Role of the hexapeptide disulfide loop present in the gamma-carboxyglutamic acid domain of human protein C in its activation properties and in the in vitro anticoagulant activity of activated protein C

In order to examine whether the structural integrity of the hexapeptide disulfide loop (residues 17-22), present in the gamma-carboxyglutamic acid (gamma) domain of human protein C (PC), and common to all vitamin K dependent coagulation proteins, is necessary for its anticoagulant properties, we employed recombinant (r) DNA technology to generate two important variants that would address this issue. One such mutein contained aspartic acid for gamma-residue substitutions at sequence positions 19 and 20 ([gamma 19D, gamma 20D]r-PC) in the light chain of the mature protein, and the other possessed a serine for cysteine substitution at position 22 ([C22S]r-PC of the same light chain. A subpopulation of molecules of these mutant proteins, containing the maximum levels of gamma-residues in each, has been purified by fast-protein anion-exchange liquid chromatography and affinity chromatography on an anti-human PC column. A study of the kinetic characteristics of the inhibition by Ca2+ of the thrombin-catalyzed activation rates of these variants, and the corresponding stimulation by Ca2+ of the thrombin/thrombomodulin-catalyzed activation rates of the same recombinant PC molecules, demonstrated that higher concentrations of Ca2+ were required to display these effects, when compared to wild-type (wt) r-PC and human plasma PC. This suggested that the kinetically relevant Ca2+ site responsible for these effects on activation of PC, and known to be present in another domain of PC, was affected by both mutations in the gamma-domain. The recombinant PC variants were converted to their activated forms ([gamma 19D, gamma 20D]r-APC and [C22S]r-APC) and assayed for their Ca(2+)-dependent anticoagulant activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of specific gamma-carboxyglutamic acid residues on the integrity of the calcium-dependent conformation of human protein C.

The concentration of Ca2+ that produced 50% of the saturable intrinsic fluorescence change (C50) of wild-type (wt) recombinant (r) human protein C (PC) was 0.40 mM. The C50 for Ca2+ increased < 2.5-fold for the following r-PC variants (Gla is gamma-carboxyglutamic acid): [Gla6-->Asp]r-PC, [Gla7-->Asp]r-PC, [Gla14-->Asp]r-PC, [Gla19-->Asp]r-PC, or [Gla25-->Asp]r-PC, and approximately 4-6-fold for [Gla20-->Asp]r-PC and [Gla29-->Asp]r-PC. Much more dramatic increases in the C50 for Ca2+ were observed for [Gla16-->Asp]r-PC (> 75-fold) and [Gla26-->Asp]r-PC (ca. 30-fold). A substantially larger maximum fluorescence change (> 3-fold) as compared to that for wtr-PC, was also found in the case of the Ca2+/[Gla16-->Asp]r-PC complex, suggesting that the final Ca(2+)-induced conformation for this variant is dissimilar to that for wtr-PC and the above mutants. When a mutation was constructed at Arg15 ([Arg15-->Leu]r-PC), a residue conserved in all Gla-containing coagulation proteins, no fluorescence alteration occurred upon addition of Ca2+. The C50 for Ca2+ for promotion of the binding of the Ca(2+)-dependent, Gla-domain-directed, conformational monoclonal antibodies, JTC-1 and JTC-3, to wtr-PC was 3.0 and 4.0 mM, respectively. A similar C50 value was found for [Gla25-->Asp]r-PC. In the case of each antibody, approximately 4-6-fold higher C50 values for Ca2+ were found for the mutants; [Gla14-->Asp]r-PC, [Gla19-->Asp]r-PC, and [Gla29-->Asp]r-PC. Ca2+ did not promote binding of either of these antibodies to the following variants; [Gla6-->Asp]r-PC, [Gla7-->Asp] r-PC, [Arg15-->Leu]r-PC, [Gla16-->Asp]r-PC, [Gla20-->Asp]r-PC, and [Gla26-->Asp]r-PC. The results of this study suggest that adoption of the Ca(2+)-dependent conformation of PC is greatly dependent upon the presence of specific essential Gla residues, particularly those, namely Gla16 and Gla26, shown in the crystal structure of the prothrombin Gla domain/Ca2+ complex to be involved with coordination of Ca2+ ions not exposed to the surface. Of similar importance is Arg15. On the other hand, Gla residues at positions 14 and 19 are much less important in directing this same conformation. This finding is readily reconciled with the above crystal structure, which shows that these latter 2 residues are mainly responsible for coordination of a surface-exposed Ca2+ that is present at the end of the Ca(2+)-ion channel.     

