulate mainly in the mineralized dentin or in enameloblastic processes, confirming in situ hybridization results. In this study, we examined for the first time the localization of both BGP and MGP gene expression and protein accumulation within the different regions of the vertebrate tooth. We clearly demonstrated that although the overall pattern of BGP and MGP gene expression and protein accumulation in A. regius teeth was in general agreement to what is known for other vertebrates such as rats or rodents, our study provided novel information and highlighted some species-differences between fish and higher vertebrates.     

Cloning of Rat Vitamin K-Dependent γ-Glutamyl Carboxylase and Developmentally Regulated Gene Expression in Postimplantation Embryos

Vitamin K-dependent carboxylase catalyzes the posttranslational modification of glutamate to γ-carboxyglutamate (Gla) in its substrates, the vitamin K-dependent proteins (VKDPs). This modification is required for the activities of the VKDPs. Recent evidence demonstrates previously unrecognized roles for VKDPs as signaling molecules important in the regulation of cell growth, adhesion, and apoptosis, suggesting developmental functions for VKDPs and hence the carboxylase. The tissue distribution and functions of carboxylase in development are unknown. In this study, we isolated and characterized the full-length cDNA encoding the rat carboxylase and analyzed, at the cellular level, the expression of this gene in rat embryos byin situhybridization. We demonstrate that the expression of this gene is highly regulated in a developmental and tissue-specific manner. Hepatocytes, the major site of synthesis of VKDPs of blood coagulation, express carboxylase mRNA late in gestation, in contrast to the central nervous system, mesenchymal, and skeletal tissues which express carboxylase mRNA early during rat embryogenesis. The tissue-specific temporal expression of the carboxylase gene during embryogenesis indicates that vitamin K-dependent carboxylation and the formation of Gla is developmentally regulated. These studies suggest that vitamin K-dependent carboxylation is an important modulator of embryonic VKDP function.     

Expression of Gla‐rich protein (GRP) in newly developed cartilage‐derived cell cultures from sturgeon (Acipenser naccarii)

Sturgeons are representative of an ancient fish group, and present mainly an internal cartilaginous skeleton, with bone found essentially in the ganoid plaques forming the exogenous skeleton. Because of its archaic genetics, sturgeon represents an important model organism to understand the role of bone and cartilage‐related Gla proteins and determine if their molecular mechanisms of action were maintained throughout evolution. Of particular relevance is understanding the regulation, in sturgeon, of those proteins known to be involved in tissue mineralization in mammals, as well as unveiling the function of newly identified calcification‐related genes such as the one encoding the recently discovered Gla‐rich protein (GRP), thus contributing to understand the poor calcification observed in sturgeon endoskeleton. However, regulation of gene expression and promoter functional analysis of sturgeon cartilage and bone‐specific genes has been hampered by lack of suitable in vitro cell systems. We have recently developed the first sturgeon vertebra (VAn2H) and branchial arches (BAAn1F) derived cell cultures, and here we report their inability to mineralize their ECM under mineralizing culture conditions, as detected by von Kossa staining. Although a more extensive characterization of these systems is ongoing, our first data indicate that these cells represent a valuable tool for expression analysis of sturgeon bone and cartilage genes.     

Characterization of the cDNA encoding bullfrog, Rana catesbeiana, osteocalcin and two forms of the protein isolated from bone

A full-length cDNA clone encoding osteocalcin from the bullfrog, Rana catesbeiana (bone Gla-protein, BGP) has been isolated, and the complete coding sequence for the 100-amino-acid pre-pro-osteocalcin protein was determined. The amino acid sequence of Rana catesbeiana osteocalcin, especially the mature 49-amino acid sequence, is closer to the mammalian than to the fish, Sparus osteocalcin. Rana mature osteocalcin has a similarity of 67% with human or 59% with rat osteocalcin, and only 42% with fish mature osteocalcin. The 51-amino-acid pre-pro-peptide contains the expected hydrophobic leader sequence and the dibasic Arg-Arg sequence preceding the NH2-terminal Ser of the mature 49-amino-acid Rana osteocalcin. The pro-peptide sequence also contains the expected motif of polar and hydrophobic residues, which targets vitamin K-dependent gamma-carboxylation of three specific Glu residues at positions 17, 21, and 24 in the mature protein. At the native protein expression levels, extraction from Rana cortical bone in the presence of protease inhibitor cocktail resulted in the isolation of two distinct forms of osteocalcin, P-1 and P-2, with a 3:2 distribution. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and amino acid sequence analysis of the N-terminal domain, we confirmed that P-1 is the intact 49-residue osteocalcin with N-terminal SNLRNAVFG., and that P-2 lacks four amino acids from the N-terminus, (NAVFG.). These results demonstrate the existence of a form of osteocalcin lacking four N-terminal amino acids in Rana bone, and that mature Rana osteocalcins remained highly conserved in their molecular evolution, especially with respect to the conservation of the C-terminal domain (residues 14-49).     

