Search

The blood coagulation and regulatory proteins that contain gamma-carboxyglutamic acid are a part of a unique class of membrane binding proteins that require calcium for their interaction with cell membranes. Following protein biosynthesis, glutamic acids on these proteins are converted to gamma-carboxyglutamic acid (Gla) in a reaction that requires vitamin K as a cofactor. The vitamin K-dependent proteins undergo a conformational transition upon metal ion binding, but only calcium ions mediate protein-phospholipid interaction. To identify the site on Factor IX that is required for phospholipid binding, we have determined the three-dimensional structure of the Factor IX Gla domain bound to magnesium ions by NMR spectroscopy. By comparison of this structure to that of the Gla domain bound to calcium ions, we localize the membrane binding site to a highly ordered structure including residues 1-11 of the Gla domain. In the presence of Ca2+, Factor IX Gla domain peptides that contain the photoactivatable amino acid p-benzoyl-L-phenylalanine at positions 6 or 9 cross-link to phospholipid following irradiation, while peptides lacking this amino acid analog or with this analog at position 46 did not cross-link. These results indicate that the NH2 terminus of the Gla domain, specifically including leucine 6 and phenylalanine 9 in the hydrophobic patch, is the contact surface on Factor IX that interacts with the phospholipid bilayer.

Multiple sclerosis is generally believed to involve an autoimmune syndrome in which affected individuals experience degradation of the myelin sheath in the central nervous system. In this study, rainbow trout were immunized with myelin basic protein and myelin in an attempt to induce a myelin-specific autoimmune response using the experimentally induced autoimmune response in Lewis rats as a model. Subsequent to immunization, rainbow trout antibodies to myelin basic protein were detected via an ELISA. Although clinical signs of nerve dysfunction were never apparent, disease due to demyelinization cannot be ruled out until histological studies are performed. It is shown that both rainbow trout immune and normal sera exert an inhibitory affect on the mitogenic stimulation of isolated mouse splenic T-cells. This in vitro inhibitory activity, which is clearly greater in immune serum, is hypothesized to be derived from a nonspecific antiinflammatory factor. While the identity of the factor(s) is not yet known, likely possibilities are discussed.

We have determined the Ca(II)-bound structure of factor IX, residues 1-47, by nuclear magnetic resonance (NMR) spectroscopy. The amino-terminal 47 residues include the gamma-carboxyglutamic acid-rich and aromatic amino acid stack domains, and this region is responsible for Ca(II)-dependent phospholipid binding in factor IX. Protons in the 1-47 amino acid sequence were assigned using standard two-dimensional homonuclear NMR experiments. A total of 851 distance restraints and 57 torsion angle restraints were used to generate 17 final structures by distance geometry and simulated annealing methods. The backbone RMSD to the geometric average is 0.6 +/- 0.1 A. The Ca(II)-bound structure is substantially more ordered with increased helical content compared to the apo-factor IX (1-47) structure. The global fold is similar to the crystal structure of the Ca(II)-bound Gla domain of prothrombin fragment I from residues 12 to 47 (RMSD approximately 1.3 A), but the backbone conformation differs in the first 11 residues, particularly between residues 3 and 6. The amino-terminal nine Gla residues are oriented to the interior of the protein and suggest an internal Ca(II) binding pocket. The carboxyl-terminal three Gla residues are exposed to solvent. The majority of hydrophobic residues are required to stabilize a globular core in the carboxyl-terminal three-quarters of the molecule. However, a hydrophobic surface patch in the amino-terminal region may represent a phospholipid binding site in factor IX.

The gamma-carboxyglutamic acid-rich domain of blood coagulation Factor IX is required for the binding of the protein to phospholipid membranes. To investigate the three-dimensional structure of this domain, a synthetic peptide corresponding to residues 1-47 of Factor IX was studied by 1H NMR spectroscopy. In the absence of metal ions, the proton chemical shift dispersion in the one-dimensional NMR spectrum indicated that the peptide contains regular structural elements. Upon the addition of Ca(II) or Mg(II), large chemical shift changes were observed in the amide proton and methyl proton regions of the spectrum, consistent with the conformational transitions that metal ions are known to induce in native Factor IX. The apopeptide was studied by two-dimensional NMR spectroscopy at 500 MHz to determine its solution structure. Protons were assigned using total correlation spectroscopy, nuclear Overhauser effect spectroscopy, and double quantum-filtered correlation spectroscopy experiments. Intensities of cross-peaks in the nuclear Overhauser effect spectrum were used to generate a set of interproton distance restraints. The structure of the apopeptide was then calculated using distance geometry methods. There are three structural elements in the apopeptide that are linked by a flexible polypeptide backbone. These elements include a short amino-terminal tetrapeptide loop (amino acids 6-9), the disulfide-containing hexapeptide loop (amino acids 18-23), and a carboxyl-terminal alpha helix (amino acids 37-46). Amide hydrogen exchange kinetics indicate that the majority of the peptide is solvent accessible, except in the carboxyl-terminal element. The structured regions in the apopeptide are insufficient to support phospholipid binding, indicating the importance of additional structural features in the Ca(II)-stabilized conformer.

