Structural studies of a human gamma 3 myeloma protein (Jir) bearing the allotypic marker Gm(st)

The authors established the amino acid substitutions determining G3m(s) and G3m(t) specificities, which characterize Mongoloid populations, by sequence analysis of the Fc region of a myeloma protein (Jir). By comparing the amino acid sequences of the IgG3 (Jir) and the other IgG subclasses analyzed to date, it was found that G3m(s) was an isoallotype specified by an amino acid substitution at position 435; i.e., whereas the subclasses IgG1, IgG2, and IgG4 had histidine in common, G3m(s-) had arginine in this position. This was also confirmed by the observation that the Fc fragment in question bound to protein A. It was also established that the amino acid at position 379 of G3m(t-) IgG3 and the other subclasses was valine, whereas methionine in this position was specific for G3m(t+). In addition, the amino acids at position 339 of G3m(u-) IgG3 Jir was threonine, and at position 296 of G3m(g-) IgG3 Jir was tyrosine. These findings are not in accord with the hitherto postulated relations of alanine and phenylalanine to G3m(u-) and G3m(g-), respectively. Finally, this study showed that a large number of substitutions occurred at positions 384 through 389, which suggests that many specificities of the G3m(b) group occur on IgG3 proteins.     

The modification of human immunoglobulin binding to staphylococcal protein A using diethylpyrocarbonate

Human IgG subclasses 1, 2, and 4, as well as proteins of the IgG3 subclass that are allotype G3m (s+t+), bind avidly to staphylococcal protein A by means of their Fc portion. Proteins of the IgG3 subclass that are allotype G3m (s-t-) do not bind. The importance of a histidine residue at position 435 has been implicated from comparison of amino acid sequences of immunoglobulins that bind with those that do not bind to staphylococcal protein A, as well as from crystallographic data. Modification of histidines at a low concentration of diethylpyrocarbonate successfully and reversibly alters the binding of immunoglobulins to staphylococcal protein A with only minimal change in the antigenic properties. This method provides strong evidence for the critical importance of histidine in the binding of immunoglobulins to staphylococcal protein A.     

An amino acid substitution determining G3m(g)

An allotypic G3m(g) marker-specific substitution was studied by sequence analysis of glycopeptides derived from myeloma proteins Ba (G3m(g+)) and Bu (G3m(g-)). The experimental results indicate that glutamic acid at position 295 is responsible for the specificity. Based on the results of chemical modification (Arg, Tyr, and Glu), this antigenic epitope is presumed to involve five sequential residues from Arg-292 to Tyr-296.     

The amino acid sequence of a human immunoglobulin G3m(g) pFc' fragment.

Amino acid sequence analysis of the pFc' fragment obtained by pepsin digestion of an IgG3; G3m(g) human myeloma protein HER shows it to consist of 112 residues. It starts at position 334 (gamma1 numbering), contains eight residues of the Cgamma2 region, and the whole Cgamma3 domain. Comparison with the sequence of gamma1 shows five differences including an extra Met at 397. Each is accountable by a single base substitution. The sequence is identical to that of a G3m(b0) molecule except for the previously noted allotype related Tyr/Phe exchange at position 436. The high degree of homology (95%) among gamma-chain subclasses suggests a recent diversification.     

Modification of the gene 5 DNA unwinding protein with ruthenium

A chemical modification of the gene 5 DNA binding protein (G5BP) from bacteriophage fd was investigated using X-ray diffraction and difference Fourier analysis. The crystalline protein was reacted with pentaammineruthenium (III) trichloride, Ru(NH₃)₅Cl₃, a reagent believed specific for histidine residues and useful in NMR and chemical modification studies of proteins. The major ruthenium site was found by difference Fourier analysis to be 4 Å from histidine 64, the only histidine residue in the molecule. A second bipartite site, believed to be a ruthenium-anion pair, appeared to be salt-bridged to glutamic acid 40 and arginine 16. Indications were present in the difference Fourier results to suggest that the loop containing tyrosine 41 had undergone a slight conformational rearrangement to accommodate this interaction.     

Gm typing by immunoglobulin heavy-chain gene RFLP analysis.