Sequence of the precursor to rat bone gamma-carboxyglutamic acid protein that accumulates in warfarin-treated osteosarcoma cells

A biosynthetic precursor to rat bone gamma-carboxyglutamic acid protein (BGP) was isolated from warfarin-treated ROS 17/2 osteosarcoma cells by antibody affinity chromatography followed by reverse phase high performance liquid chromatography. Thirty-two residues of its NH2-terminal sequence were determined by gas-phase protein sequence analysis. Comparison of this sequence with the known structure of rat BGP established that the intracellular precursor is a 76-residue molecule of Mr = 9120 that differs from 6000-Da bone BGP in having an NH2-terminal extension of 26 residues. This precursor appears to be generated from the primary translation product by cleavage of a hydrophobic signal peptide and is the probable substrate for gamma-carboxylation by virtue of its accumulation in the presence of warfarin. The putative targeting region for gamma-carboxylation previously identified in the leader sequences of vitamin K-dependent proteins is found in the propeptide portion of the precursor. Since the immunoreactive component secreted by warfarin-treated cells is identical in sequence to the 6000-Da BGP from bone, propeptide cleavage from the precursor is independent of gamma-carboxylation and precedes secretion of BGP from the cell.     

No evidence of association of the osteocalcin gene HindIII polymorphism with bone mineral density in Chinese women

Osteoporosis is a major health problem, mainly characterized by low bone mineral density (BMD). Osteocalcin (also known as BGP, for bone Gla protein) is a significant biomarker of bone turnover and thus the BGP gene has been considered as an important candidate gene for osteoporosis. A few studies on the relationship between variants of the BGP gene and BMD variation, via traditional association and/or linkage methods, have yielded conflicting results. In the present study, we simultaneously tested linkage and/or association of the BGP HindIII polymorphism with BMD in a large cohort of pre-menopausal Chinese women. A total of 1,263 subjects from 402 Chinese nuclear families were examined. Each family consists of both parents and at least one daughter aged between 20-45 years. BMDs at the lumbar spine and hip were measured by dual-energy X-ray absorptiometry (DXA). Using the QTDT (quantitative transmission disequilibrium test) program, we did not detect significant evidence of linkage or association between the BGP HindIII polymorphisms and the BMD variation at any skeletal site. Our data do not support the BGP gene having a major effect on BMD variation in pre-menopausal Chinese women.     

The vitamin K-dependent bone protein is accumulated within cultured osteosarcoma cells in the presence of the vitamin K antagonist warfarin

Osteosarcoma cells grown in normal culture medium secrete bone gamma-carboxyglutamic acid protein (BGP, osteocalcin) which is identical with BGP purified from the bone matrix. Two tests indicate that the secreted medium protein contains the full complement of three gamma-carboxyglutamate residues present on BGP purified from the bone matrix. First, the secreted protein from ROS 17/2 and bone matrix BGP have identical isoelectric points (pI = 4.0). Second, they have identical hydroxyapatite binding behavior. If warfarin is added to the culture medium, the secreted protein has a higher isoelectric point (pI = 4.6) and a lower affinity for hydroxyapatite characteristic of thermally decarboxylated or non-gamma-carboxylated BGP. The observed shift in isoelectric point of secreted BGP after warfarin treatment from pH 4.0 to 4.6 is also reflected in the presence of pI = 4.1 and pI = 4.6 species intracellularly. These isoelectric species correspond to fully carboxylated BGP and noncarboxylated BGP, which are in the process of secretion. Addition of 10 micrograms/ml of warfarin causes a specific 47% reduction in secretion rate of BGP, while at the same time, the intracellular BGP concentration increases 3-fold. These phenomena appear related to the interruption by warfarin of the normal sequence of processing of precursor BGP proteins, as a new, immunoreactive species with a higher isoelectric pH not present in control cells appears to be responsible for the increased intracellular antigen within warfarin-treated cells. Our results show that vitamin K-dependent processing is important for normal secretion of BGP from the cell.