Probing the activation of protein C by the thrombin-thrombomodulin complex using structural analysis, site-directed mutagenesis, and computer modeling

Protein C (PC) is activated to an essential anticoagulant enzyme (activated PC or APC) by thrombin (T) bound to thrombomodulin (TM), a membrane receptor present on the surface of endothelial cells. The understanding of this complex biological system is in part limited due to the lack of integration of experimental and structural data. In the work presented here, we analyze the PC-T-TM pathway in the context of both types of information. First, structural analysis of the serine protease domain of PC suggests that a positively charged cluster of amino acids could be involved in the activation process. To investigate the importance of these basic amino acids, two recombinant PC mutants were constructed using computer-guided site-directed mutagenesis. The double mutant had the K62[217]N/K63[218]D substitution and in the single mutant, K86[241] was changed to S. Both mutants were activated by free thrombin at rates equivalent to that of wild-type PC (wt-PC) and they demonstrated similar calcium-dependent inhibition of their activation. The K86[241]S mutant and wt-PC were activated by thrombin bound to soluble TM at a similar rate. In contrast, the K62[217]N/ K63[218]D mutant was activated by the T-TM complex at a 10-fold lower catalytic efficiency due to a lowering in k(cat) and increase in Km. Molecular models for PC and thrombin bound to a segment of TM were developed. The experimental results and the modeling data both indicate that electrostatic interactions are of crucial importance to orient PC onto the T-TM complex. A key electropositive region centered around loops 37[191] and 60[214] of PC is defined. PC loop 37[191] is located 7-8 A from the TM epidermal growth factor (EGF) 4 while the loop 60[214] is about 10 A away from TM EGF4. Both loops are far from thrombin. A key function of TM could be to create an additional binding site for PC. The Gla domain of PC points toward the membrane and away from thrombin or the EGF modules of TM during the activation process.     

Crosstalk between ARF6 and protein kinase Calpha in Fc(gamma)RI-mediated activation of phospholipase D1

Fc receptors play a pivotal role linking the cellular and humoral arms of the immune system [1-3]. Our previous studies have shown that the human high-affinity immunoglobulin G receptor Fc(gamma)RI couples to a novel intracellular signaling pathway requiring phospholipase D activation [4]. The mechanisms that regulate receptor coupling to phospholipase D in intact cells are poorly understood but involve small molecular weight GTPases and protein kinase C [5-7]. Here, we show that immune complex aggregation of Fc(gamma)RI stimulates the association of phospholipase D1 with ARF6 and protein kinase Calpha. Surprisingly, PKCalpha activity per se is not required. Rather, all of the Fc(gamma)RI-mediated increase in PKC activity requires phospholipase D1, as treatment of cells with butan-1-ol (0.3%) or specific downregulation of phospholipase D1 using antisense oligonucleotides inhibits Fc(gamma)RI-coupled PKC activation. Moreover, treatment of cells with butan-1-ol or phospholipase D1 antisense oligonucleotides inhibits translocation of PKCdelta, -epsilon, and -zeta but had no effect on the association of PKCalpha or ARF6 with phospholipase D1. These data indicate that association with ARF6 and PKCalpha plays a role in coupling Fc(gamma)RI to phospholipase D1 activation and that PLD1 lies upstream of all Fc(gamma)RI-mediated PKC activity.     

