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J E Coligan

Publications and source records attributed to J E Coligan.

At least 253 records · Page 14Linked to original sources
PubMed1978

Isolation and characterization of a high molecular weight glycoprotein from human blood platelets.

A high molecular weight glycoprotein consisting of three disulfide-linked 142,000 molecular weight chains has been isolated from human blood platelets. The glycoprotein, designated thrombospondin, is released by platelets in response to thrombin treatment and is proteolyzed when left in the presence of platelets after liberation. It is relatively insensitive to degradation by thrombin. Thrombospondin is a filamentous protein of dimensions approximately 7 X 70 nm and contains 1.9% neutral sugars, 1.4% amino sugars, 0.7% sialic acid, and no hexuronic acid. Amino acid analysis reveals that the level of cysteine is approximately 260 residues per molecule. Thrombospondin binds to immobilized heparin but is released by 0.45 M sodium chloride. A single band is obtained by isoelectric focusing, indicating a pI of 4.7 as well as a relatively high degree of purity. Degradation of the intact molecule with trypsin yields a stable core particle of molecular weight 210,000 comprised of three 70,000 chains.

Alkylation↗
PubMed1978

Primary structure of murine major histocompatibility complex alloantigens: isolation, biochemical characterization, and preliminary alignment of CNBr fragments from the H-2Ib glycoprotein.

Radiochemical methodology has been applied in studies directed toward the determination of the amino acid sequence of the murine H-2Kb gene product. Five major CNBr fragments, which comprise the NH2-terminal 80% of the intact glycoprotein and include the H-2Kb fragment isolated by papain cleavage, were isolated and characterized. By means of amino-terminal sequence analysis, homology to other products of the major histocompatibility complex, identification of partial CNBr cleavage products, and localization of the site of papain cleavage, it was possible to align the fragments obtained from the molecule in the following order: the NH2-terminal fragment, designated IIIn, contains 23 residues and is followed by fragment IIIa, which contains 29 residues. Two peptides Ib (Mr 11,500) and Ia (Mr 12,000), each of which contains a carbohydrate moiety, are followed by fragment Ic (Mr 5800), which includes the site of papain cleavage. The data indicate the presence of disulfide bonds between fragments Ib and Ia and between Ia and Ic.

Amino Acid Sequence↗
PubMed1978

Primary structure of murine major histocompatibility complex alloantigens: amino acid sequence studies of the cyanogen bromide fragments of the H-2Kb glycoprotein.

Radiochemical microtechniques have been used in the amino acid sequence analysis of five major CNBr fragments of the glycoprotein specified by the murine major histocompatibility complex gene H-2k(b). These fragments have been tentatively aligned and represent the NH(2)-terminal 80% of the intact molecule. All amino acids except Asp, Asn, and Gln have been assigned in 128 out of 149 possible positions in the NH(2)-terminal portions of each of these fragments. These assignments, which represent approximately 50% of the total sequence from these fragments, are listed below in the order of their alignment in the intact H-2K(b) molecule: IIIn, -PHSLRYFVTAVSRP(G)L(G)(E)PRYM; IIIa, EVGYV--TEFVRF-S-AE(A)PRYEPR(A)--M; Ib, E-EGPEYWERET-KAK(G)-E-SFR--LRTLL(G)YY--TK; Ia, AALITK-KWE-AGEAERLRAYLEGTC-E-L; Ic, ELVETRPAG-GTF-KWAS-VVPLGKE-YY(T). The unassigned positions represented by dashes in the above sequences may be tentatively assigned as Asp, Asn, or Gln. The NH(2)-terminal sequence obtained for the H-2K(b) molecule was compared to the limited sequence information available for other major histocompatibility complex gene products. An 84% homology (16 of 19 residues) to the H-2K(q) and H-2K(k) molecules, which are identical to one another in the positions compared, was observed. A similar comparison with 28 of the 31 NH(2)-terminal residues of HLA-B7 indicated 68% homology. Furthermore, significant homology was observed between H-K(b) and HLA-B7 in a region of glycosylation, which occurs between positions 85 and 100 in the two molecules.

Amino Acid Sequence↗
PubMed1978

Immunochemistry of streptococcal group C polysaccharide and the nature of its crossreaction with the Forssman glycolipid.

Acid hydrolysis of streptococal Group C polysaccharide yields a disaccharide, 3-O-alpha-N-acetylgalactosaminosyl-N-acetylgalactosamine (3-O-alpha-GalNAc-GalNAc) which expresses Group C antigenic activity. This disaccharide, which exists as a side chain in the intact polysaccharide, can completely inhibit the binding between Group C polysaccharide and most Group C antibodies, indicating that this unit is the immunodominant feature of the intact polysaccharide. The alpha anomeric configuration and N-acetylation are required for the expression of the antigenic activity by the haptenic disaccharide. Also obtained from the acid hydrolysis of the Group C polysaccharide are rhamnose oligosaccharides with structural identity to the Group A-variant polysaccharide and with Group A-variant antigenic activity. It is inferred from these data that the Group A-varient polysaccharide structure is the core unit of the Group C polysaccharide. The nature of the immunologic crossreactivity between the Forssman glycolipid and Group C polysaccharide, which possess identical nonreducing terminal digalactosamine units, was investigated. Rabbit anti-Group C antibodies bound the Forssman glycolipid with approximately the same affinity as 3-O-alpha-GalNAc-GalNAc and were capable of mediating lysis of sheep red blood cells (SRBC). Antibody fractions isolated from anti-sheep hemolysin were likewise able to bind Group C polysaccharide. The heterologous reactions were in most assay systems weaker than reactions with the immunizing antigen.