This study was undertaken to investigate a means of assigning Gm allotypes to Caucasians by RFLP analysis. A single immunoglobulin heavy-chain gamma-4 cDNA probe (HU gamma 4) was hybridized with genomic DNA digested separately with two restriction enzymes, TaqI and PvuII. Results showed excellent correlation (P less than .001) between serologically defined Gm allotypes G1m(1), G1m(2), G2m(23), and G1m;G3m (3;5,10) and RFLPs identified with the (HU gamma 4) probe. We conclude that it is now possible to define common Gm haplotypes in Caucasians by RFLP analysis. This method provides a useful adjunct to serological allotyping and indeed has several important advantages over traditional serology: it allows confident Gm assignment and the definition of homozygous and heterozygous Gm arrangements, is highly reproducible, and is readily executed in any molecular genetic laboratory.     

Receptor- and nucleotide exchange-independent mechanisms for promoting G protein subunit dissociation

Mechanisms for heterotrimeric G protein activation that do not rely on G protein coupled receptor activation are becoming increasingly apparent. We recently identified beta gamma subunit-binding peptides that we proposed bound to a "hot spot" on beta gamma subunits, stimulating G protein dissociation without stimulating nucleotide exchange and activating G protein signaling in intact cells. AGS3, a member of the activators of G protein signaling family of proteins, also activates G protein signaling in a nucleotide exchange-independent manner, and AGS3 homologues are involved in asymmetric cell division during development. Here we demonstrate that a consensus G protein regulatory (GPR) peptide from AGS3 and related proteins is sufficient to induce G protein subunit dissociation and that both the GPR and hot spot-binding peptides promote dissociation to extents comparable with a known G protein activator, AMF. Peptides derived from adenylyl cyclase 2 and GRK2 prevented formation of the heterotrimeric complex but did not alter the rate of alpha subunit dissociation from beta gamma subunits. These data indicate that these nucleotide exchange-independent G protein activator peptides do not simply compete for alpha interactions with beta gamma subunits, but actively promote subunit dissociation. Thus, we propose two novel mechanisms for nucleotide exchange independent activation of G protein signaling, one that involves conformational changes in the alpha subunit and one that involves conformational changes in the beta gamma subunits.     

Monoclonal antibodies directed against gonococcal protein I vary in bactericidal activity

Monoclonal antibodies (Mab) with specificity for protein I (PI) from Neisseria gonorrhoeae (GC) were examined for bactericidal activity. Mab 4G5 (gamma 3), ID3 (gamma 2a), and 1G6 (gamma 2a) bound to surface-exposed epitopes on PI of GC strain R11 (IA serotype) as assessed by co-agglutination and 125I protein A uptake. Mab 2H1 (gamma 3) that were directed against IB serotype strains and Mab 2E9 (gamma 2a) were negative in co-agglutination and protein A uptake assays and served as controls for some experiments. Only 4G5 and 1D3 were bactericidal for R11 when presensitized organisms were incubated in 10% absorbed, pooled normal human serum (PNHS) or 10% hypogammaglobulinemic serum (H gamma S) despite binding of nearly equivalent numbers of 4G5, 1D3, and 1G6 to R11 during presensitization, as assessed by 125I-protein A uptake. These Mab activated complement to a similar extent on GC R11, leading to deposition of 56.4 X 10(3), 61.9 X 1093), and 47.1 X 10(3) molecules of C3/organism during incubation in 10% C8-deficient serum. Deposition occurred almost exclusively via the classical complement pathway. Measurement of complement component C9 binding to R11 during incubation in H gamma S showed 35,700 molecules of C9/organism with 4G5, 32,600 C9/organism with 1D3, and surprisingly, 29,600 C9/organism with 1G6. Eight thousand four hundred molecules of C9/organism bound to 2E9-coated organisms, 6000 C9/organism to 2H1-coated bacteria, and 3600 C9/organism to nonpresensitized organisms. The C5b-9 complex deposited by 4G5 had a different sedimentation profile by sucrose density gradient analysis from the C5b-9 complex deposited by 1G6, consistent with a different molecular configuration of the bound complex. Mab 1G6 and 1D3, but not 2E9 or 2H1, were able to compete with 125I-4G5 for binding to GC R11. A Mab (2E6) directed against protein III of GC competed weakly with 125I-4G5 for binding to GC R11. Mab 1G6, but not 1D3, blocked 4G5-dependent killing in a dose-related fashion. Both 4G5 and IG6 reacted weakly with native PI of GC R11 by immunoblotting, but neither Mab recognized the 34,800 m.w. fragment of PI generated by trypsin and chymotrypsin treatment of outer membranes. In contrast, 2E9 reacted strongly by immunoblot with both native and cleaved PI of GC R11, suggesting binding to buried determinants of PI. These experiments show that Mab directed against identical or closely associated, surface-exposed epitopes on gonococcal PI differ markedly in bactericidal activity, despite leading to deposition of nearly equivalent numbers of C3 and C9 molecules per organism.     

