Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0393754 (HSA)
 2,996 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

HPLC analyses of GM-CSF in solution mixtures containing both GM-CSF and HSA showed losses of GM-CSF which could not be accounted for using conventional electrophoretic and/or RP-HPLC techniques. Further investigation of these mixtures by immunoblotting and by immunoaffinity chromatography demonstrated the presence of high molecular weight (> 67,000 GM-CSF related species. No such species was detectable in solutions of GM-CSF alone. This experiment pointed to the formation of an adduct between GM-CSF and HSA in the solution mixtures. To probe further the hypothesis of a GM-CSF/HSA adduct, an immunologically based test was conceived which could react only with this type of hybrid molecule. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed using two antibodies, anti-GM-CSF (capture antibody) and anti-HSA (detection antibody), as part of the quantitation of GM-CSF/HSA adducts. After confirming its existence by ELISA, a GM-CSF/HSA adduct was isolated from the solution mixture containing both GM-CSF and HSA. This isolate served as a primary reference standard in the ELISA assay. The immunoassay has a subnanogram sensitivity and is highly specific for GM-CSF/HSA adducts in the presence of either free GM-CSF or free HSA. As a verification, conjugates of GM-CSF/HSA were synthesized using a cross-linking reagent. These covalent conjugates reacted positively in the ELISA and are employed as a convenient alternative reference standard.
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PMID:An enzyme-linked immunosorbent assay (ELISA) for quantitation of adducts of granulocyte-macrophage colony stimulating factor (GM-CSF) and human serum albumin (HSA) in stressed solution mixtures. 800

Soluble MHC antigens are detected in body fluids but their role and origin are still unclear. This study examined whether serum IA antigens, isolated from BALB/c mice (sIA(d)), could modulate the immune response. Specific purification procedures isolated intact IA molecules, which were thereafter applied to functional assays. Thus, sIA(d) were shown to stimulate spleen cell proliferation and the major target was identified to be the CD4(+) cell population. Inhibition of the CD4 co-receptor using specific neutralizing antibodies destroyed the sIA(d)-mediated proliferative activity, while sIA(d) successfully antagonized surface IA(d) antigens for binding to anti-IA(d) antibody. Serum-IA(d) stimulated BALB/c versus C3H/HeN but not C3H/HeN versus BALB/c mixed lymphocyte reactions, while increasing responsiveness to Legionella pneumophila. However, sIA(d) displayed an inhibitory activity during the effector phase of the humoral response, since they inhibited the anti-DNP-specific IgM production to a DNP-HSA hapten-carrier system. Furthermore, sIA(d) molecules increased Th1/Th2 cytokines during the L. pneumophila stimulus, while decreasing IL-2, GM-CSF and increasing IL-4, IL-15 during the DNP-HSA stimulus. These results suggest that sIA(d), following steps similar to surface class II antigen binding mechanisms, stimulate the initiation of a humoral or cellular immune response but rather inhibit the effector phase of the reactions, attributing thus soluble class II MHC antigens important immunomodulatory roles.
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PMID:Serum-derived MHC class II molecules: potent regulators of the cellular and humoral immune response. 1945 70

Glycoproteins secreted by the yeast Kluyveromyces lactis are usually modified by the addition at asparagines-linked glycosylation sites of heterogeneous mannan residues. The secreted glycoproteins in K. lactis that become hypermannosylated will bear a non-human glycosylation pattern and can adversely affect the half-life, tissue distribution and immunogenicity of a therapeutic protein. Here, we describe engineering a K. lactis strain to produce non-hypermannosylated glycoprotein, decreasing the outer-chain mannose residues of N-linked oligosaccharides. We investigated and developed the method of two-step homologous recombination to knockout the OCH1 gene, encoding alpha1,6-mannosyltransferase and MNN1 gene, which is homologue of Saccharomyces cerevisiae MNN1, encoding a putative alpha1,3-mannosyltransferase. We found the Kloch1 mutant strain has a defect in hyperglycosylation, inability in adding mannose to the core oligosaccharide. The N-linked oligosaccharides assembled on a secretory glycoprotein, HSA/GM-CSF in Kloch1 mutant, contained oligosaccharide Man(13-14)GlcNAc(2), and in Kloch1 mnn1 mutant, contained oligosaccharide Man(9-11)GlcNAc(2), whereas those in the wild-type strain, consisted of oligosaccharides with heterogeneous sizes, Man(>30)GlcNAc(2). Taken together, these results indicated that KlOch1p plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in K. lactis. The KlMnn1p, was proved to be certain contribution to the outer hypermannosylation, most possibly encodes alpha1,3-mannosyltransferase. Therefore, the Kloch1 and Kloch1 mnn1 mutants can be used as a foundational host to produce glycoproteins lacking the outer-chain hypermannoses and further maybe applicable to be a promising system for yeast therapeutic protein production.
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PMID:Disruption of the OCH1 and MNN1 genes decrease N-glycosylation on glycoprotein expressed in Kluyveromyces lactis. 1955 61

Glycosylation is vital for activity, higher structure and function of protein. Glycoproteins derived from yeast contain N-glycan of high mannose type and are usually hyperglycosylated, while those from mammalian cells contain N-glycan of hybrid or complex type. We introduced the alpha-1,2-mannosidase I (MDSI) into yeast cells, which catalyzed an essential proceeding of N-glycan structures from Man8GlcNAc2 to Man5GlcNAc2. The plasmids contained MDSI genes from Homo sapiens [HMDSI(delta185)] or Arabidopsis thaliana [ATMDSI(delta48)], and three ER-signals were used to be transformed a mutant Pichia pastoris GJK01, respectively. The reporter protein HSA/GM-CSF (human serum albumin and granulocyte-macrophage colony stimulating factor fusion protein) was expressed and its N-glycans were analyzed by DSA-FACE (DNA sequencer assisted fluorophore-assisted carbohydrate electrophoresis). The plasmid contained ER-ScMnsI-ATMDSI(delta48) was expressed in Pichia pastoris, the Man5GlcNAc2 N-glycan on secreted glycoprotein HSA/GM-CSF was observed. The research reported here provided basic substrate to obtain the hybrid- and complex-type glycans in mammalian cell.
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PMID:[Construction of yeast Pichia pastoris to produce Man5GlcNAc2 mammalian mannose-type glycoprotein]. 2155 96