Amino Sugars↗
PubMed1977

The use of idiotypes as markers for antibody variable regions in the rabbit.

This article reviews the use of idiotypes as variable region genetic markers in the rabbit. Topics discussed include reagents and assays for the detection of idiotypes, evidence concerning the association of idiotypes with specific antigen binding sites on antibodies, and the inheritance of idiotypes in the rabbit. Several points are emphasized in this review. First, interpretation of idiotypic phenomenons are strongly dependent on the reagents and assays employed. Second, while strong evidence exists that a given idiotype is a marker for a specific antigen binding site, exceptions to this association have been reported. Third, the inheritance of identical or similar idiotypes has been demonstrated in several instances, but it is not always demonstrable, perhaps because an idiotype is a complex phenotype. Several reasons for this complexity are pointed out. Fourth, idiotypes are linked to group a allotypes and VL subgroups but exceptions to these associations have been described for antibodies isolated from single individuals. The significance of these exceptions is discussed. Current areas of interest in rabbit idiotypy include the relationship of idiotypes to other V region markers, the genetics of idiotypes specific to H or L chains, and the relationships among the idiotypes of antibodies isolated from a single rabbit.

Animals↗
PubMed1977

A disaccharide hapten from streptococcal group C carbohydrate that cross-reacts with the Forssman glycolipid.

A disaccharide hapten was isolated in approximately 20% yield from an acid hydrolysate of the streptococcal Group C carbohydrate. This disaccharide was assigned the structure 3-0-alpha-N-acetylgalactosaminosyl-N-acetylgalactosamine on the basis of chemical and immunochemical data. The ability of the hapten to inhibit completely the binding between Group C carbohydrate and most Group C antibodies indicated that this disaccharide is the immunodominant feature of the Group C carbohydrate. Fractionation of antisera on an alpha-N-acetylgalactosamine immunoadsorbent column yielded several populations of anti-Group C antibodies. The reaction between labeled Group C carbohydrate and antibodies which strongly bound the immunoadsorbent column was 50% inhibitable by the disaccharide hapten when the hapten was added in approximately 15-fold excess (w/w) of the labeled antigen. On the other hand, the binding of those antibodies which did not bind to the immunoadsorbent column was poorly inhibited under these conditions. The Forssman glycosphingolipid, which has a common terminal digalactosamine unit, was shown to likewise inhibit Group C carbohydrate binding reactions.

Acetylgalactosamine↗
PubMed1976

Methylation analysis of the carbohydrate portion of carcinoembryonic antigen.

The carbohydrate structural units of carcinoembryonic antigen samples isolated from four different tumors were quantitated using gas chromatography-mass spectrometery after methylation and subsequent conversion to their alditol acetates. Different carcinoembryonic antigen preparations showed some quantitative but no qualitative differences in the structural units present. The results indicate that a large portion of the fucose residues in the glycoprotein were linked to N-acetylglucosamine and that most of the branching mannose residues were probably linked to three N-acetylglucosamine residues.

Acetylglucosamine↗
PubMed1976

Characterization of carcinoembryonic antigen fractionated by concanavalin A chromatography.

A small percentage (15%) of particles morphologically distinct from carcinoembryonic antigen (CEA) cruller-like particles (averaging 9 x 40 nm) as determined by electron microscopy, and previously presumed to be impurities, has been removed from CEA preparations by concanavalin A (Con A) affinity chromatography. The first of four affinity fractions constituted 45% of the recovered weight; the last, which constituted 17% of the recovered weight, was obtained only after a 2-day soak in 20% alpha-D-mannopyranoside. The affinity fractions were of essentially equivalent specific activity, morphological appearance, and composition. They all demonstrated markedly different chemical compositions and an approximately 100-fold higher specific activity than the bulk of the nonaffinity material. Thus, the bulk of the Con A-nonbound fractions apparently is not CEA but contaminating particles of several different varieties. Compositional analyses further indicated that the mannose content of various CEA fractions was directly correlated with Con A binding affinity, amino acid content, morphological type, and CEA specific activity. This led to the conclusion that the protein chain of the various CEA preparations fractionated by us is characteristic. An attempt to fractionate very impure crude CEA by Con A affinity chromatography indicated that this scheme cannot by itself produce as pure CEA as standard methods. Thus, for the first time the effectiveness of the purification of a glycoprotein has been successfully monitored by electron microscopy, demonstrating that the cruller-like appearance of the CEA particle is closely related to antigenic specificity. Whether antisera to our affinity fractions will improve the specificity and sensitivity of the clinical assay is currently being investigated.

