PubMed Health⌕ Search

Biomedical subjects

R B Parekh

Publications and source records attributed to R B Parekh.

At least 19 recordsLinked to original sources
PubMed1998

Human T cell IgD receptors react with O-glycans on both human IgD and IgA1.

Previous studies on murine T cell IgD-R have shown that these receptors recognize N-glycans of murine IgD, and not of other Ig isotypes. We have now studied the specificity of IgD-R on human T cells. Human IgD digested with proteinase K to fragments of < 5 kDa inhibit the ability of T cells to form rosettes with IgD-coated ox erythrocytes. The same amount of digested IgG does not. We tested all the human Ig isotypes: IgG1, -2, -3, -4, IgA2, IgE and IgM fail to inhibit significantly at 20 microg/assay. However, IgA1 is as effective as IgD itself, showing approximately 60 % and 80 % inhibition at 5 microg and 10 microg/assay. Human IgA1 and IgD both contain Gal-1 --> 3-GalNac-rich O-linked glycans, and on this basis are both bound to ricin and jacalin. The O-linked glycans may therefore also represent the common moiety binding to IgD-R. Disaccharides Gal-1 --> 3-GalNac, and Gal-1 --> 4-Glc at 10 microg/assay blocked IgD rosetting while Gal-1 --> 6-Glc did not. We conclude that the human IgD-R is a lectin, differing from the murine IgD-R in that it has both IgA1 and IgD as ligands.

Animals↗
PubMed1997

Post-translational modification of proteins and the discovery of new medicine.

Post-translational modifications are fundamental to processes controlling behaviour, including cellular signaling, growth and transformation. As the molecular basis of protein modifications in normal and disease processes are becoming better defined, so new strategies for designing therapeutic entities to control complex disease processes are emerging.

Drug Design↗
PubMed1996

The inter-locus recombinant HLA-B*4601 has high selectivity in peptide binding and functions characteristic of HLA-C.

The vast majority of new human HLA class I alleles are formed by conversions between existing alleles of the same locus. A notable exception to this rule is HLA-B*4601 formed by replacement of residues 66-76 of the alpha 1 helix of B*1501 by the homologous segment of Cw*0102. This inter-locus recombination, which brings together characteristic elements of HLA-B and HLA-C structure, is shown here to influence function dramatically. Naturally processed peptides bound by B*4601 are distinct from those of its parental allotypes B*1501 and Cw*0102 and dominated by three high abundance peptides. Such increased peptide selectivity by B*4601 is unique among HLA-A,B,C allotypes. For other aspects of function, presence of the small segment of HLA-C-derived sequence in an otherwise HLA-B framework converts B*4601 to an HLA-C-like molecule. Alloreactive cytotoxic T lymphocytes (CTL), natural killer (NK) cells, and cellular glycosidases all recognize B*4601 as though it were an HLA-C allotype. These unusual properties are those of an allotype which has frequencies as high as 20% in south east Asian populations and is associated with predisposition to autoimmune diseases and nasopharyngeal carcinoma.

Amino Acid Sequence↗
PubMed1996

Unusual uniformity of the N-linked oligosaccharides of HLA-A, -B, and -C glycoproteins.

MHC class I glycoproteins possess an invariant site for N-linked oligosaccharide addition at position 86 of the heavy chain. For human HLA-A, -B, and -C class I glycoproteins, position 86 is the only site of N-linked glycosylation. Comparison of the size and relative abundance of oligosaccharides associated with nine HLA-A, -B, or -C allotypes isolated from EBV-transformed B cell lines and mixtures of HLA-A, -B, and -C allotypes isolated from pooled PBLs revealed a very restricted set of structures. Allotypes encoded by the HLA-A and -B loci have two predominant glycan structures that were almost exclusively di-sialylated. In contrast, HLA-C allotypes have four glycan structures, comprising those associated with HLA-A and -B and two additional glycans. Identical oligosaccharides were present on different allotypes of a class I HLA locus, and in particular, HLA-C allotypes defining two inhibitory specificities for NK cells were shown to possess the same set of oligosaccharides. The uniformity of oligosaccharide structure associated with different HLA-A, -B, and -C products and the relative lack of heterogeneity for any given allotype are unusual features for a mammalian glycoprotein. Particularly striking is that such conserved oligosaccharide structures juxtapose the major regions of amino acid sequence variation within the Ag recognition site, including the polymorphisms of the alpha 1 helix that determine the inhibitory ligands for human NK cells.

