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.     

Discovery of a gamma-carboxyglutamic acid-containing protein in human spermatozoa

Here we describe the identification of a gamma-carboxyglutamic acid-containing protein in human spermatozoa. After thermal decarboxylation the protein is a good substrate for vitamin K-dependent carboxylase from various origins. A quick purification procedure for the decarboxylated protein is presented and in a preliminary characterization we have established its Mr (28 000-30 000) and its amino acid composition.     

Processing and transport of matrix gamma-carboxyglutamic acid protein and bone morphogenetic protein-2 in cultured human vascular smooth muscle cells: evidence for an uptake mechanism for serum fetuin

Matrix gamma-carboxyglutamic acid protein (MGP) is a member of the vitamin K-dependent protein family with unique structural and physical properties. MGP has been shown to be an inhibitor of arterial wall and cartilage calcification. One inhibitory mechanism is thought to be binding of bone morphogenetic protein-2. Binding has been shown to be dependent upon the vitamin K-dependent gamma-carboxylation modification of MGP. Since MGP is an insoluble matrix protein, this work has focused on intracellular processing and transport of MGP to become an extracellular binding protein for bone morphogenetic protein-2. Human vascular smooth muscle cells (VSMCs) were infected with an adenovirus carrying the MGP construct, which produced non-gamma-carboxylated MGP and fully gamma-carboxylated MGP. Both forms of MGP were found in the cytosolic and microsomal fractions obtained from the cells by differential centrifugation. The crude microsomal fraction was shown to contain an additional, more acidic Ser-phosphorylated form of MGP believed to be the product of Golgi casein kinase. The data suggest that phosphorylation of MGP dictates different transport routes for MGP in VSMCs. A proteomic approach failed to identify a larger soluble precursor of MGP or an intracellular carrier protein for MGP. Evidence is presented for a receptor-mediated uptake mechanism for fetuin by cultured human VSMCs. Fetuin, shown by mass spectrometry not to contain MGP, was found to be recognized by anti-MGP antibodies. Fetuin uptake and secretion by proliferating and differentiating cells at sites of calcification in the arterial wall may represent an additional protective mechanism against arterial calcification.     

Atherocalcin, a gamma-carboxyglutamic acid containing protein from atherosclerotic plaque.

The specialized calcium binding amino acid, γ-carboxyglutamic acid (Gla) is quantitated in developing atherosclerotic plaque relative to progression of the disease, and a Gla-containing protein isolated from calcified atherosclerotic plaque is partially characterized. Low levels of Gla are found in fatty streak and fibrous plaque lesions, and a marked increase in Gla content occurs in calcified plaque. A unique Gla-containing protein is purified from 0.5M EDTA (pH 8.0) extracts of calcified plaque, named atherocalcin. The protein containing 19 Gla residues/1000 amino acids is 80,000 molecular weight, with a pI of 4.16 – 4.3 and is uniquely different from other known Gla-containing proteins. The implications of this work for the further understanding of the pathogenesis and therapy of atherosclerosis are discussed.     

The inhibition of calcium phosphate precipitation by fetuin is accompanied by the formation of a fetuin-mineral complex

The present studies show that the previously reported ability of fetuin to inhibit the precipitation of hydroxyapatite from supersaturated solutions of calcium and phosphate in vitro is accompanied by the formation of the fetuin-mineral complex, a high molecular mass complex of calcium phosphate mineral and the proteins fetuin and matrix Gla protein that was initially discovered in the serum of rats treated with etidronate and that appears to play a critical role in inhibiting calcification in vivo. Rat serum potently inhibited the precipitation of calcium phosphate mineral when the concentration of calcium and phosphate were increased by 10 mm each, and the modified serum was incubated at 37 degrees C for 9 days; in the absence of serum, precipitation occurred in seconds. Large amounts of the fetuin-mineral complex were generated in the first 3 h of this incubation and remained throughout the 9-day incubation. Purified bovine fetuin inhibited the precipitation of mineral for over 14 days in a solution containing 5 mM calcium and phosphate at pH 7.4 at 22 degrees C, whereas precipitation occurred in minutes without fetuin. There was a biphasic drop in ionic calcium in the fetuin solution, however, from 5 to 3 mM in the first hour and from 3 to 0.9 mM between 20 and 24 h; these changes in ionic calcium are due to the formation of complexes of calcium, phosphate, and fetuin. The complex found at 24 h to 14 days is identical to the fetuin-mineral complex found in the serum of etidronate-treated rats, whereas the complex found between 1 and 20 h is less stable.     