Chemical modification of bovine prothrombin fragment 1 in the presence of Tb3+ ions. Sequence studies on 3-gamma-MGlu-fragment

Chemical modification of the gamma-carboxyglutamyl (Gla) residues of bovine prothrombin fragment 1 using the formaldehyde-morpholine method in the presence of 100 Kappm Tb3+ ions at pH 5.0 provided a modified protein containing 3 gamma-methyleneglutamyl residues (gamma-MGlu) and 7 Gla residues (bovine 3-gamma-MGlu-fragment 1). The modified protein bound the same number of Ca2+ ions as the native protein (six to seven), exhibited 28Mg2+-binding properties identical to native fragment 1 (five Mg2+ ions bound), exhibited the metal ion-promoted quenching of the intrinsic fluorescence in a manner similar to the native protein, but did not bind to phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles in the presence of Ca2+ ions. Modification of the bovine protein using [14C]formaldehyde-morpholine provided a 14C-labeled 3-gamma-MGlu-fragment 1 suitable for sequence analysis. Edman sequencing of the peptides released by a tryptic digest of the reduced and carboxymethylated bovine [14C]3-gamma-MGlu-fragment 1 indicated that Gla residues at positions 7, 8, and 33 had been converted to [14C]gamma-methyleneglutamyl residues. In addition Lys97 was found to contain a 14C label. Similar analysis of the human [14C]3-gamma-MGlu-fragment 1 indicated that Gla residues at positions 7 and 32 were major modification sites and that Gla residues at positions 6 and 14 were partially modified. Lysine 96 was also modified in the human protein. The incorporation of a 14C label at Lys97 in bovine 3-gamma-MGlu-fragment 1 protein is not responsible for the loss of Ca2+-promoted binding to PS/PC vesicles. We suggest that Gla residues 7, 8, and 33 are elements of the first Ca2+-binding site; occupancy of this site establishes the Ca2+-specific conformation which is essential for the Ca2+-promoted interaction of the bovine protein with PS/PC vesicles. These studies also suggest that the loss of Gla residues at positions 7 and 32 prevents the formation of the initial Ca2+-binding site in the human protein.     

Novel regulation of p38gamma by dopamine D2 receptors during hypoxia

The p38 signalling pathway is part of the MAPK superfamily and is activated by various stressors. Our previous results have shown that two p38 isoforms, p38alpha and p38gamma, are activated by hypoxia in the neural-like PC12 cell line. PC12 cells also synthesize and secrete catecholamines, including dopamine, in response to hypoxia. We have now used this system to study the interaction between D2-dopamine receptor signalling and the p38 stress-activated protein kinases. Our results show that two D2 receptor antagonists, butaclamol and sulpiride, enhance hypoxia-induced phosphorylation of p38gamma, but not p38. This effect persists in protein kinase A (PKA)-deficient PC12 cells, demonstrating that p38gamma modulation by the D2 receptor is independent of the cAMP/PKA signalling system. We further show that removal of extracellular calcium blocks the hypoxia-induced increase in p38gamma activity. These results are the first to demonstrate that p38gamma can be regulated by the D2 receptor and calcium following hypoxic exposure.     

Primary structure of human gamma 3 immunoglobulin deletion mutant: gamma 3 heavy-chain disease protein Wis