Polymorphism of immunoglobulin Gm- and Km-allotypes in northern Altaians (western Sibiria)]

In two small adjacent groups-kumandinias and chelkanians-the distribution of G1m(a), G1m(x), G1m(f), G3m(b1), G3m(b5) and Km(1) serum allotypic markers was studied. Gmf;b haplotype frequency in kumandinians was found to be 0.310; in chelkanians living eastward it was 0.212. Gma and Gmax frequencies were observed to be 0.606 and 0.084 in the former and 0.687 and 0.101 in the latter. Km1 frequency was found to be 0.074 in kumandinians and 0.087 in chelkanians. Unusual Gm(a, f) henotype was observed in both groups with a frequency lesser than 5%.     

Soluble antigens of IgG receptor Fc gamma RIII in human seminal plasma

Human seminal plasma (SP) has been shown to affect several immunologic reactions in vitro. This might be due in part to the presence of proteins that specifically bind the Fc domain of IgG. By using mAb Leu 11a, Leu 11b, Leu 11c, and 3G8 we showed that the Fc binding of SP is associated with a molecule that antigenically resembles Fc gamma RIII. This molecule manifests specific affinity for solid phase-coupled IgG-Fc, and appears not be be cell membrane-associated. When compared with serum or blood plasma, its highest concentration was found in SP. Western blot analysis of SP performed with mAb Leu 11a, Leu 11b, Leu 11c, and 3G8 showed distinct bands at approximately 70 and 35 kDa, which contrasts with the broad area of electrophoretic mobility reported for membrane-bound Fc gamma RIII. These molecules in SP could influence maternal immune responses to paternal Ag during fertilization and pregnancy.     

Monoclonal antibodies to protein kinase C gamma. Functional relationship between epitopes and cofactor binding sites

Four monoclonal antibodies (mAbs), derived from high-responder Biozzi mice immunized with purified protein kinase C, were selected by ELISA and further characterized by immunoblot: 3G12, 5A2, 36G9 are of isotype gamma 1, kappa and 15G4 is of isotype gamma 2b, kappa. Competition analysis between 15G4 and the three other mAbs showed that 15G4 and 3G12 are directed against either the same or overlapping epitope(s). All four mAbs are specific for the bovine gamma isoform of protein kinase C and cross-react with protein kinase C gamma from a variety of animal species. Immunoblot analysis of protein kinase C tryptic fragments revealed that the mAbs recognize the regulatory domain and not the catalytic domain. Two of the mAbs, 36G9 and 5A2, inhibit protein kinase C gamma cofactor-dependent activity (80% and 50% respectively). Consistent with the epitope mapping, none of mAbs inhibit the cofactor-independent catalytic activity of protein kinase C gamma. Competition analysis between these mAbs and phosphatidylserine, 12-O-tetradecanoylphorbol 13-acetate and Ca2+ showed that 36G9 and 5A2 block cofactor binding to protein kinase C gamma. These four mAbs thus interact with at least three distinct epitopes in the regulatory domain of protein kinase C gamma.     

Molecular cloning and characterization of the cDNA coding for C4b-binding protein, a regulatory protein of the classical pathway of the human complement system.