Claudin-2 forms homodimers and is a component of a high molecular weight protein complex

Tight junctions are multiprotein complexes that form the fundamental physiologic and anatomic barrier between epithelial and endothelial cells, yet little information is available about their molecular organization. To begin to understand how the transmembrane proteins of the tight junction are organized into multiprotein complexes, we used blue native-PAGE (BN-PAGE) and cross-linking techniques to identify complexes extracted from MDCK II cells and mouse liver. In nonionic detergent extracts from MDCK II cells, the tight junction integral membrane protein claudin-2 was preferentially isolated as a homodimer, whereas claudin-4 was monomeric. Analysis of the interactions between chimeras of claudin-2 and -4 are consistent with the transmembrane domains of claudin-2 being responsible for dimerization, and mutational analysis followed by cross-linking indicated that the second transmembrane domains were arranged in close proximity in homodimers. BN-PAGE of mouse liver membrane identified a relatively discrete high molecular weight complex containing at least claudin-1, claudin-2, and occludin; the difference in the protein complex sizes between cultured cells and tissues may reflect differences in tight junction protein or lipid composition or post-translational modifications. Our results suggest that BN-PAGE may be a useful tool in understanding tight junction structure.     

Proton, calcium, and magnesium binding by peptides containing gamma-carboxyglutamic acid.

gamma-Carboxyglutamic acid (Gla) is believed to bind Ca [II] ions and Mg [II] ions in prothrombin and other coagulation proteins. Binding constants for H+, Ca [II] ions, and Mg [II] ions to Gla-containing peptides are determined using pH and ion selective electrode titrations. The binding constants for peptides containing a single Gla residue are similar to the constants for malonic acid. Peptides containing two Gla residues in sequence (di-Gla peptides) bind Ca [II] ions and Mg [II] ions more strongly. KMgL for the di-Gla peptides is similar to the site-binding constant for Ca [II] ions in denatured BF1. These di-Gla peptides may be useful analogs for metal binding by the disordered Gla domain in BF1.     

Characterization of protein S, a gamma-carboxyglutamic acid containing protein from bovine and human plasma.

Protein S is a vitamin K dependent protein of unknown function, which is present in mammalian plasma. It was isolated from bovine plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, and column chromatography on DEAE-Sephadex, heparin-agarose, and polyhomoarginine-Sepharose. Bovine Protein S (Mr 64,200) is a single-chain glycoprotein with an amino-terminal sequence of Ala-Asn-Thr-Leu-Leu-. It contains 7.0% carbohydrate and 10 residues of gamma-carboxyglutamic acid per mol of protein. Human Protein S (Mr 69,000) is also a single-chain glycoprotein with an amino-terminal sequence of Ala-Asn-Ser-Leu-Leu-. It contains 7.8% carbohydrate and 10 residues of gamma-carboxyglutamic acid per mol of protein. These results indicate that Protein S from bovine or human plasma shows many similarities to the other vitamin K dependent proteins present in plasma.     

Complete amino acid sequence of a subunit from rapeseed high molecular weight protein

A subunit (Mr 15,600) from the high molecular weight protein from rapeseed was separated and isolated; its purity and homogeneity were ascertained. The subunit was cleaved with cyanogen bromide, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease. The fragments were separated and isolated by polyacrylamide gel electrophoresis, gel filtration, column chromatography on Dowex 1 x 2, and paper electrophoresis. The amino acid compositions of the intact subunit and different fragments obtained from enzymatic and chemical cleavages were determined. The subunit and its fragments were sequenced by manual Edman method. The phenylthiohydantoin amino acids obtained after each step were identified by thin-layer chromatography and ultraviolet spectroscopy. The complete amino acid sequence of the subunit consisting of 125 amino acid residues has been established by the overlapping method.     