The complete sequence of a gamma 3 heavy-chain disease (HCD) protein Wis is presented. The molecule is a dimer of a 289-residue chain linked by 12 disulfide bonds. Protein Wis has an unusual amino terminus, followed by a deletion of most of the VH domain. After a small stretch homologous to the VC joining region, there is a second deletion which ends at the beginning of the quadruplicated hinge. Two carbohydrate groups are linked to Asn-6 and -140. the molecule has an extra interchain disulfide bridge at position 7 in addition to the 11 normally present in the quadruplicated hinge. The previously noted homology to the gamma 1 heavy chain is striking; from positions 224 to 234, protein Wis resembles gamma 1 Nei [Ponstingl, H., & Hilschmann, N. (1976) Hoppe-Seyler's Z. Physiol. Chem. 357, 1571--1604] except for a serine which replaces Asn at position 227. The results, taken together with studies of other immunoglobulin heavy-chain deletion mutants, support the suggestion that the different domains and interdomain regions of human H chains are coded for by different gene segments and that the deleted proteins reflect alterations in the recombination of different genes and/or the splicing of heterogeneous nuclear messenger ribonucleic acid (hn mRNA).     

Human liver alcohol dehydrogenase. 2. The primary structure of the gamma 1 protein chain

The primary structure of the gamma 1 subunit of human liver alcohol dehydrogenase isoenzyme gamma 1 gamma 1 was deduced by characterization of 36 tryptic and 2 CNBr peptides. The polypeptide chain is composed of 373 amino acid residues. gamma 1 differs from the beta 1 subunit of human liver alcohol dehydrogenase at 21 positions, and from the E subunit of horse liver alcohol dehydrogenase at 43 positions including a gap at position 128 as in the beta 1 subunit. All zinc-liganding residues from the E subunit of the horse protein and the beta 1 subunit of the human enzyme are conserved, but like beta 1, gamma 1 also has an additional cysteine residue at position 286 (in the positional numbering system of the horse enzyme) due to a Tyr----Cys exchange. Most amino acid exchanges preserve the properties of the residues affected and are largely located on the surface of the molecules, away from the active site and the coenzyme binding region. However, eight positions with charge differences in relation to the E subunit of the horse enzyme are noticed. These result in a net positive charge increase of one in gamma 1 versus E, explaining the electrophoretic mobilities on starch gels. Of functional significance is the conservation of Ser-48 in gamma 1 relative to E. The residue is close to the active site but different (Thr-48) in the beta 1 subunit of the human enzyme. Thus, the closer structural relationship between human gamma 1 and horse E enzyme subunit than between beta 1 and E is also reflected in functionally important residues, explaining a greater similarity between gamma 1 gamma 1 and EE than between beta 1 beta 1 and EE.     

Identification of a sequence of human activated protein C (residues 390-404) essential for its anticoagulant activity.

Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. To identify the regions on the surface that mediate anticoagulant activity, 26 synthetic peptides were prepared representing 90% of the human protein C heavy chain primary structure and tested for their ability to inhibit APC anticoagulant activity. Peptide-(390-404) specifically inhibited APC activity in activated partial thromboplastin time and Xa-1-stage coagulation assays in normal, in protein S-depleted and Factor VIII-deficient plasma with 50% inhibition at 5 microM peptide. Polyclonal antibodies raised against this peptide and immunoaffinity-purified on a protein C-Sepharose column inhibited APC anticoagulant activity in activated partial thromboplastin time and Xa-1-stage assays in normal, protein S-depleted, and Factor VIII-deficient plasma with half-maximal inhibition at 30 nM anti-(390-404) antibody. Neither the peptide-(390-404) nor the anti-(390-404) antibodies inhibited APC amidolytic activity or the reaction of APC with recombinant [Arg358] alpha 1-antitrypsin. Furthermore, in a purified system, peptide-(390-404) inhibited APC-catalyzed inactivation of Factor Va in the presence as well as in the absence of phospholipids with 50% inhibition at 4 microM peptide. These data suggest that the region containing residues 390-404 in APC is essential for anticoagulant activity and is available to interact with antibodies or with other proteins such as the macromolecular substrates Factors Va or VIIIa.     

Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells

Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VIIa, participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca2+ and tissue factor. Three types of potential posttranslational modifications exist in the human factor VIIa molecule, namely, 10 gamma-carboxylated, N-terminally located glutamic acid residues, 1 beta-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VIIa as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VIIa. By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradations, the protein backbone of recombinant factor VIIa was found to be identical with human factor VIIa. Neither recombinant factor VIIa nor human plasma factor VIIa was found to contain beta-hydroxyaspartic acid. In human plasma factor VIIa, the 10 N-terminally located glutamic acid residues were found to be fully gamma-carboxylated whereas 9 full and 1 partial gamma-carboxylated residues were found in the corresponding positions of the recombinant factor VIIa molecule. Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VIIa. In the recombinant factor VIIa, asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VIIa and human plasma factor VIIa.(ABSTRACT TRUNCATED AT 250 WORDS)     

The cytoplasmic region of mouse Fc gamma RIIb1, but not Fc gamma RIIb2, binds phospholipid membranes

The cytoplasmic regions of the mouse low-affinity Fc gamma RII isoforms, Fc gamma RIIb1 and Fc gamma RIIb2, play key roles in signal transduction by mediating different cellular functions. The Fc gamma RIIb1 (94 residues) and Fc gamma RIIb2 (47 residues) cytoplasmic regions are generated by differential mRNA splicing in which a single aspartic acid residue in Fc gamma RIIb2 is replaced by a 48-residue insert in Fc gamma RIIb1. In previous work, quantities of the mFc gamma RIIb1 and mFc gamma RIIb2 cytoplasmic regions were generated, and their secondary structures were examined in different solutions with circular dichroism [Chen, L., Thompson, N. L., and Pielak, G. J. (1997) Protein Sci. 6, 1038-1046]. In the work described here, steady-state light scattering was used to investigate possible interactions of the two isolated cytoplasmic regions with phospholipid vesicles. Three phospholipid compositions were examined: phosphatidylserine/phosphatidylcholine (PS/PC) (25/75, mol/mol); phosphatidylinositol bisphosphate/phosphatidylcholine (PIP2/PC) (25/75, mol/mol); and pure phosphatidylcholine (PC). Binding was examined in the presence and absence of Ca2+. The mFc gamma RIIb1 cytoplasmic peptide binds PS/PC vesicles weakly in the absence of Ca2+ and more strongly in the presence of Ca2+. For PIP2/PC vesicles, the behavior is reversed; binding is weak in the presence of Ca2+ and stronger in its absence. The mFc gamma RIIb1 peptide also weakly binds pure PC vesicles, in a Ca2+-independent manner. The mFc gamma RIIb2 cytoplasmic peptide does not bind, in the presence or absence of Ca2+, to PS/PC, PIP2/PC, or PC vesicles. The implications of these results for the mechanisms of signal transduction mediated by the two mFc gamma RII cytoplasmic regions are discussed.     

Multifunctional specificity of the protein C/activated protein C Gla domain

Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that are mediated in part by its interactions with its cofactor protein S and the endothelial cell protein C receptor (EPCR). The protein C/APC Gla domain is implicated in both interactions. We sought to identify how the protein C Gla domain enables specific protein-protein interactions in addition to its conserved role in phospholipid binding. The human prothrombin Gla domain, which cannot bind EPCR or support protein S cofactor activity, has 22/45 residues that are not shared with the human protein C Gla domain. We hypothesized that the unique protein C/APC Gla domain residues were responsible for mediating the specific interactions. To assess this, we generated 13 recombinant protein C/APC variants incorporating the prothrombin residue substitutions. Despite anticoagulant activity similar to wild-type APC in the absence of protein S, APC variants APC(PT33-39) (N33S/V34S/D35T/D36A/L38D/A39V) and APC(PT36/38/39) (D36A/L38D/A39V) were not stimulated by protein S, whereas APC(PT35/36) (D35T/D36A) exhibited reduced protein S sensitivity. Moreover, PC(PT8/10) (L8V/H10K) displayed negligible EPCR affinity, despite normal binding to anionic phospholipid vesicles and factor Va proteolysis in the presence and absence of protein S. A single residue variant, PC(PT8), also failed to bind EPCR. Factor VIIa, which also possesses Leu-8, bound soluble EPCR with similar affinity to wild-type protein C, collectively confirming Leu-8 as the critical residue for EPCR recognition. These results reveal the specific Gla domain residues responsible for mediating protein C/APC molecular recognition with both its cofactor and receptor and further illustrate the multifunctional potential of Gla domains.     