The gamma-carboxylase recognition site in the propeptide of profactor IX signals the gamma-carboxylation of specific glutamic acid residues in the adjacent Gla domain during factor IX biosynthesis. To study posttranslational processing of the vitamin K-dependent blood coagulation factors and the properties of processing intermediates, we have isolated an incompletely processed factor IX species, profactor IX, from the medium of heterologous mammalian cells expressing the human factor IX cDNA. Profactor IX was purified by sequential immunoaffinity chromatography using antibodies specific for the propeptide and antibodies specific for the well-carboxylated factor IX species. This purified profactor IX preparation was fully gamma-carboxylated and contained the N-terminal propeptide, but it exhibited no factor IX procoagulant activity. Profactor IX was not cleaved following incubation with factor XIa. In contrast to mature factor IX, profactor IX did not demonstrate Ca(II)-dependent binding to acidic phospholipid vesicles, nor can the membrane binding surface be expressed, as detected by antibodies specific for this epitope. The propeptide of profactor IX can be removed in vitro by a specific endopeptidase, furin/PACE, yielding factor IX, which can be converted to fully active factor IXa by factor XIa and which binds normally to acidic phospholipid vesicles. These results indicate that fully gamma-carboxylated profactor IX is biologically inactive due to the presence of the propeptide.

The fully gamma-carboxylated peptides based upon the complete and truncated Gla/aromatic amino acid stack domains of human Factor IX were prepared by solid phase peptide synthesis using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. A 47-residue peptide Factor IX-(1-47) and a 42-residue peptide Factor IX-(1-42), both containing 12 residues of L-gamma-carboxyglutamic acid, were purified by high performance liquid chromatography and oxidized to form the disulfide bond. Quantitative gamma-carboxyglutamic acid analysis of Factor IX-(1-47) and Factor IX-(1-42) indicated the presence of 12.1 and 11.2 gamma-carboxyglutamic acid residues/mol of peptide, respectively; no glutamic acid was detected. As monitored by fluorescence quenching, calcium ions induced the prototypical conformational transition in Factor IX-(1-47), but not in Factor IX-(1-42), that is observed with Factor IX. Half-maximal quenching of the intrinsic fluorescence of Factor IX-(1-47) was observed at Ca(II) concentrations of about 50 microM. Factor IX-(1-47) bound to the conformation-specific antibodies, anti-Factor IX:Mg(II) and anti-Factor IX:Ca(II)-specific in the presence of metal ions. Factor IX-(1-47) bound to phospholipid membranes, as monitored by energy transfer from intrinsic fluorophores to dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-phosphatidylethanolamine incorporated into a lipid bilayer composed of phosphatidylserine:phosphatidylcholine. In contrast, Factor IX-(1-42) bound poorly to these same membranes. Factor IX-(1-47) did not inhibit Factor XIa activation of Factor IX but did inhibit the activation of Factor X by Factor IXa bound to Factor VIII in the presence of calcium ions and phospholipid. These results show that phospholipid membrane binding is a property of the Gla/aromatic amino acid stack domain and that the Factor IX-(1-47) peptide, prepared by chemical synthesis, preserves the membrane binding properties and the metal-induced conformational transitions observed in native Factor IX. These results indicate that Factor IX-(1-47) but not Factor IX-(1-42) is a suitable model for structural studies of Factor IX-membrane interaction.

1. The molecular basis for the high survival rate of rainbow trout, Oncorhynchus mykiss, infected with furunculosis was investigated. 2. Alpha 2-macroglobulin (alpha 2M), a major serum protease inhibitor, was partially purified from rainbow trout and brook trout, Salvelinus fontinalis, sera; the latter species shows marked disease susceptibility. 3. It is shown that a 10-fold species-based difference in alpha 2M inhibitory activity exists against a furunculosis associated bacterial protease. 4. A possible basis for the observed disparity is discussed. 5. Results suggest that the high mol. wt form of teleost (trout) albumin is a dimer composed of two 85,000 subunits.

The effect of heat shock on the expression of the nuclear protooncogenes c-fos, c-jun, and c-myc was studied in human lymphoid cells. Heat shock caused an increase in c-fos and c-jun mRNA levels and a decrease in c-myc mRNA levels in pre-B (Hyon) and T (DND-41) cell lines as well as in freshly isolated normal human thymocytes. The changes in the mRNA levels of these protooncogenes in Hyon cells were most pronounced at 42 and 43 degrees C; kinetic analysis demonstrated that the changes could be detected within 30 min of heat shock. Altered transcription of c-fos and c-myc genes was the primary effect of heat shock. Secondarily, heat shock of Hyon cells stabilized the c-myc mRNA level by increasing its half-life from 24 to 45 min. The overall effect of heat shock on c-myc mRNA level, however, was a marked inhibition of its transcription. These results demonstrate that the transcription of nuclear protooncogenes is regulated by heat shock indicating a role for nuclear protooncogenes in the stress response of lymphoid cells.