By using synthetic oligonucleotides as probes, plasmid clones containing portions of cDNA coding for human C4b-binding protein were isolated from a liver cDNA library. The entire amino acid sequence of the C4b-binding protein can be predicted from this study of the cloned cDNA when allied to a previous sequence study at the protein level [Chung, Gagnon & Reid (1985) Mol. Immunol. 22, 427-435], in which over 55% of the amino acid sequence, including the N-terminal 62 residues, was obtained. The plasmid clones isolated allowed the unambiguous determination of 1717 nucleotides of cDNA sequence between the codon for the 32nd amino acid in the sequence of C4b-binding protein and the 164th nucleotide in the 3' non-translated region. The sequence studies show that the secreted form of C4b-binding protein, found in plasma, is composed of chains of apparent Mr 70 000 that contains 549 amino acid residues. Examination of the protein and cDNA sequence results show that there are at least two polymorphic sites in the molecule. One is at position 44, which can be glutamine or threonine, and the other is at position 309, which can be tyrosine or histidine. Northern-blot analysis indicated that the mRNA for C4b-binding protein is approx. 2.5 kilobases long. The N-terminal 491 amino acids of C4b-binding protein can be divided into eight internal homologous regions, each approx. 60 amino acids long, which can be aligned by the presence in each region of four half-cystine, one tryptophan and several other conserved residues. These regions in C4b-binding protein are homologous with the three internal-homology regions that have been reported to be present within the Ba region of the complement enzyme factor B and also to the internal-homology regions found in the non-complement beta 2-glycoprotein I.     

DNA sequences specific for Caucasian G3m(b) and (g) allotypes: allotyping at the genomic level

Assignment of the G3m(g) and (b) correlative amino acid residues was performed at the genomic level by direct sequencing of DNA from nine Caucasian individuals. Two oligonucleotide primers were used for subclass-specific enzymatic amplification of a DNA segment comprising a major portion of the second and third constant region domains (C_H2 and C_H3) fo the human IgG3 heavy chain gene. Comparison of the sequences of amplified DNA from individuals serologically typed as homozygous for G3m(b) or G3m(g) or as heterozygous, G3m(b,g), revealed differences in the codons for the amino acid residues 291, 296, and 384. Proline, phenylalanine, and serine at these positions corresponded to G3m(b), and leucine, tyrosine, and asparagine to G3m(g). Heterozygotic individuals, typed G3m(b,g), displayed both the G3m(b) and G3m(g) codons at these three positions. The polymorphism at each of these three codons could be identified either as the appearance, or the loss, of recognition sites for the two restriction endonucleases, Nsp BII and Rsa I. This allowed the development of a polymerase chain reaction (PCR)-based assay permitting the distinction of G3m ^b and G3m ^g alleles by analyzing the electrophoretical mobility of the DNA fragments generated by digestion of the PCR-products with Nsp BII and Rsa I. Correspondence to: R. Grubb.     

Rabbit intestine contains a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to rhoB p20, a ras p21-like GTP-binding protein

A novel regulatory protein for rhoB p20, a ras p21-like GTP-binding protein (G protein), was partially purified from the cytosol fraction of rabbit intestine. This protein, designated as rhoB p20 GDP dissociation inhibitor (GDI), inhibited the dissociation of GDP from rhoB p20. rhoB p20 GDI also inhibited the binding of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to the GDP-bound form of rhoB p20 but not of that to the guanine nucleotide-free form. GDI did not affect the GTPase activity of rhoB p20 and by itself showed no GTP gamma S-binding activity. GDI was inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, smg p21 and smg p25A. The Mr value of GDI was estimated to be about 27,000 from the S value. These results indicate that rabbit intestine contains a novel regulatory protein that inhibits the dissociation of GDP from and thereby the subsequent binding of GTP to rhoB p20.     

Mapping of sites on the Src family protein tyrosine kinases p55blk, p59fyn, and p56lyn which interact with the effector molecules phospholipase C-gamma 2, microtubule-associated protein kinase, GTPase-activating protein, and phosphatidylinositol 3-kinase.