Adult↗
PubMed1996

Getting the glycosylation right: implications for the biotechnology industry.

Glycosylation is the most extensive of all the posttranslational modifications, and has important functions in the secretion, antigenicity and clearance of glycoproteins. In recent years major advances have been made in the cloning of glycosyltransferase enzymes, in understanding the varied biological functions of carbohydrates, and in the accurate analysis of glycoprotein heterogeneity. In this review we discuss the impact of these advances on the choice of a recombinant host cell line, in optimizing cell culture processes, and in choosing the appropriate level of glycosylation analysis for each stage of product development.

Animals↗
PubMed1995

Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid.

Reaction conditions for conjugation of two fluorescent ortho-substituted aniline derivatives, 2-amino benzamide (2-AB) and 2-anthranilic acid (2-AA), to N- and O-glycans have been investigated. Conjugation conditions for attaching 2-AB and 2-AA to core-fucosylated and non-fucosylated glycans were developed using complex N-glycans radiolabeled at the nonreducing terminus with [3H]C6-galactose. Optimal conditions for each of the following reaction parameters were experimentally defined: [glycans], [2-AB] or [2-AA], solvent and acid composition, temperature and time of Schiff's base formation, nature of reductant, and temperature and time of reduction. Using the optimized reaction conditions it has been shown with several standard glycans and glycoprotein-derived glycan libraries that (i) molar labeling efficiencies are high and essentially independent of the amount of glycans; (ii) negligible (< 2 mol%) desialylation occurs during conjugation; (iii) glycan labeling is nonselective, i.e., independent of glycan structure; and (iv) insignificant fluorescent or chemical "blank" is recovered during the glycan-labeling and purification protocol. Labeling glycan pools with 2-AB or 2-AA therefore allows representative glycan profiles to be obtained and also allows relative molar quantitation of individual glycans in a pool. The 2-AB label is compatible with several chromatographic means for separation of carbohydrates including Bio Gel P4 gel permeation, high-performance anion-exchange chromatography with fluorescence detection, and a variety of HPLC procedures, as well as with mass spectrometric methods including matrix-assisted laser desorption-mass spectrometry and electrospray-mass spectrometry. The 2-AA label is particularly well-suited for electrophoretic separations by polyacrylamide gel electrophoresis. These fluorophores show high intrinsic sensitivity and thus facilitate very sensitive analysis of protein glycosylation.

Chromatography, High Pressure Liquid↗
PubMed1995

Identification of endogenous protein-associated carbohydrate ligands for E-selectin.

A comparative analysis of carbohydrate libraries derived from cell lines binding E-selectin was used to identify endogenous protein-associated carbohydrate ligands for E-selectin. Three structures, which together constitute less than 1% of the total cell surface protein-associated carbohydrate, were unique to cell lines capable of binding E-selectin, including neutrophils and the monocytic cell line U937. All are tetra-antennary N-linked structures, with a sialic acid alpha 2 --> 3 galactose beta 1 --> 4 (fucose alpha 1 --> 3) N-acetyl glucosamine beta 1 --> 3 galactose beta 1 --> 4 (fucose alpha 1 --> 3) N-acetyl glucosamine lactosaminoglycan extension (sialyl-di-Lewis X [S-diLe(x)]) on the arm linked through the C4 residue on the mannose. While all contained the expected 3-SLe(x) sub-structure, these native structures have an additional fucosylated lactosamine unit. Direct evidence that these S-di-Lex-containing structures are high-affinity ligands for E-selectin came from the use of recombinant soluble E-selectin-agarose affinity chromatography. These three carbohydrate structures bound specifically to the E-selectin column, while 3-SLe(x) itself does not bind under identical conditions.

Binding Sites↗
PubMed1994

Isolation and characterization of natural protein-associated carbohydrate ligands for E-selectin.