Elucidation of the Biosynthesis of Eicosapentaenoic Acid in the Microalga Porphyridium cruentum (II. Studies with Radiolabeled Precursors)

In the course of the study of the biosynthesis of the fatty acid eicosapentaenoic acid (EPA) in the microalga Porphyridium cruentum, cells were pulse-labeled with various radiolabeled fatty acid precursors. Our data show that the major end products of the biosynthesis are EPA-containing galactolipids of a eukaryotic and prokaryotic nature. The prokaryotic molecular species contain EPA and arachidonic acid at the sn-1 position and C16 fatty acids, mainly 16:0, at the sn-2 positions, whereas in the eukaryotic species both positions are occupied by EPA or arachidonic acid. However, we suggest that both the eukaryotic and prokaryotic molecular species are formed in two pathways, [omega]6 and [omega]3, which involve cytoplasmic and chloroplastic lipids. In the [omega]6 pathway, cytoplasmic 18:2-phosphatidylcholine (PC) is converted to 20:4[omega]6-PC by a sequence that includes a [delta]6 desaturase, an elongation step, and a [delta]5 desaturase. In the minor [omega]3 pathway, 18:2-PC is presumably desaturated to 18:3[omega]3, which is sequentially converted by the enzymatic sequence of the [omega]6 pathway to 20:5[omega]3-PC. The products of both pathways are exported, as their diacylglycerol moieties, to the chloroplast to be galactosylated into their respective monogalactosyldiacylglycerol molecular species. The 20:4[omega]6 in both eukaryotic and prokaryotic monogalactosyldiacylglycerol can be further desaturated to EPA by a chloroplastic [delta]17 ([omega]3) desaturase.     

Isolation and properties of gamma protein,the major transmembrane sialoglycoprotein of the HeLa cell

The surface of the HeLa cell is composed of a heterogeneous population of sialogly coproteins which undergo lectin-mediated endocytosis (Kramer and Canellakis, Biochim Biophys Acta 551:328, 1979). One such sialoglyco-protein, gamma protein, is the major periodate-Schiff-reactive and [³H]-glucosamine-labeled component of the plasma membrane; it has an apparent molecular weight of 165,000. Gamma protein is also the major [¹²⁵I]-wheat germ agglutinin-binding component in sodium dodecyl sulfate gels. Neuraminidase digestion of HeLa cells abolishes binding of [¹²⁵I]-wheat germ agglutinin to gamma protein, and pretreatment of cells with wheat germ agglutinin protects gamma protein from desialation by neuraminidase. suggesting that wheat germ agglutinin binds to the sialic acid residues of gamma protein at the cell surface. Gamma protein can be extracted with various detergents but not with high-salt, chelating, or chaotropic agents. Intact inside-out plasma membrane vesicles have been prepared from HeLa cells that had phagocytosed latex particles. Treatment of these isolated vesicles with trypsin reduces the molecular weight of gamma protein. These results suggest that gamma protein is an integral membrane protein that spans the plasma membrane. Gamma protein can be purified to homogeneity by sequential lithium diiodosalicylate-phenol extraction, wheat germ agglutinin-agarose affinity chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

A point mutation of human interferon gamma abolishes receptor recognition.