Engagement of the B-cell antigen receptor complex induces immediate activation of receptor-associated Src family tyrosine kinases including p55blk, p59fyn, p53/56lyn, and perhaps p56lck, and this response is accompanied by tyrosine phosphorylation of distinct cellular substrates. These kinases act directly or indirectly to phosphorylate and/or activate effector proteins including p42 (microtubule-associated protein kinase) (MAPK), phospholipases C-gamma 1 (PLC gamma 1) and C-gamma 2 (PLC gamma 2), phosphatidylinositol 3-kinase (PI 3-K), and p21ras-GTPase-activating protein (GAP). Although coimmunoprecipitation results indicate that the Src family protein tyrosine kinases interact physically with some of these effector molecules, the molecular basis of this interaction has not been established. Here, we show that three distinct sites mediate the interaction of these kinases with effectors. The amino-terminal 27 residues of the unique domain of p56lyn mediate association with PLC gamma 2, MAPK, and GAP. Binding to PI 3-K is mediated through the Src homology 3 (SH3) domains of the Src family kinases. Relatively small proportions of cellular PI 3-K, PLC gamma 2, MAPK, and GAP, presumably those which are tyrosine phosphorylated, bind to the SH2 domains of these kinases. Comparative analysis of binding activities of Blk, Lyn, and Fyn shows that these kinases differ in their abilities to associate with MAPK and PI 3-K, suggesting that they may preferentially bind and subsequently phosphorylate distinct sets of downstream effector molecules in vivo. Fast protein liquid chromatography Mono Q column-fractionated MAPK maintains the ability to bind bacterially expressed Lyn, suggesting that the two kinases may interact directly.     

His-384 allotypic variant of factor H associated with age-related macular degeneration has different heparin binding properties from the non-disease-associated form

A polymorphism in complement factor H has recently been associated with age-related macular degeneration (AMD), the leading cause of blindness in the elderly. A histidine rather than a tyrosine at residue position 384 in the mature protein increases the risk of AMD. Here, using a recombinant construct, we show that amino acid 384 is adjacent to a heparin-binding site in CCP7 of factor H and demonstrate that the allotypic variants differentially recognize heparin. This functional alteration may affect binding of factor H to polyanionic patterns on host surfaces, potentially influencing complement activation, immune complex clearance, and inflammation in the macula of AMD patients.     

Tyrosines 1021 and 1009 are phosphorylation sites in the carboxy terminus of the platelet-derived growth factor receptor beta subunit and are required for binding of phospholipase C gamma and a 64-kilodalton protein, respectively.

Binding of platelet-derived growth factor (PDGF) to the PDGF receptor (PDGFR) beta subunit triggers receptor tyrosine phosphorylation and the stable association of a number of signal transduction molecules, including phospholipase C gamma (PLC gamma), the GTPase activating protein of ras (GAP), and phosphatidylinositol-3 kinase (PI3K). Previous reports have identified three PDGFR tyrosine phosphorylation sites in the kinase insert domain that are important for stable association of GAP and PI3K. Two of them, tyrosine (Y) 740, and Y-751 are required for the stable association of PI3K, while Y-771 is required for binding of GAP. Here we present data for two additional tyrosine phosphorylation sites, Y-1009 and Y-1021, that are both in the carboxy-terminal region of the PDGFR. Characterization of PDGFR mutants in which these phosphorylation sites are substituted with phenylalanine (F) indicated that Y-1021 and Y-1009 were required for the stable association of PLC gamma and a 64-kDa protein, respectively. An F-1009/F-1021 double mutant selectively failed to bind both PLC gamma and the 64-kDa protein, whereas all of the carboxy-terminal mutants bound wild-type levels of GAP and PI3K. The carboxy terminus encodes the complete binding site for PLC gamma, since a phosphorylated carboxy-terminal fusion protein selectively bound PLC gamma. To determine the biological consequences of failure to associate with PLC gamma, we measured PDGF-dependent inositol phosphate production and initiation of DNA synthesis. The PDGFR mutants that failed to associate with PLC gamma were not able to mediate the PDGF-dependent production of inositol phosphates. Since tyrosine phosphorylation of PLC gamma enhances its enzymatic activity, we speculated that PDGFR mutants that failed to activate PLC gamma were unable to mediate its tyrosine phosphorylation. Surprisingly, the F-1021 receptor mediated readily detectable levels of PDGF-dependent PLC gamma tyrosine phosphorylation. Thus, the production of inositol phosphates requires not only PLC gamma tyrosine phosphorylation but also its association with the PDGFR. Comparison of the mutant PDGFRs' abilities to initiate PDGF-dependent DNA synthesis indicated that failure to associate with PLC gamma and produce inositol phosphates diminished the mitogenic response by 30%. In contrast, preventing the PDGFR from binding the 64-kDa protein did not compromise PDGF-triggered DNA synthesis at saturating concentrations of PDGF. Thus, it appears that phosphorylation of the PDGFR at Y-1021 is required for the stable association of PLC gamma to the receptor's carboxy terminus, the production of inositol phosphates, and initiation of the maximal mitogenic response.     