Identification of a high molecular weight actin-binding protein in skeletal muscle.

A large polypeptide having a molecular weight of 240,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate has been identified in whole cell homogenates from chick skeletal muscle myoblasts and the rat myoblast L6 cell line. A similar polypeptide was identified in both thigh and breast chicken skeletal muscle, but the latter contained less of this protein per g of tissue. Antibodies made to gizzard filamin (an actin-binding protein having a molecular weight of 240,000) cross-reacted with the partially purified Mr = 240,000 protein from chicken skeletal muscle. With use of the indirect immunofluorescence technique, the filamin antibody localized in the Z-line region of chicken skeletal muscle myofibrils. These results indicate that skeletal muscle contains a filamin-like protein that may form an integral part of the myofibril structure.     

Transference of the high molecular weight carbohydrate sequences of fetuin to the surface of carrot embryo protoplasts

A method is described for the removal of the carbohydrate sequences of glycoproteins, and their covalent attachment to hydrocarbon chains. These synthetic membrane components may then be incorporated into liposome and cell membranes. Pronase-liberated glycopeptides derived from fetuin were linked by a reduced Schiff's base linkage to tetradecyl aldehyde. The resulting glycolipid was incorporated by external addition, into phosphatidylcholine liposomes. Glycolipid transfer to these liposomes rendered them suseptible to agglutination by wheat germ lectin, which binds N-acetylneuraminic acid, the terminal carbohydrate of the high molecular weight fetuin sugar sequence. Sequential removal of the terminal sugars, and subsequent agglutination behaviour towards various lectins, suggests that the carbohydrate sequence had been transfered intact. The glycolipid was incorporated into plant protoplast membranes by incubation with glycolipid-containing liposomes for 2 h at 37°C. These synthetic glycolipids may find a use in the study of carbohydrate-based recognition systems in animal and plant membranes. In addition they may prove useful in the development of cell and membrane tagging and handling techniques, by the insertion of sugar groups not normally present in these membranes.     

Cancer resistance,high molecular weight hyaluronic acid,and longevity

Longevity varies greatly among mammals. The naked mole rat is among the longest-lived rodents, having an average lifespan of 32 years, compared to the similarly-sized house mouse with lifespan of 4 years. The rate of cancer also varies widely among mammals and interestingly, the naked mole rat is essentially cancer-free (Gorbunova et al., Nat Rev Genet 15(531):540, 2014). A series of elegant studies (Tian et al. Nature 499:346–349, 2013) has revealed that this cancer resistance derives from the abundant production of high molecular weight hyaluronic acid. Remarkably, high molecular weight hyaluronic acid, which accumulates within the extracellular matrix, stimulates an intracellular pathway that induces expression of p16^ink4a and suppresses oncogenic transformation.     

Heavy chain of human high molecular weight and low molecular weight kininogens binds calcium ion

An antibody subpopulation, anti high molecular weight (anti-HMW) kininogen-Ca2+ antibody able to bind specifically to the HMW kininogen-Ca2+ complex, was isolated from anti-HMW kininogen antiserum. Partially purified anti-HMW kininogen antibody was applied to a HMW kininogen-Sepharose column equilibrated with 40 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 7.5, containing 1.0 M NaCl and 1 mM CaCl2, and anti-HMW kininogen-Ca2+ antibody was eluted with 5 mM ethylenediaminetetraacetic acid. As a result of characterization by enzyme-linked immunosorbent assay, this antibody specifically recognized the cyanogen bromide cleaved fragment 1 (CB-1) region (1-160 amino acid sequence) of the heavy chain of kininogen molecules in the presence of Ca2+ or Mg2+. Furthermore, circular dichroism (CD) experiments showed that the conformational changes of HMW kininogen and heavy chain were induced by metal ions such as Ca2+ and Mg2+ and that these changes were due to the conformational change of the CB-1 region of the heavy chain. The dissociation constant (Kd) for the heavy chain-Ca2+ measured by CD analysis at 214 nm was found to be 0.33 +/- 0.09 mM (mean +/- SD). The number of Ca2+-binding sites of heavy chain calculated from the Hill plot was 1.15 +/- 0.04 (mean +/- SD). Then, a possible Ca2+-binding site was found in the amino-terminal portion of the heavy chain of kininogen molecules.     