We identified a single amino acid mutation that abolished the bioactivity of human IFN gamma. The mutation was identified by screening a mutagenized IFN gamma expression library for molecules with altered biological activity. The mutant protein was expressed at high levels in Escherichia coli, and remained soluble upon purification. However, the protein was completely inactive in all IFN gamma assays investigated, exhibiting less than 0.0006% of the specific activity of native IFN gamma antiviral activity. Sequencing the plasmid DNA encoding this mutant protein showed that the histidine at position 111 of native human IFN gamma is changed to aspartic acid (IFN gamma/H111D). Other mutations at this site showed that only hydrophobic amino acids at position 111 maintain significant, though low, biological activity. Structural characterization of the IFN gamma/H111D protein by NMR as well as CD spectroscopy demonstrated that the protein has limited conformational differences from native IFN gamma. Models of the X-ray crystal structure of human IFN gamma [Ealick, P.E., W.J. Cook, S. Vijay-Kumar, M. Carson, T.L. Nagabhushan, P.P. Trotta and C.E. Bugg (1991) Science, 252, 698-702] suggest that this histidine residue is located at a severe 55 degrees bend in the C-terminal F helix. We conclude that H111 lies within or affects the receptor binding domain of human IFN gamma.     

High-level expression of a functional human fibrinogen gamma chain in Escherichia coli

Human fibrinogen gamma chain has been expressed intact at high levels in Escherichia coli. The construction of the expression plasmid, p253, is described. Synthesis of the recombinant protein is isopropyl-beta-D-thiogalactopyranoside-dependent and is driven by the tac promoter. Western analysis of E. coli lysates demonstrates a novel protein of approx. 45 kDa which cross-reacts with antisera to human fibrinogen gamma chains. The protein is not soluble in common E. coli lysis buffers and becomes soluble in 6 M guanidine.HCl or 6 M urea. Initial insolubility is due to interchain disulfide bond formation and to noncovalent interactions. Induced cells examined by phase-contrast microscopy contain dense inclusion bodies. A known function of the gamma chains of human fibrinogen is the clumping of Staphylococcus aureus Newman D2C cells [Hawiger et al., Biochemistry (1982) 1407-1413]. We demonstrate that suspensions of recombinant gamma chains retain the ability to clump cells from this strain of S. aureus.     

Chemical evidence for the separation of G gamma and A gamma chains by polyacrylamide gel electrophoresis in urea and triton X-100

Polyacrylamide gel electrophoresis in urea and Triton X-100 of a hemolysate from human fetal red blood cells produces four major protein bands: alpha, beta, and 2 gamma globin chains. We have verified that the latter two are the G gamma and A gamma globin chains which have respectively glycine or alanine at position 136. After incorporation of either [3H] alanine or [3H] glycine into newly synthesized globin each gamma chain was isolated by preparative electrophoresis. The chains were cleaved with cyanogen bromide at methionines 55 and 133, then subjected to automated sequencing, and the residues from each sequencer turn counted. Glycine incorporation was detected for the third turn (position 136) of the G gamma chain and alanine for the A gamma. Substantial metabolic conversion of [3H] glycine to serine and proline was also noted.     

Fc gamma 2b receptor-mediated prostaglandin synthesis by a murine macrophage cell line (P388D1)

The nature of signals transmitted by two types of Fc gamma receptors (one specific for IgG2b and the other for IgG2a) present on the surface of a murine macrophage cell line (P388D1) was investigated. Specific binding of IgG2b (presented as EA2b) to cell surface Fc gamma 2br triggered the release of 3H-arachidonic acid and 3H-prostaglandins (PG) from P388D1 cells that were prelabeled with 3H-arachidonate. The release of 3H-arachidonic acid, which increased in a dose-dependent manner, was enhanced by exogenous Ca++ (1.25 mM) and was completely blocked by ethylenediaminetetraacetate (EDTA) (4 mM) or a phospholipase A2 inhibitor, p-bromophenacylbromide (7 microgram/ml). A cyclooxygenase inhibitor, indomethacin (9 microgram/ml), reduced the 3H-arachidonic acid release and completely blocked the conversion of arachidonate into PG. Cytochalasin D (1 microgram/ml), which inhibited the phagocytosis of immune complexes by 90% of P388D1 cells, did not affect the Fc gamma 2bR-triggered release of arachidonic acid. Specific binding of IgG2a (presented as EA2a) to cell surface Fc gamma 2aR did not trigger the release of either 3H-arachidonic acid or 3H-PG from P388D1 cells. Our data demonstrate a signal for the activation of the arachidonic acid metabolic cascade is transmitted by Fc gamma 2bR, but not by Fc gamma 2aR, on the surface of P388D1 cells, probably through the initial activation of the phospholipase A2 activity associated with Fc gamma 2bR.     

