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G J Hughes

Publications and source records attributed to G J Hughes.

At least 19 recordsLinked to original sources
PubMed1992

Disulfide bond assignment in human J chain and its covalent pairing with immunoglobulin M.

The assignment of disulfide bonds in human J chain and its covalent pairing with immunoglobulin M was determined under conditions which minimize disulfide bond interchange. We show that in J chain the three intradisulfide bridges are formed between Cys 12 and 100, Cys 71 and 91, and Cys 108 and 133. Previous reports [reviewed by Koshland, M. E. (1985) Annu. Rev. Immunol. 3, 425-453] have proposed that cysteines 12, 14, or 68 were linked to the penultimate cysteine 575 of two mu chain tails. In this work, we demonstrate that cysteines 14 and 68 are disulfide-bridged to mu chains. A revised, albeit putative, model of J chain folding is presented which takes into account the correct disulfide pairing and the predictive secondary structure assignment.

Amino Acid Sequence↗
PubMed1992

Polypeptide marker and disease patterns found while mapping proteins in ascitis.

To assess the protein composition of ascitis, 28 samples of ascitic fluid were obtained from patients admitted to Geneva University Hospital. The samples were analysed randomly and blindly by high-resolution two-dimensional polyacrylamide gel electrophoresis. The final visual evaluation was compared with the discharged summary and diagnosis. The protein pattern of ascitis was, as expected, very similar to normal or diseased plasma, with the exception of two spots which were present in ascitic fluids but not in the 200 plasma samples analyzed in parallel. After microsequencing, they proved to be beta-fibrinogen fragments. Several diseases showed distinct patterns, especially acute pancreatitis. A group of intense spots with an apparent molecular mass between those of alpha 1-antitrypsin and beta-haptoglobin were found in all ascitic fluid from pancreatitis cases (six patients). These spots had isoelectric points similar to those of alpha 1-antitrypsin and beta-haptoglobin and microsequencing revealed that they were three different fragments of alpha 1-antitrypsin.

Acute Disease↗
PubMed1992

Structural and electron-microscopic studies of jacalin from jackfruit (Artocarpus integrifolia) show that this lectin is a 65 kDa tetramer.

The 133-amino-acid sequences of the alpha-subunit of jacalin (a lectin from Artocarpus integrifolia) and of the slightly larger alpha'-subunit were determined. The alpha'- and alpha-subunits, in the approximate ratio of 1:3, were found to be virtually identical in their primary structures, except for one valine for isoleucine substitution at position 113. Although both alpha'- and alpha-chains were glycosylated, the extent of glycosylation in the alpha'-chain was much greater than that in the alpha-subunit. In the alpha'-polypeptide, all molecules contained an N-linked oligosaccharide at position 74 and some contained sugar at position 43. The alpha- and alpha'-subunits were found to be strongly non-covalently associated with three distinct beta-subunits containing 20 amino acids each. Electron-microscopic visualization of native jacalin disclosed a structure composed of four alpha-type subunits with a clear-cut 4-fold symmetry. Analytical-ultracentrifugation studies of jacalin revealed an average molecular mass of 65 kDa, a value compatible with a tetrameric structure of the alpha(alpha')-subunits. The recalculated number of sugar-binding sites per jacalin molecule, given a molecular mass of 65 kDa, would yield 0.8 sites per alpha(alpha')-promoter, i.e. about twice the value previously determined [Appukutan & Basu (1985) FEBS Lett. 180, 331-334; Ahmed & Chatterjee (1989) J. Biol. Chem. 264, 9365-9372].

Amino Acid Sequence↗
PubMed1992

Human menopausal gonadotrophins in the treatment of unexplained infertility.

Ninety one patients with unexplained infertility were treated with human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG). The overall pregnancy rates were 29% per patient and 11% per cycle. In patients under the age of 37, the rates were 40% per patient and 15% per cycle. Many cycles (15%) were cancelled because of poor response to stimulation and by definition these women had normal cycles prior to stimulation. Within 1 year of ceasing HMG treatment, 9 patients had conceived spontaneously and another 4 conceived by GIFT.

Adult↗
PubMed1991

Parvalbumin isoforms in chicken muscle and thymus. Amino acid sequence analysis of muscle parvalbumin by tandem mass spectrometry.