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.     

Inhibition of human collagenase activity by a small molecular weight serum protein.

Fractionation of human serum proteins by gel filtration in Sephadex G-200 revealed two regions of collagenase inhibition which corresponded to α₂-macroglobulin and a smaller serum component which eluted after α₁-antitrypsin. The smaller collagenase inhibitor, having a molecular weight of 40,000 was separated from α₁-antitrypsin by chromatography in Sephadex DEAE A.50. It was found to inhibit human collagenases derived from skin, rheumatoid synovium, gastric mucosa and granulocytes, but not the neutral proteases trypsin and papain. Purified preparations of α₁-antitrypsin inhibiting trypsin and papain had no effect on the collagenase activities. The small collagenase inhibitor may have importance as a regulatory factor in the control of collagenase activity in vivo.     

Epstein-Barr virus induction by a serum factor. Purification of a high molecular weight protein that is responsible for induction

Serum contains a factor that induces Epstein-Barr virus antigens in latently infected human lymphoblastoid cell lines and that cooperates with chemical inducers. Here we report the purification of a protein that is responsible for the effect. Using ammonium sulphate precipitation, chromatography on DEAE-agarose and cellulose, molecular sieve chromatography on Bio-Gel A-5, and glycerol gradient centrifugation, the factor was enriched about 300,000-fold compared to calf serum. Under neutral conditions, the protein chromatographed as a high molecular weight protein of about 5.5 X 10(5) both in its active and inactive form. Both forms of the molecule sedimented at about 7 s. The factor is an acidic protein with a pI of 5. It seems to be composed of high molecular and low molecular weight subunits. Nanogram amounts of purified factor are sufficient for measurable inducing effects.     

Isolation and identification of a high molecular weight protein in sow milk

A high molecular weight protein (HMWP) was isolated and purified from sow milk, and some of its biochemical characteristics and biological functions were identified. The origin of HMWP was also investigated. The molecular weight of HMWP was determined to be about 115 000 and 114 800 by SDS-PAGE and gel filtration, respectively. The sequence of 10 amino acids in N-terminal of HMWP was Ala-Leu-Val-Gln-Ser-Cys-Leu-Asn-Leu-Val. The sequence was blasted against GenBank. No protein showed significant similarity with this sequence suggesting the HMWP may be novel. The result of liquid chromatography mass spectrometry (LC-MS) also proved HMWP could be a novel protein. By amino acid assay, HMWP was rich in glutamate (including glutamine), cysteine, glycine, aspartic acid (including asparagines) and proline. The content of hydrophobic amino acids (Ala, Val, Leu, Ile, Met, Phe and Pro) was lower at 18.59% of the total amino acids suggesting HMWP has high solubility in water. Western blots of lectins were used to identify the kinds of carbohydrate residues attached to HMWP qualitatively. The result showed that HMWP was a kind of glycoprotein containing N-acetylneuraminic acid (NeuNAc), mannose (Man) and/or N-acetylglucosamine (GlcNAc). By isoelectric focusing, HMWP pI was found to be 5.1. Compared with milk fat globule membrane protein (MFGMP) isolated from the sow milk in SDS-PAGE, MFGMP did not contain HMWP. HMWP was assumed to be a secretory milk protein. HMWP was not found in bovine, goat, rabbit or human milk in SDS-PAGE gel suggesting HMWP may be unique to sow milk. By Western blot, HMWP could be detected in sow milk, not in sow serum, which suggests it is synthesized and secreted by the mammary gland. HMWP concentrations in sows milk were the lowest in the first day of lactation, rose significantly during lactation 1 to 7 days. The HMWP content of sows milk remained relatively constant ((1.95±0.13) g/l) during lactation 7 to 20 days. HMWP significantly inhibited Escherichia coli in a dose related manner in vitro. Overall, HMWP could be a novel sow milk protein with implications for the mammary gland and the piglet.