Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

A monoclonal antibody (mAb; A10) raised against murine Ehrlich tumor cell surface carbohydrates was tested for reactivity with human normal and malignant tissues. A10 reacted strongly, with a high proportion of adenocarcinomas arising from colon and other tissues but not with breast carcinomas or other malignant tumors. Normal tissues were virtually A10 unreactive, except for the duct cells from breast and pancreas and some bronchial mucosae. Ultrastructural studies showed mAb A10 immunolabeling of both microvilli and mucin droplets in colon cancer cells but not in normal absorptive or globet cells. A10 reacted strongly with mucin-enriched fractions from colon cancer tissues and HT-29 xenografts but not from normal colon tissues. A10 epitope was carried on MUC1 derived from colon adenocarcinomas and probably on other mucin species, although not on MUC2 molecules. A10 epitope was resistant to exoglycosidases and periodate oxidation but sensitive to the Smith's degradation and beta-elimination, suggesting the involvement of O-linked carbohydrates in nonterminal reducing positions. A mucin-type glycosidic linkage was supported because of the lack of A10 reactivity with HT-29 cells grown with phenyl-N-acetyl-alpha-D-galactosaminide. Deglycosylation studies with trifluoromethanesulfonic acid pointed to the involvement of core mucin glycans in the A10 epitope. This epitope was resistant to protease, O- and N-glycanase treatments carried out on trifluoromethanesulfonic acid-deglycosylated mucins. Inhibition studies with core 1, core 2, core 3, and core 6 suggested the latter [GlcNAcbeta(1-6)GalNAc] as being involved in A10 epitope. Taken together, the present results point to A10 defining a core 6-related epitope on core mucin glycans expressed by colon cancer MUC1 not previously associated with human cancer.
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PMID:Human colon adenocarcinomas express a MUC1-associated novel carbohydrate epitope on core mucin glycans defined by a monoclonal antibody (A10) raised against murine Ehrlich tumor cells. 1007 Sep 64

Haemagglutinin (HA) activity of Clostridium botulinum type A 19S and 16S toxins (HA-positive progenitor toxin; HA(+)-PTX) was characterized. HA titres against human erythrocytes of HA(+)-PTX were inhibited by the addition of lactose, D-galactose, N-acetyl-D-galactosamine and D-fucose to the reaction mixtures. A direct glycolipid binding test demonstrated that type A HA(+)-PTX strongly bound to paragloboside and some neutral glycolipids, but did not bind to gangliosides. Type A HA(+)-PTX also bound to asialoglycoproteins (asialofetuin, neuraminidase-treated transferrin), but not to sialoglycoproteins (fetuin, transferrin). Although glycopeptidase F treatment of asialofetuin abolished the binding of HA(+)-PTX, endo-alpha-N-acetylgalactosaminidase treatment did not. Thus these results can be interpreted as indicating that type A HA(+)-PTX detects and binds to Gal beta 1-4GlcNAc in paragloboside and the N-linked oligosaccharides of glycoproteins. Regardless of neuraminidase treatment, type A HA(+)-PTX bound to glycophorin A which is a major sialoglycoprotein on the surface of erythrocytes. Both native glycophorin A and neuraminidase-treated glycophorin A inhibited the binding of erythrocytes to type A HA(+)-PTX. Since the N:-linked oligosaccharide of glycophorin A is di-branched and more than 50% of this sugar chain is monosialylated, type A HA(+)-PTX probably bound to the unsialylated branch of the N-linked oligosaccharide of glycophorin A and agglutinated erythrocytes. One subcomponent of HA, designated HA1, did not agglutinate native erythrocytes, although it did bind to erythrocytes, paragloboside and asialoglycoproteins in a manner quite similar to that of HA(+)-PTX. These results indicate that type A HA(+)-PTX binds to oligosaccharides through HA1.
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PMID:Clostridium botulinum type A haemagglutinin-positive progenitor toxin (HA(+)-PTX) binds to oligosaccharides containing Gal beta1-4GlcNAc through one subcomponent of haemagglutinin (HA1). 1128 77

The lectin from the mushroom Polysporus squamosus (PSL) has an extended carbohydrate combining site, which exhibits a high specificity and affinity toward the NeuAc5alpha2,6Galbeta1,4Glc/GlcNAc trisaccharide sequence of asparagine-linked oligosaccharides. Therefore, PSL should be a superior reagent to the lectin from Sambucus nigra (SNA), which does not discriminate between alpha2,6-linked NeuAc5 present either in asparagine- or serine/threonine-linked oligosaccharides. We have prepared a digoxigenin-conjugated PSL and applied it for histochemistry and blotting. We observed a more restricted staining pattern by PSL as compared to SNA in paraffin sections from different rat organs. Pretreatment of sections with N-glycanase F abolished PSL staining indicating that it interacts only with asparagine-linked oligosaccharides. Furthermore, PSL staining was neuraminidase sensitive. In contrast, SNA staining was only partially sensitive to N-glycanase F pretreatment demonstrating that it was in part due to alpha2,6-linked NeuAc5 present in serine/threonine-linked oligosaccharides. The most striking observation in this regard was that PSL, in contrast to SNA, did not stain the mucus of sheep submandibular gland, which is extremely rich in serine/threonine-linked Neu5Acalpha2,6N-acetylgalactosamine. Furthermore, in some tissues neuraminidase pretreatment resulted in increased intensity of SNA staining probably due to binding to exposed terminal N-acetylgalactosamine residues. Collectively, these results indicate that PSL is a useful tool for the histochemical detection of alpha2,6-linked NeuAc5 in asparagine-linked oligosaccharides.
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PMID:Application of a lectin from the mushroom Polysporus squamosus for the histochemical detection of the NeuAcalpha2,6Galbeta1,4Glc/GlcNAc sequence of N-linked oligosaccharides: a comparison with the Sambucus nigra lectin. 1168 46