Parvalbumins are high-affinity Ca(2+)-binding proteins characterized by an EF-hand structure. Muscles of lower vertebrates contain up to five isoparvalbumins whereas higher vertebrates were believed to contain only one isoform per species. Recently Brewer et al. [Brewer, J.M., Wunderlich, J.K., & Ragland, W. (1990) Biochimie 72, 653-660] purified and sequenced a protein that they named avian thymic hormone, from chicken thymus. This protein, promoting immunological maturation of bone marrow cells in culture, was identified as a parvalbumin. The amino acid composition of this thymic parvalbumin was, however, considerably different from those of chicken muscle parvalbumin [Strehler, E.E., Eppenberger, H.M., & Heizman, C.W. (1977) FEBS Lett. 75, 127-133], suggesting the existence of two tissue-specific parvalbumins in chicken. We purified parvalbumin from chicken muscle, determined its complete amino acid sequence by tandem mass spectrometry, and showed that this protein is rather homologous to muscle parvalbumins from other species but different in 45 positions from the thymic parvalbumin. We discuss the possibility that a parvalbumin gene family might exist in higher vertebrates, expressed in a tissue-specific and developmentally regulated manner.

Amino Acid Sequence↗
PubMed1991

A nodulin specifically expressed in senescent nodules of winged bean is a protease inhibitor.

Nodule senescence is one aspect of nitrogen fixation that is important to study from the perspective of improving the host-bacteroid interaction. In winged bean nodules, a 21-kilodalton protein is specifically expressed when senescence begins. Using subcellular fractionation, we observed that this plant protein interacts with the bacteroids. Microsequencing of the protein allowed us to obtain a specific oligonucleotide that was used to isolate the corresponding nodule cDNA. Sequence analysis of this cDNA revealed that the 21-kilodalton protein has all of the features of a legume Kunitz protease inhibitor. Subsequent analysis confirmed that this nodulin is indeed a protease inhibitor. Immunocytochemical study showed that the protease inhibitor is exclusively localized in infected senescent cells of the nodule, particularly in disorganized bacteroids, the peribacteroid membrane, vacuole membranes, and in the vacuole fluid. The specific expression of a protease inhibitor at senescence may be of particular interest if the targeted proteolytic activity is important for the symbiotic relationship. This point is discussed in relation to the known nodule proteases.

Aging↗
PubMed1990

The amino acid sequence of rabbit J chain in secretory immunoglobulin A.

The primary structure of rabbit J chain, which occurs covalently bound to secretory IgA, was determined. J chain was isolated in its S-carboxymethylated form, in one step, by SDS/PAGE followed by electro-elution; 5 nmol of protein (approx. 75 micrograms), in all, was necessary for the determination of the complete sequence by the 'shot-gun' microsquencing technique; with the use of several site-specific endoproteinases, the various digests of S-carboxymethylated J chain were separated by micro-bore reverse-phase h.p.l.c. and the partial N-terminal sequences of all peptides were analysed. From the sequence alignment, gaps were filled by further extensive sequencing of the relevant overlapping fragments isolated from selected digests. Rabbit J chain comprises 136 amino acid residues, out of which eight are conserved cysteine residues, and is more closely similar to the human sequence (73.5% identify) than to the mouse sequence (68% identity). There is one unique glycosylation site at asparagine-48.

Amino Acid Sequence↗
PubMed1990

Immunological heterogeneity of autoreactive T lymphocytes against the nicotinic acetylcholine receptor in myasthenic patients.

The response of human T lymphocytes against the nicotinic acetylcholine receptor (AChR) was studied in five patients with myasthenia gravis (MG) and in six healthy donors using either native Torpedo AChR or recombinant protein derived from the mammalian AChR alpha subunit (X4, residues 6-216 of mouse AChR alpha subunit). The present study demonstrates that (a) AChR-specific T helper cell lines can be generated from MG patients [either from peripheral blood lymphocytes (PBL) or from thymocytes] as well as from PBL of normal controls, (b) lymphocytes from MG patients, but not from controls, recognize the mammalian AChR but not the Torpedo receptor, (c) in humans, the HLA-DR2-associated T cell epitope is probably located in the region of residues 162-216 of the AChR alpha subunit and (d) there is a considerable heterogeneity of autoreactive T cell responses: (i) T cell lines from different HLA-type donors have distinct epitope profiles; (ii) the epitope specificity of the PBL-derived T cell line is different from that of the thymocyte-derived line; (iii) the epitope specificities of patient-derived T cell lines are different from those generated from normal controls who share the same HLA phenotype.

Animals↗
PubMed1990

Chemical and immunochemical identification of the second major rabbit immunoglobulin lambda chain polypeptide bearing c7 epitopes.