A comparative analysis of carbohydrate 'libraries' derived from cell lines binding E-selectin with differing avidity identified endogenous protein-associated carbohydrate ligand candidates for E-selectin. Three unusual structures, which constitute less than 3% of cell surface protein-associated carbohydrate, were unique to the E-selectin-binding cells, including neutrophils and the monocytic cell line U937. All are tetraantennary N-linked structures with a NeuAc alpha 2-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3Gal beta 1-->4- (Fuc alpha 1-->3)GlcNAc lactosaminoglycan extension (diSLex) on the arm linked through the C4 residue on the mannose. While all contained the expected SLex [NeuAc alpha 2-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] moiety, these structures have an additional fucosylated lactosamine unit. Direct evidence that these diSLex-containing structures are, indeed, high-affinity ligands for E-selectin came from the use of recombinant soluble E-selectin-agarose affinity chromatography. We found that these three carbohydrate structures bound specifically to the E-selectin column. SLex itself does not bind under identical conditions. In summary, these related structures: (1) all possess an unusual 3-sialyl di-Lewis x extension on one arm of an N-linked tetraantennary glycan; (2) of the cells tested, are present only on E-selectin-binding leukocytes and leukocytic cell lines; (3) bind to E-selectin with a relatively high affinity (Kd < microM) and one greater than that of 3-sialyl Lewis x or 3-sialyl Lewis a; and (4) represent a very small percentage of the protein-associated carbohydrate. These carbohydrate structures appear to be present on only a very small number of cell surface proteins and may alone be responsible for the specificity of E-selectin-dependent adhesion.

Carbohydrate Sequence↗
PubMed1994

Selectins--glycoprotein targets for therapeutic intervention in inflammation.

Inflammation can be a beneficial response in the host for the control of infection and injury. However, occasionally, the inflammatory response can result in acute systemic collapse or, more frequently, a chronic reaction such as that observed in autoimmune disease, Crohn's disease and asthma. Injury to tissues or organs results in leukocyte adhesion to the endothelial cell surface, followed by diapedesis. Investigation of the initial leukocyte-endothelium adhesion processes has clearly shown the involvement of an inducible set of molecules, called selectins, on the endothelial and leukocyte cell surfaces. These molecules are of interest as the interactions with their respective ligands appear to involve carbohydrates. The exact nature of these interactions is still being elucidated. Therapeutic intervention using carbohydrate small-molecule mimetics may be beneficial in the modification of the inflammatory process.

Carbohydrate Sequence↗
PubMed1994

A human T-cell receptor recognizes 'O'-linked sugars from the hinge region of human IgA1 and IgD.

A receptor which binds secretory IgA (sIgA) is expressed on human T cells from patients with systemic lupus erythematosus, rheumatoid arthritis, Behcet's syndrome and IgA nephropathy and on normal T cells following phytohaemagglutinin (PHA) stimulation. The specificity of this receptor was initially probed with a panel of normal serum immunoglobulins in competitive inhibition assays with sIgA using two-colour immunofluorescence. While the receptor showed the strongest affinity for IgA1 (IC(50)10(-6) M), IgD which has a similarly glycosylated hinge region to IgA1, also bound to the receptor (IC50 10(-5) M). IgA2, which lacks the 'O'-glycosylated hinge region, did not significantly inhibit the binding at these concentrations suggesting that the IgA determinants for this receptor might be the oligosaccharides present in the hinge region of IgA1. IgA1 has up to 10 'O'-linked oligosaccharides and four N-linked oligosaccharides per molecule. In order to probe the role of the 'O'-linked hinge sugars in the binding event, a sugar library was prepared from IgA1 by a procedure designed to release 'O'-linked oligosaccharides preferentially, and to retain them in the natural closed ring formation. The sugars were released by hydrazinolysis at 65 degrees and the resulting oligosaccharide library analysed by high voltage paper electrophoresis (HVE) and P4 gel permeation chromatography. Competitive inhibition studies demonstrated that both the library and the individual 'O'-linked sugars associated with IgA1 were implicated in the binding of IgA1 to this receptor (IC50 between 1 x 10(-5) M and 6 x 10(-5) M). Within this range the individual sugars showed small differences in their affinity for the receptor in the following order: Gal beta 3GalNAc = NeuNAc2 alpha 3(6)Gal beta 3GalNAc > NeuNAc2 alpha 3(6)Gal beta 3[NeuNAc2 alpha 6]GalNAc > or = GalNAc.

Binding, Competitive↗
PubMed1993

Glycosylation of CD4 and Thy-1.

The site-specific glycosylation of soluble recombinant variants of human and rat CD4 (sCD4) expressed in Chinese hamster ovary (CHO) cells has been characterized. The presence of identical oligosaccharides at the conserved glycosylation site in domain 3 of rat and human sCD4 and the greater abundance of oligomannose and hybrid type glycans at the non-conserved glycosylation site of rat sCD4 clearly indicate that the protein structure influences oligosaccharide processing. Comparisons of rat sCD4 glycopeptides with mutant molecules with only single glycosylation sites and with a truncated form containing only the two NH2-terminal domains, indicate that independent processing occurs at each glycosylation site and that domain interactions can also affect oligosaccharide processing. These and other analyses of sCD2 expressed in CHO cells and Thy-1 purified from various tissues suggest that the diversity of oligosaccharide structures on a protein is regulated by the location of the glycosylation sites and the nature of the target protein, cell and tissue. The functional significance of this control remains to be determined.

