Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Antibodies against Leishmania major wheat germ agglutinin-binding glycoproteins were used to select from a genomic lambda gt11 expression library a clone coding for a L. major glycoprotein. The partial DNA sequence indicated the presence of a mosaic of repetitive sequences. Southern blot hybridisation on genomic DNA using the cloned gene as a probe at high stringency suggested a single gene, which was localised to chromosome band 18. Northern blot analysis of L. major mRNA detected a major transcript of 7.5 kb and a minor 4.0-kb transcript. Antibodies affinity-purified on the fusion protein identified a complex of two water-soluble cytoplasmic polypeptides of approximately 96 kDa and 92 kDa in L. major promastigotes and amastigotes. They also recognised polypeptides in other Leishmania species, in Crithidia lucilliae and very weakly in Leptomonas. The apparent molecular weight of these polypeptides, while conserved within each species, varied between species. A peptide map of the two polypeptides from L. major generated an identical pattern suggesting a close relatedness at the protein level. This protein complex was not hydrolysed by N-glycanase and was not affected by tunicamycin, but treatment with anhydrous hydrogen fluoride suggested that it is O-glycosylated. The glycan moiety appears to be N-acetylglucosamine, and N-acetylglucosamine beta-1,4-galactosyltransferase was capable of adding [3H]galactose to it. This was susceptible to beta elimination and beta-galactosidase treatment. Taken together, the data indicates that gp96/92 belongs to the newly described class of cytoplasmic and nuclear glycoproteins containing O-linked N-acetylglucosamine.
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PMID:Identification, characterisation and genomic cloning of a O-linked N-acetylglucosamine-containing cytoplasmic Leishmania glycoprotein. 811 27

Therapeutic glycoproteins produced in different host cells by recombinant DNA technology often contain terminal GlcNAc and Gal residues. Such glycoproteins clear rapidly from the serum as a consequence of binding to the mannose receptor and/or the asialoglycoprotein receptor in the liver. To increase the serum half-life of these glycoproteins, we carried out in vitro glycosylation experiments using TNFR-IgG, an immunoadhesin molecule, as a model therapeutic glycoprotein. TNFR-IgG is a disulfide-linked dimer of a polypeptide composed of the extracellular portion of the human type 1 (p55) tumor necrosis factor receptor (TNFR) fused to the hinge and Fc regions of the human IgG(1) heavy chain. This bivalent antibody-like molecule contains four N-glycosylation sites per polypeptide, three in the receptor portion and one in the Fc. The heterogeneous N-linked oligosaccharides of TNFR-IgG contain sialic acid (Sia), Gal, and GlcNAc as terminal sugar residues. To increase the level of terminal sialylation, we regalactosylated and/or resialylated TNFR-IgG using beta-1,4-galactosyltransferase (beta1,4GT) and/or alpha-2,3-sialyltransferase (alpha2,3ST). Treatment of TNFR-IgG with beta1,4GT and UDP-Gal, in the presence of MnCl(2), followed by MALDI-TOF-MS analysis of PNGase F-released N-glycans showed that the number of oligosaccharides with terminal GlcNAc residues was significantly decreased with a concomitant increase in the number of terminal Gal residues. Similar treatment of TNFR-IgG with alpha2,3ST and CMP-sialic acid (CMP-Sia), in the presence of MnCl(2), produced a molecule with an approximately 11% increase in the level of terminal sialylation but still contained oligosaccharides with terminal GlcNAc residues. When TNFR-IgG was treated with a combination of beta1,4GT and alpha2,3ST (either in a single step or in a stepwise fashion), the level of terminal sialylation was increased by approximately 20-23%. These results suggest that in vitro galactosylation and sialylation of therapeutic glycoproteins with terminal GlcNAc and Gal residues can be achieved in a single step, and the results are similar to those for the stepwise reaction. This type of in vitro glycosylation is applicable to other glycoproteins containing terminal GlcNAc and Gal residues and could prove to be useful in increasing the serum half-life of therapeutic glycoproteins.
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PMID:Glycoengineering of therapeutic glycoproteins: in vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues. 1146 48

Lectin blot analysis of membrane glycoprotein samples from Sf-9 cells upon transfection of individual human beta-1,4-galactosyltransferase (beta-1,4-GalT) I, II, III, IV, V et VI cDNAs showed that the endogenous N-linked oligosaccharides are galactosylated (Guo et al., Glycobiology (2001), in press). Further analysis revealed that membrane glycoprotein samples from all the gene-transfected cells are also reactive to Lycopersicon esculentum agglutinin (LEA) et Datura stramonium agglutinin (DSA), both of which bind to oligosaccharides with poly-N-acetyllactosamine chains while no lectin reactive protein bands are detected when blots are pretreated with a mixture of diplococcal beta-1,4-galactosidase et jack bean beta-N-acetylhexosaminidase or N-glycanase. Analysis of endo-beta-galactosidase-digestion products revealed the presence of the Gal1-->GlcNAc1-->Gal and/or GlcNAc1-->Gal structures in the gene-transfected cells. When the homogenates of the gene-transfected cells were used as enzyme sources towards oligosaccharides with the GlcNAc beta 1-->(3Gal beta 1-->4GlcNAc)(1-3) structures, human recombinant beta-1,4-GalTs I et II galactosylated these oligosaccharides more effectively than other beta-1,4-GalTs. These results indicate that beta-1,4-GalTs I-VI can synthesize poly-N-acetyllactosamine chains with beta-1,3-N-acetylglucosaminyltransferase.
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PMID:Occurrence of poly-N-acetyllactosamine synthesis in Sf-9 cells upon transfection of individual human beta-1,4-galactosyltransferase I, II, III, IV, V and VI cDNAs. 1153 Feb 3

Several studies showed that Sf-9 cells can synthesize the galactosylated N-linked oligosaccharides if beta-1,4-galactosyltransferase (beta-1,4-GalT) is supplied. The full-length human beta-1,4-GalT I, II, III, IV, V, and VI cDNAs were independently transfected into Sf-9 cells, and the galactosylation of endogenous membrane glycoproteins was examined by lectin blot analysis using Ricinus communis agglutinin-I (RCA-I), which preferentially interacts with oligosaccharides terminated with Galbeta1-->4GlcNAc group. Several RCA-I-reactive bands appeared in all of the gene-transfected cells, and disappeared on treatment of blots with beta-1,4-galactosidase or N-glycanase prior to incubation with lectin. Introduction of the antisense beta-1,4-GalT II and V cDNAs separately into human colorectal adenocarcinoma SW480 cells, in which beta-1,4-GalT I, II, and V genes were expressed, resulted in the reduction of RCA-I binding toward N-linked oligosaccharides of the membrane glycoproteins. Differences were found in their K(m) values toward UDP-Gal and GlcNAcbeta-S-pNP and in their acceptor specificities toward oligosaccharides with the GlcNAcbeta1-->4(GlcNAcbeta1-->2)Man branch and with the GlcNAcbeta1-->6(GlcNAcbeta1-->2)Man branch. These results indicate that beta-1,4-GalTs II, III, IV, V, and VI are involved in the N-linked oligosaccharide biosynthesis cooperatively but not in a redundant manner with beta-1,4-GalT I within cells.
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PMID:Galactosylation of N-linked oligosaccharides by human beta-1,4-galactosyltransferases I, II, III, IV, V, and VI expressed in Sf-9 cells. 1158 57