The purification of lambda chain-containing IgG fraction from pooled sera of Basilea rabbits, which were bred and selected for the expression of a high level of lambda chains positive for c7 but negative for c21, was carried out. On the basis of specific binding to anti-c7 antiserum, the c7 lambda chain fraction in serum IgG was shown to account for up to 70% of the total immunoglobulin light chains, the remaining 30%, bearing the expected k2 bas isotype. Peptide mapping of the mixed light chains (lambda + k2 bas) followed by microsequencing of the constant region fragments indicated that the C lambda region originated from the cDNA sequence derived from a c7+,c21- Basilea rabbit (i.e. identical to gene C lambda 6), as described in the preceding report (Hayzer, D. J. et al., Eur. J. Immunol. 1990. 20: 2707). In addition, a synthetic peptide encompassing residues 139-159 of the constant region derived from the predicted sequence of gene C lambda 6 was shown to partially inhibit the c7-anti-c7 binding reaction in a sensitive enzyme-linked immunosorbent assay. Taken together, the chemical and immunochemical data clearly demonstrate that gene C lambda 6 indeed encodes c7 epitopes.

Amino Acid Sequence↗
PubMed1990

Identification of T-cell epitopes of autoantigens using recombinant proteins; studies on experimental autoimmune myasthenia gravis.

In the Lewis rat, T-cell lines from animals immunized with native or denatured Torpedo nAChR recognize the Torpedo-derived recombinant protein T alpha X1 omega (alpha-2-200) but not the equivalent mouse- or chick-derived recombinant proteins X4 omega or C alpha X1 omega (alpha 6-216 and alpha 35-216, respectively). T-cell lines derived from animals immunized with T alpha X1 omega, X4 omega or C alpha X1 omega are specific for the homologous protein. This lack of cross-species reactivity suggests caution in the use of Torpedo nAChR-selected lines generated from human patients. Proteolysis and fractionation of the products by reverse-phase HPLC was effective in localization of a T-cell epitope of X4 omega, a mouse-derived recombinant protein. With Lewis rats, the major epitope of T alpha X1 omega is alpha 97-112. However, the major epitope of the mouse-derived protein, X4 omega, as determined by proteolytic digestion and fractionation of the products by reverse-phase HPLC, is alpha 14-22. This shift in T-cell epitope between closely related proteins may result from the conservation of sequence of alpha 97-112 between mammalian species.

Animals↗
PubMed1988

Rabbit secretory components of different allotypes vary in their carbohydrate content and their sites of N-linked glycosylation.

The asparagine-linked glycosylation sites in rabbit high and low Mr secretory components (SC) have been determined for the three known allotypes, t61, t62, and t63. Purified SC polypeptides were subjected to mild periodate oxidation of terminal nonreducing sugars followed by selective reduction with [3H]sodium borohydride, SC polypeptides were further proteolytically cleaved, and the 3H-labeled peptides were isolated and characterized. Both high and low Mr SCs of the three allotypes possess a common glycosylation site at the asparagine residue position 400, whereas the second site, in the amino-terminal domain of SC, was found to be variable: the t61 and t63 allotypes contained a glycosylation site at positions 70 and 90, respectively. Moreover, although the t62 allotype was found to contain a triplet acceptor site (N-X-S) at positions 90-92, analyses showed that less than 30% of the t62 allotype peptides encompassing this region were glycosylated at residue 90. Furthermore, the amino acid sequence of the t61 SC in the region of residues 69-90 varies by 8 and 10 amino acid substitutions when compared with the t62 and t63 allotype sequences, respectively. However, neither the variation in amino acid sequence nor the variation in degree or site of glycosylation measurably affected the non-covalent binding of domain 1 to dimeric IgA.

Animals↗
PubMed1988

Monoclonal antibodies to the main immunogenic region of the nicotinic acetylcholine receptor bind to residues 61-76 of the alpha subunit.

Monoclonal antibodies (mAbs) to the main immunogenic region (MIR) bind to fusion proteins containing region 37-200 of the alpha chain of Torpedo, mouse, and chicken nicotinic acetylcholine receptor. In the case of the mouse alpha chain, these mAbs react with sequence 61-216 but not with 74-216. A synthetic peptide M1, containing residues 61-76 of the mouse alpha chain, also binds these anti-MIR mAbs, showing that all or part of their binding site is included in this region. The conformational dependence and epitope specificity of the mAbs are discussed.

Amino Acid Sequence↗
PubMed1988

Detection of the major epitopes of human protamine P1 recognized by rabbit and mouse antibodies.

