Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.1.53 (sialidase)
 2,694 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of ammonium on the glycosylation pattern of the recombinant immunoadhesin tumor necrosis factor-IgG (TNFR-IgG) produced by Chinese hamster ovary cells is elucidated in this study. TNFR-IgG is a chimeric IgG fusion protein bearing one N-linked glycosylation site in the Fc region and three complex-type N-glycans in the TNF-receptor portion of each monomer. The ammonium concentration of batch suspension cultures was adjusted with glutamine and/or NH(4)Cl. The amount of galactose (Gal) and N-acetylneuraminic acid (NANA) residues on TNFR-IgG correlated in a dose-dependent manner with the ammonium concentration under which the N-linked oligosaccharides were synthesized. As ammonium increased from 1 to 15 mM, a concomitant decrease of up to 40% was observed in terminal galactosylation and sialylation of the molecule. Cell culture supernatants contained measurable beta-galactosidase and sialidase activity, which increased throughout the culture. The beta-galactosidase, but not the sialidase, level was proportional to the ammonium concentration. No loss of N-glycans was observed in incubation studies using beta-galactosidase and sialidase containing cell culture supernatants, suggesting that the ammonium effect was biosynthetic and not degradative. Several biosynthetic mechanisms were investigated. Ammonium (a weak base) is known to affect the pH of acidic intracellular compartments (e.g., trans-Golgi) as well as intracellular nucleotide sugar pools (increases UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine). Ammonium might also affect the expression rates of beta1, 4-galactosyltransferase (beta1,4-GT) and alpha2,3-sialyltransferase (alpha2,3-ST). To separate these mechanisms, experiments were designed using chloroquine (changes intracellular pH) and glucosamine (increases UDP-GNAc pool [sum of UDP-GlcNAc and UDP-GalNAc]). The ammonium effect on TNFR-IgG oligosaccharide structures could be mimicked only by chloroquine, another weak base. No differences in N-glycosylation were found in the product synthesized in the presence of glucosamine. No differences in beta1, 4-galactosyltransferase (beta1,4-GT) and alpha2,3-sialyltransferase (alpha2,3-ST) messenger RNA (mRNA) and enzyme levels were observed in cells cultivated in the presence or absence of 13 mM NH(4)Cl. pH titration of endogenous CHO alpha2,3-ST and beta-1,4-GT revealed a sharp optimum at pH 6.5, the reported trans-Golgi pH. Thus, at pH 7.0 to 7.2, a likely trans-Golgi pH range in the presence of 10 to 15 mM ammonium, activities for both enzymes are reduced to 50% to 60%. Consequently, ammonium seems to alter the carbohydrate biosynthesis of TNFR-IgG by a pH-mediated effect on glycosyltransferase activity.
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PMID:Ammonium alters N-glycan structures of recombinant TNFR-IgG: degradative versus biosynthetic mechanisms. 1079 88

Severe neurological deficits and mental retardation are frequently associated with disrupted ganglioside metabolism in a variety of gangliosidoses and lysosomal storage disorders. Accumulation of glycosphingolipids (GSLs) in the central nervous system (CNS) of humans and animals affected with several types of mucopolysaccharidoses (MPS) also correlates with the severity of neurological dysfunction. Mucopolysaccharidosis type IIID (MPS IIID) is characterized by deficiency in lysosomal N-acetylglucosamine 6-sulfatase activity and the accumulation and excretion of heparan sulfates and N-acetylglucosamine 6-sulfate. We investigated the metabolism of GSLs in the prenatal, neonatal, and adult MPS IIID caprine brains and an MPS experimental cell culture model. The amounts of total glycolipids in prenatal, neonatal, and adult MPS IIID caprine brains were about 2-fold higher than those in control samples. GM3, GD3, and lactosyl ceramide were the principal GSLs which abnormally accumulated in caprine MPS IIID brains. These changes may be, in part, due to the reduction of sialidase and UDP-N-acetylgalactosamine:GM3 N-acetylgalactosaminyltransferase (GalNAc-T) activities in MPS IIID caprine brain. To further examine the possible mechanism of GSL accumulation in MPS IIID brains, we employed a cell culture model using suramin-treated neuronal cultures of differentiated P19 cells. HPTLC analysis showed elevated GSLs in suramin-treated cells. Metabolic pulse-chase labeling study revealed that the GSL accumulation in suramin-treated cells may be attributed to both disturbed biosynthesis and significantly slower degradation of GSLs. In addition, the consistency of observations in the cell culture and caprine models supports the cell culture system as a means of evaluating GSL metabolic perturbations.
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PMID:Metabolic studies of glycosphingolipid accumulation in mucopolysaccharidosis IIID. 1124 30