Interaction of C3 with antigen-antibody complexes in the process of solubilization of immune precipitates

The binding properties of activated C3 to immune complexes were studied by using solubilization phenomenon as a model system. IgG or F(ab')2 immune precipitates were solubilized by the six isolated alternative pathway proteins, and the solubilized complexes were analyzed by SDS-PAGE. As a result of solubilization, we observed some high m.w. bands. Under reducing conditions, the bands with m.w. of 150,000 and 115,000 appeared in the case of IgG and F(ab')2 complexes, respectively. Two-dimensional SDS-PAGE revealed that hydroxylamine treatment resulted in the dissociation of the 150,000-m.w. polypeptide into the C3 alpha-65 and the heavy chain of IgG. Similarly, the 115,000-m.w. polypeptide was dissociated into the C3 alpha-65 and the Fd chain. Therefore, it is likely that iC3b binds covalently to the Fd region of the heavy chain of IgG via an ester bond. Under nonreducing conditions, iC3b-IgG and iC3b-F(ab')2 complexes had apparent m.w. of 340,000 and 270,000, respectively, corresponding to one iC3b molecule bound to one antibody molecule. In addition, a considerable amount of iC3b also binds to antigen molecules via an ester bond. The findings that C3 binds to the F(ab')2 molecules and bovine serum albumin, which contain only a small amount of carbohydrate, suggest that C3 may not bind to the carbohydrate moiety of antibody molecules. Indeed, various carbohydrate molecules did not inhibit the solubilization even at high concentrations. In contrast, acetyl tyrosine having an aromatic ring and a hydroxyl group produced the best inhibition of the solubilization. Furthermore, we demonstrated that generation of C3b in the presence of 3H-tyrosine resulted in covalent binding of the tyrosine specifically to the C3 alpha' chain, indicating that the inhibition of solubilization may be due to the competition between tyrosine and immune complexes for the covalent binding of C3. Thus, it could be concluded that C3 binds covalently to the amino acid residues of antigen and antibody molecules during solubilization.     

Sequence of a human immunoglobulin gamma 3 heavy chain constant region gene: comparison with the other human C gamma genes.

We report the first and complete nucleotide sequence of a human gamma 3 heavy chain constant region gene (C gamma 3). This gene displays the same organization than the others C gamma genes and exhibits normal RNA splice and polyadenylation sites. A comparison of its primary sequence with those of C gamma 1, C gamma 2 and C gamma 4 genes confirms the high degree of homology (95%) of the human family in both coding and non-coding regions, and the divergence of the hinge region. The C gamma 3 gene we sequenced codes for a Gm(b) gamma 3 chain (EZZ). Comparison with other known protein sequences reveals that only two specific aminoacids are involved in the Gm(b) and Gm(g) allotypes, which suggests an important part of the spatial configuration in the allotypic specificities.     

Staurosporine-based binding assay for testing the affinity of compounds to protein kinases

Staurosporine is a broad-spectrum inhibitor of both tyrosine and serine/threonine protein kinases. Excitation of staurosporine and its analogues at 296 nm results in major emission bands centered at 378 and 396 nm. The intensity of the emission bands is enhanced on binding to the adenosine triphosphate (ATP) site of many protein kinases. This property was used to develop a competitive displacement assay for evaluating the binding affinity of small molecules to protein kinases. The assay was validated in both cuvette and plate formats for several phosphorylated and non-phosphorylated protein kinases. The throughput of the assay is high enough to be used in drug discovery for screening as well as lead optimization.