The characterization of the major antigenic determinants present in human protamine P1 has been carried out by the use of specific rabbit polyclonal and mouse monoclonal antisera raised against protamine P1. This basic protein, the full amino acid sequence of which has been determined here, has been cleaved by cyanogen bromide and/or by pepsin to generate a discrete number of peptides. These have been purified, characterized by partial amino acid sequencing and used for the determination of their antigenic reactivities with antisera to native protamine P1. Both rabbit polyclonal and mouse monoclonal antibodies were able to recognize the NH2-terminal CNBr peptide encompassing residues 1-36 to the same extent as the intact protamine. A minor epitope present on the COOH-terminal peptide 37-50 could be detected only with the polyclonal rabbit antisera. Attempts to further cleave the P1 molecule in order to isolate peptides shorter than fragments 1-36 whilst retaining full antigenic reactivities, were unsuccessful. This suggests that the epitopes in P1 are conformation-dependent and located for the most part on the amino-terminal half of the molecule, which comprises the characteristic central arginine cluster. The implication of these findings for the studies of the specificities of autoantibodies in sera from infertile and vasectomized individuals is discussed.

Amino Acid Sequence↗
PubMed1987

Rabbit secretory components: identification of a third allotype, t63.

A third allotype of rabbit secretory component has been identified. The allotype previously referred to as t62 by our laboratory can now be subdivided into two allotypes, t62 and t63, with alloantisera capable of discriminating between the two. Results of family studies are consistent with a three allele system (t61, t62 and t63) at the t-locus. By SDS PAGE, electrophoretic mobilities of the multiple SC bands for each of the three allotypes are characteristic of the allotype; the apparent molecular sizes of the bands of the t62 allotype are 2 to 3 kDa lower than those for the t61 allotype. The banding patterns of the t61 and t63, although similar, are not identical to each other. Results of serologic cross-reaction studies and of tryptic peptide mapping studies suggest multiple structural differences between the allotypes as well as a closer relationship between t62 and t63 than between either of these allotypes and t61.

Alleles↗
PubMed1987

A modified nicotinic acetylcholine receptor lacking the 'ion channel amphipathic helices'.

Antibodies to a synthetic peptide from the 'amphipathic helix' of the alpha-chain of the nicotinic acetylcholine receptor (nAChR) bound both to detergent-solubilised and membrane-bound nAChR, indicating that this region, suggested as a component of the transmembrane ion channel in one model, is not buried in the membrane. Trypsinisation of membranes prior to affinity purification yielded preparations lacking the amphipathic helices of the alpha- and beta-chains and probably also of the gamma- and delta-chains. Such material should allow direct testing, by reconstitution experiments, of the importance of these regions for channel activity.

Animals↗
PubMed1987

Structural variability of rabbit secretory components. Allotype-associated differences in the third, fourth, and fifth domains.

We have previously shown (Frutiger, S., Hughes, G. J., Hanly, W. C., Kingzette, M., and Jaton, J.-C. (1986) J. Biol. Chem. 261, 16673-16681) that limited tryptic digestion of the high Mr form of rabbit secretory component of allotypes t61, t62, and t63 generates two major fragments, the NH2-terminal domain and a 40-kDa fragment encompassing domains 3, 4, and 5. Similarly, from the low Mr form of secretory component, (SC) the NH2-terminal domain, together with a 30-kDa fragment containing domains 4 and 5, were released. These fragments were used as inhibitors in a sensitive competitive binding radioimmunoassay with noncross-reactive rabbit alloantisera to study the distribution and localization of the major allotype-specific allotopes within the SC polypeptide. The 40-kDa fragments were shown to inhibit the 125I-labeled intact SC/anti-SC allotype reaction to the extent of 90%, i.e. nearly as well as the intact homologous high Mr SC form. In contrast, the NH2-terminal fragments (domain 1) were not inhibitory. The low Mr SC of each allotype was less inhibitory on a molar basis than the homologous high Mr SC polypeptide, an observation compatible with the deletion of domains 2 and 3 in the smaller polypeptide (Deitcher, D. L., and Mostov, K. E. (1986) Mol. Cell. Biol. 6, 2712-2715; Frutiger, S., Hughes, G. J., Fonck, Ch., and Jaton, J.-C. (1987) J. Biol. Chem. 262, 1712-1715). The structural correlates of the allotypic specificities were evaluated by comparative peptide mapping of the 40-kDa fragments (allotypes t61, t62, and t63). The data suggest that the t61 allotype structure differs significantly from the t62 and t63 structures, the latter two being much more related to each other than to t61. These findings are in full agreement with the serological data. The inhibition results suggest that the major allotype-specific, noncross-reactive allotopes of SC are distributed throughout domains 3, 4, and 5, even though domain 4 appears to be more conserved than domains 3 and 5 between the allotypes t61 and t63. Seven amino acid substitutions between t61 and t63 have been detected within domains 3, 4, and 5.

Amino Acid Sequence↗