Des-Tyr1-gamma-endorphin (DT gamma E) and des-enkephalin-gamma-endorphin (DE gamma E): plasma profile and brain uptake after systemic administration in the rat

The plasma disappearance, metabolism and uptake in the brain of [3H-Phe4]-DT gamma E and [3H-Lys9]-DE gamma E were investigated following systemic administration of these neuroleptic-like peptides to rats. 3H-DT gamma E, 3H-DE gamma E and their radioactive metabolites in plasma and brain extracts were determined by reversed-phase HPLC. Plasma disappearance of DT gamma E upon intravenous (IV) dosing followed a biphasic pattern with half-lives of 0.7 min (distribution phase) and 5.5 min (elimination phase). For DE gamma E the plasma disappearance curve was best characterized by a one-compartment model since a second elimination phase was hardly detectable by our methods. The corresponding half-life was 0.6 min, probably representative for the initial distribution phase of DE gamma E. Both neuropeptides distributed rapidly over the larger part of the extracellular fluid. Following the IV route of administration, brain uptake of DT gamma E and DE gamma E appeared to be low. Brain levels of DT gamma E decreased from 0.0075% to 0.0031% of the administered dose/g tissue at 2-15.5 min after injection, whereas those of DE gamma E decreased very rapidly from 0.0174% of the dose/g brain tissue to below the detection limit at 2-4.5 min after injection. As compared to the IV route of administration, subcutaneous (SC) injection of DE gamma E resulted into lower but remarkably longer-lasting peptide concentrations in plasma as well as in brain, possibly because of a sustained release from the SC site of injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and functional importance of tyrosine sulfate residues within recombinant factor VIII.

Sulfated tyrosine residues within recombinant human factor VIII were identified by [35S]sulfate biosynthetic labeling of Chinese hamster ovary cells which express human recombinant factor VIII. Alkaline hydrolysis of purified [35S]sulfate-labeled factor VIII showed that greater than 95% of the [35S]sulfate was incorporated into tyrosine. [3H]Tyrosine and [35S]sulfate double labeling was used to quantify the presence of 6 mol of tyrosine sulfate per mole of factor VIII. Amino acid sequence analysis of thrombin and tryptic peptides isolated from [35S]sulfate-labeled factor VIII demonstrated tyrosine sulfate at residue 346 in the factor VIII heavy chain and at residues 1664 and 1680 in the factor VIII light chain. In addition, the carboxyl-terminal half of the A2 domain contained three tyrosine sulfate residues, likely at positions 718, 719, and 723. Interestingly, all sites of tyrosine sulfation border thrombin cleavage sites. The functional importance of tyrosine sulfation was examined by treatment of cells expressing factor VIII with sodium chlorate, a potent inhibitor of tyrosine sulfation. Increasing concentrations of sodium chlorate inhibited sulfate incorporation into factor VIII without affecting its synthesis and/or secretion. However, factor VIII secreted in the presence of sodium chlorate exhibited a 5-fold reduction in procoagulant activity, although the protein was susceptible to thrombin cleavage. These results suggest that tyrosine sulfation is required for full factor VIII activity and may affect the interaction of factor VIII with other components of the coagulation cascade.