Animals↗
PubMed1993

Comparative analysis of the N-glycans of rat, mouse and human Thy-1. Site-specific oligosaccharide patterns of neural Thy-1, a member of the immunoglobulin superfamily.

Protein structure and tissue type are known to influence glycosylation of proteins. We have previously investigated the N-glycans at each of the three glycosylation sites of the cell surface glycoprotein Thy-1 when isolated from rat brain and thymocytes. Here we report a comparative analysis of the site-specific N-glycosylation patterns from rat (Asn 23, 74, 98), mouse (Asn 23, 75, 99) and human (Asn 23, 60, 100) neural Thy-1. Despite considerable differences in amino acid sequence, the results show a remarkable conservation of the pattern of N-glycans at corresponding sites between the three species, as judged by chromatographic comparisons and glycosidase susceptibility. This is particularly marked for sites at Asn 74/75 in rat/mouse and the equivalent site at 60 in human Thy-1, as well as for sites at Asn 98/99 and 100, respectively. The sites at Asn 23 in rat/mouse also contained almost identical glycosylation patterns, but at this site human Thy-1 showed significantly different glycosylation patterns. These site glycosylation patterns are discussed in relation to the likely accessibility of the oligosaccharides for processing. It is known that within a species, the glycosylation of Thy-1 is tissue specific; therefore, this degree of conservation of glycosylation of Thy-1 expressed in the same tissue in different species is all the more striking, given the known variation between species in the amino acid sequence of Thy-1. It is therefore proposed that neural cells have a particular requirement for specific surface carbohydrates and that the Thy-1 polypeptide serves as an appropriate carrier for these structures.

Amino Acid Sequence↗
PubMed1992

The mapping by high-pH anion-exchange chromatography with pulsed amperometric detection and capillary electrophoresis of the carbohydrate moieties of human plasma alpha 1-acid glycoprotein.

The reducing oligosaccharides released from alpha 1-acid glycoprotein (AGP) by conventional hydrazinolysis have been analyzed by two different mapping techniques, using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and capillary electrophoresis (CE) with uv detection at 190 nm. The CE measurements proved about 4000 times more sensitive than the measurements by HPAE-PAD. The N-glycan pool was fractionated by Mono Q anion-exchange chromatography, and individual fractions so obtained were desialylated using Vibrio cholerae neuraminidase. The resulting asialo-N-glycans were further analyzed by HPAE-PAD, revealing 2 major, 4 intermediate, and 4 small peaks and at least 3 spikes, which counted for at least 13 different asialo-N-glycans. The carbohydrate structures were tentatively assigned by comparison of the Mono Q-separated N-glycans with the known AGP carbohydrate structures and known structures contained in a mapping database that allows structural assignment of N-glycans by mere comparison of retention times. In addition to the hitherto known AGP carbohydrate structures, we have tentatively identified a number of sulfated N-glycans that are currently being analyzed in more detail. We have also compared the glycan pools recovered from AGP using hydrazinolysis and glycopeptidase F (PNGase F). Approximately 40 distinct peaks could be detected in the hydrazinolysis-derived N-glycan pool by either technique (HPAE-PAD and CE), while about 30 distinct peaks were detected in the N-glycan pool derived by PNGase F digestion of the tryptic AGP digest of the same batch of AGP. These differences were attributed to an increased desialylation (approximately 3 mol%) during hydrazinolysis, based on the detection by HPAE-PAD and CE of free sialic acid and monosialylated oligosaccharides in the glycan pool derived by conventional hydrazinolysis. The integrity of the N-glycans' chitobiose core was examined by 500-MHz 1H NMR spectoscopy. The hydrazinolysis procedure could be optimized such that the hydrazinolysis-derived N-glycan pool was chromatographically essentially identical to the PNGase F-derived N-glycan pool. Hydrazinolysis proved best, with practically no loss of N-acetlylneuraminic acid and the closest resemblance to the PNGase F-derived N-glycan pool, using an automated apparatus. Notably, it was recognized that, in our hands, PNGase F digestion in the presence of sodium dodecyl sulfate resulted in partial desialylation of the liberated N-glycans.

Capillary Action↗