Amino Acids↗
PubMed1976

Physical characterization and structural studies of the carcinoembryonic antigen.

Knowledge of the structure of carcinoembryonic antigen (CEA) is essential if we are to understand the relationship of this molecule to similar, possibly cross-reacting, molecules present in nonmalignant states. Electron microscopy shows that at neutral pH, CEA particles consist of homogeneous, morphologically distinctive, twisted rod-shaped particles, about 9 X 40 nm. The carbohydrate structure of CEA has been studied by periodate oxidation. All the N-acetylneuraminic acid and fucose and a portion of the galactose and mannose were destroyed by the first periodate treatment without altering immunological activity. N-Acetylneuraminic acid was shown to be linked to galactose since its prior removal with neuraminidase led to an equivalent increased destruction of galactose by one treatment with periodate. Significantly, even after 100% of the fucose and N-acetylneuraminic acid, 75% of the galactose, and 50% of the N-acetylglucosamine and mannose were destroyed by serial periodate oxidation (Smith degradation), the remaining portion of the CEA molecule lost no more antigenic activity than did control samples where periodate was omitted. No carbohydrate was lost or destroyed in the control reaction.

Carbohydrates↗
PubMed1975

Electron microscopy and physical characterization of the carcinoembryonic antigen.

Carcinoembryonic antigen (CEA), a glycoprotein material purified from human tumors, has been visualized by electron microscopy. At neutral pH, it consists largely of relatively homogenous, morphologically distinctive twisted rod or cruller shaped particles, with dimensions 9 x 40 nm. The particle length is considerably diminished at pH 40.0, which correlates with a known diminution of charge. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular weight of 180,000 in the peak region of the CEA band for both 10 and 15% acrylamide. When native CEA was treated with neuraminidase, reduced, and alkylated, a relatively compact random coil was produced, whereas reduction and alkylation without neuraminidase treatment produced a less configuration, as determined by sedimentation studies and by electron microscopy. Electrophoretic migration, however, was apparently unaffected by reduction and alkylation. Thus the characteristic CEA particle appears by several lines of evidence to be substantially folded into a recognizably tertiary structural arrangement.

Amino Acids↗
PubMed1975

Structural studies on carcinoembryonic antigen. Periodate oxidation.

Periodate oxidation has been applied to examine the carbohydrate structure of carcinoembryonic antigen (CEA) and the possible role of the carbohydrate residues in its antigenic activity. Sialic acid (N-acetylneuraminic acid) and fucose were completely destroyed, and galactose and mannose were partially destroyed by a single periodate treatment. Serial periodate treatment (Smith degradation) destroyed additional amounts of galactose and mannose as well as significant amounts of N-acetylglucosamine. Prior removal of sialic acid by neuraminidase treatment led to increased destruction of galactose by periodate. Antigenic activity persisted indicating that the residues destroyed played little, if any, part in the antigenicity of CEA. These results yield an initial view of the structural arrangement of the carbohydrate residues in the CEA molecule.

Amino Acids↗
PubMed1975

The chemistry of carcinoembryonic antigen.

The structure of carcinoembryonic antigen is being determined as an approach to greater diagnostic specificity in the radioimmune assay. Examination by combined gas chromatography-mass spectrometry of derivatized saccharides from CEA has given a general outline of its carbohydrate structure. Sialic acid and fucose were completely destroyed, and galactose and mannose were partially destroyed by a single periodate treatment. Serial periodate oxidation (Smith degradation) destroyed additional amounts of galactose and mannose, as well as significant amounts of N-acetylglucosamine. Antigenic activity persisted, indicating that the residues destroyed played little, if any, part in the antigenicity of DEA.

Animals↗
PubMed1975

Immunochemical and chemical studies on streptococcal group-specific carbohydrates.

Structural studies on the carbohydrates of Groups A, C, and A-variant (AV) streptococci have utilized periodate oxidation, permethylation analysis, and immunochemical comparison of intact and periodate-oxidized polysaccharides. The data indicate that a similar 1,2- and 1,3-linked rhamnose chain is present in both the A and AV carbohydrates. The group A carbohydrate contains in addition N-acetylglucosamine residues at nonreducing terminals, whereas the AV is a homopolymer of rhamnose. There is some evidence that Group Ccarbohydrate contains the same rhamnose chain, but structural comparisons to the A and AV carbohydrates are complicated by the presence of intrachain N-acetylgalactosamine residues. Periodate oxidation and permethylation analysis show that while approximately 50% of the N-acetylgalactosamine of the Group C carbohydrate occupies terminal positions, the remainder is present as 1,3-linked units. Removal of the nonreducing terminal hexosamine units from the Group A carbohydrate by periodate treatment significantly enhanced its cross-reactivity with AV antiserum, whereas no enhancement was observed after similar treatment of the Group C carbohydrate. The data indicate the presence of an alpha-1,3-linked N-acetylgalactosamine disaccharide at the nonreducing terminal of the Group C carbohydrate.

Amino Acids↗