Carboxyl-terminal proteolytic processing of matrix Gla protein.
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The present study was undertaken to determine the extent of COOH-terminal proteolytic processing in matrix Gla protein (MGP), a 10-kDa protein which contains 5 residues of the vitamin K-dependent Ca2+ binding amino acid, gamma-carboxyglutamic acid (Gla). Two forms of MGP were isolated from demineralization and urea extracts of bovine cortical bone, one 79 residues in length with the COOH terminus Phe-Arg-Gln and the other 83 residues in length with the COOH terminus Phe-Arg-Gln-Arg-Arg-Gly-Ala. The 84-residue form of bovine MGP predicted from the message structure could not be detected in the bone extracellular matrix extracts, and it therefore seems probable that the lysine at position 84 was removed by the action of a carboxypeptidase B-like enzyme prior to secretion. A plausible sequence of proteolytic cleavages that could generate the 79-residue form of MGP would be a trypsin-like cleavage at Arg80-Arg81 or Arg81-Gly82 followed by carboxypeptidase B-like cleavage to remove COOH-terminal arginine(s). Since essentially equal amounts of the 79- and 83-residue forms of MGP were also detected in bovine articular cartilage and plasma, it seems likely that the COOH-terminal processing events identified in bone apply to many of the other tissues which synthesize this protein. Only one form of MGP was detected in human bone extracts, a 77-residue protein that lacks the COOH-terminal residues Arg-Lys-Arg-Arg-Gly-Thr-Lys. This shortened version of human MGP is consistent with the proposed model for COOH-terminal processing, since the amino acid substitution in the COOH terminus of the human protein, Lys79 for Gln79, would allow removal of the additional basic residues from the human MGP COOH terminus by the action of the carboxypeptidase B-like enzymic activity. Recent studies have shown that MGP is strongly induced by retinoic acid in fibroblasts, chondrocytes, and osteoblasts, a response which suggests that MGP mediates an action of retinoic acid on an aspect of cell growth or differentiation. If this hypothesis is true, the present evidence for complex COOH-terminal processing events could provide a means to regulate the as yet unknown activity of MGP in the extracellular environment in a mechanism similar to the activation of hormones such as anaphlotoxins and kinins. (+info)
Non-strict strand orientation of the Ca2+-induced dimerization of a conantokin peptide variant with sequence-shifted gamma-carboxyglutamate residues.
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Monoclonal antibodies for human thrombomodulin which recognize binding sites for thrombin and protein C.
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Monoclonal antibodies for human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C, were prepared and their epitopes characterized. All six antibodies (MFTM-1-MFTM-6) bound to an elastase-digested active fragment of thrombomodulin, which contains six consecutive EGF domains. Binding of thrombomodulin to these antibodies did not depend on Ca2+ concentration. MFTM-4, MFTM-5, and MFTM-6 strongly inhibited protein C activation by thrombin and thrombomodulin. MFTM-4 and MFTM-5 inhibited thrombin binding to fixed thrombomodulin and bound to a recombinant mutant EGF456 protein, which contained the fourth, fifth, and sixth EGF domains of thrombomodulin. However, MFTM-6 did not inhibit thrombin binding to thrombomodulin and did not bind to EGF456 protein. Binding of thrombomodulin to fixed MFTM-4 or MFTM-5 was competitively inhibited by a recombinant mutant EGF45 protein which contained the fifth and sixth EGF-domains. These results suggest that epitopes of MFTM-4 and MFTM-5 are located in the fifth EGF domain of thrombomodulin. Thus, the binding site for thrombin is located in the fifth EGF domain. These results also suggest that an epitope for MFTM-6 is located at a region near the binding site for gamma-carboxyglutamic acid residues of protein C via Ca2+ on thrombomodulin. (+info)
Vitamin K promotes mineralization, osteoblast-to-osteocyte transition, and an anticatabolic phenotype by {gamma}-carboxylation-dependent and -independent mechanisms.
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Solution conformations of the gamma-carboxyglutamic acid domain of bovine prothrombin fragment 1, residues 1-65.
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Molecular dynamics simulations have been performed (AMBER version 3.1) on solvated residues 1-65 of bovine prothrombin fragment 1 (BF1) by using the 2.8-A resolution crystallographic coordinates as the starting conformation for understanding calcium ion-induced conformational changes that precede experimentally observable phospholipid binding. Simulations were performed on the non-metal-bound crystal structure, the form resulting from addition of eight calcium ions to the 1-65 region of the crystal structure, the form resulting from removal of calcium ions after 107 ps and continuing the simulation, and an isolated hexapeptide loop (residues 18-23). In all cases, the 100-ps time scale seemed adequate to sample an ensemble of solution conformers within a particular region of conformation space. The non-metal-containing BF1 did not unfold appreciably during a 106-ps simulation starting from the crystallographic geometry. The calcium ion-containing structure (Ca-BF1) underwent an interesting conformational reorganization during its evolution from the crystal structure: during the time course of a 107-ps simulation, Ca-BF1 experienced a trans----cis isomerization of the gamma-carboxyglutamic acid-21 (Gla-21)-Pro-22 peptide bond. Removal of the calcium ions from this structure followed by 114 ps of additional molecular dynamics showed significant unfolding relative to the final 20-ps average structure of the 107-ps simulation; however, the Gla-21-Pro-22 peptide bond remained cis. A 265-ps simulation on the termini-protected hexapeptide loop (Cys-18 to Cys-23) containing two calcium ions also did not undergo a trans----cis isomerization. It is believed that the necessary activation energy for the transitional event observed in the Ca-BF1 simulation was largely supplied by global conformational events with a possible assist from relief of intermolecular crystal packing forces. The presence of a Gla preceding Pro-22, the inclusion of Pro-22 in a highly strained loop structure, and the formation of two long-lived salt bridges prior to isomerization may all contribute to this finding. (+info)
Structural requirements for Ca2+ binding to the gamma-carboxyglutamic acid and epidermal growth factor-like regions of factor IX. Studies using intact domains isolated from controlled proteolytic digests of bovine factor IX.
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Blood coagulation factor IX is composed of discrete domains with an NH2-terminal vitamin K-dependent gamma-carboxyglutamic acid (Gla)-containing region, followed by two domains that are homologous with the epidermal growth factor (EGF) precursor and a COOH-terminal serine protease part. Calcium ions bind to the Gla-containing region and to the NH2-terminal EGF-like domain. To be able to determine the structure and function of the Gla- and EGF-like domains, we have devised a method for cleaving factor IX under controlled conditions and isolating the intact domains in high yield, either separately or linked together. The Ca2+ and Mg2+ binding properties of these fragments were examined by monitoring the metal ion-induced changes in intrinsic protein fluorescence. A fragment, consisting of the Gla region linked to the two EGF-like domains, bound Ca2+ in a manner that was indistinguishable from that of the intact molecule, indicating a native conformation. The Ca2+ affinity of the isolated Gla region was lower, suggesting that the EGF-like domains function as a scaffold for the folding of the Gla region. The Gla-independent high affinity metal ion binding site in the NH2-terminal EGF-like domain was shown to bind Ca2+ but not Mg2+. A comparison with similar studies of factor X (Persson, E., Bjork, I., and Stenflo, J. (1991) J. Biol. Chem. 266, 2444-2452) suggests that the Ca2(+)-induced fluorescence quenching is due to an altered environment primarily around the tryptophan residue in position 42. (+info)