Bovine binucleate trophoblast giant cells (BNCs) produce large amounts of PAS-positive cytoplasmic granules. After fusion of BNCs with uterine epithelial cells, the contents of these granules are released into the maternal stroma which underlies the uterine epithelium. Histochemically, the granules can be labeled with N-acetylgalactosamine-specific lectins ( Dolichos biflorus, Vicia villosa, and Wisteria floribunda agglutinins) and with Phaseolus vulgaris leucoagglutinin. In this study, we used lectin western blot analysis of proteins from fetal cotyledons to characterize the lectin binding glycoproteins. Lectin western blots showed several bands. A main band of approximately 65 kDa was identified as pregnancy-associated glycoproteins (PAGs) and a double band at 34-35 kDa as prolactin-related protein-I (PRP-I) by their crossreactivity with specific antisera. Enzymatic cleavage of N-linked glycans with peptide- N-glycanase F abolished the lectin binding to PRP and PAGs in western blots, revealing that the lectins bound to asparagine-linked glycans. The high specificity of the lectins was used for the enrichment of PRP-I and PAGs from placental cotyledons with Vicia villosa lectin affinity chromatography. The occurrence of the relatively uncommon asparagine-linked N-acetylgalactosaminyl glycans on secretory proteins of the BNCs suggests a functional role of this specific glycosylation pattern.
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PMID:In bovine binucleate trophoblast giant cells, pregnancy-associated glycoproteins and placental prolactin-related protein-I are conjugated to asparagine-linked N-acetylgalactosaminyl glycans. 1264 35

Our previous studies showed that expression of the GalNAcbeta1-->4GlcNAc group on N-linked oligosaccharides is associated with functional differentiation of the bovine mammary gland. In the present study, the occurrence of the GalNAcbeta1-->4GlcNAc group was established in human milk proteins and membrane glycoproteins from a human breast cancer cell line, MRK-nu-1, by structural analysis of oligosaccharides released by hydrazinolysis. Whether the expression level of the disaccharide group is affected upon malignant transformation was examined in human breast cancer specimens using Wistaria floribunda agglutinin (WFA) which interacts with oligosaccharides with N-acetylgalactosamine at their termini. Lectin blot analysis of membrane glycoprotein samples from human breast cancer specimens showed that the number of protein bands reacting with WFA, as well as their intensities, are lower in samples from primary carcinoma lesions compared with samples from surrounding normal tissues. No lectin binding was observed when the blots were treated with jack bean beta-N-acetylhexosaminidase or N-glycanase, indicating that WFA-reactive oligosaccharides are N-linked. A histochemical study of tissue specimens from 92 patients with breast cancer revealed that the reduced WFA staining levels in primary carcinoma lesions correlate with advancing clinical stages and prognostic status (i.e., 58% of patients in a group showing reduced/negative staining died of disease recurrence, whereas more than 90% of those in the positive staining group survived for 5 years after surgery). These results indicate that reduced expression of beta-N-acetylgalactosaminylated N-linked oligosaccharides on primary carcinoma lesions predicts a poor prognosis for patients with breast cancer.
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PMID:Prognostic significance of reduced expression of beta-N-acetylgalactosaminylated N-linked oligosaccharides in human breast cancer. 1271 46

We found a novel human glycosyltransferase gene carrying a hypothetical beta1,4-glycosyltransferase motif during a BLAST search, and we cloned its full-length open reading frame by using the 5'-rapid amplification of cDNA ends method. It encodes a type II transmembrane protein of 999 amino acids with homology to chondroitin sulfate synthase in its C-terminal region (GenBank accession number AB089940). Its putative orthologous gene was also found in mouse (accession number AB114826). The truncated form of the human enzyme was expressed in HEK293T cells as a soluble protein. The recombinant enzyme transferred GalNAc to GlcNAc beta-benzyl. The product was deduced to be GalNAc beta 1-4GlcNAc beta-benzyl based on mass spectrometry and NMR spectroscopy. We renamed the enzyme beta1,4-N-acetylgalactosaminyltransferase-III (beta 4GalNAc-T3). beta 4GalNAc-T3 effectively synthesized N,N'-diacetylgalactosediamine, GalNAc beta 1-4GlcNAc, at non-reducing termini of various acceptors derived not only from N-glycans but also from O-glycans. Quantitative real time PCR analysis showed that its transcript was highly expressed in stomach, colon, and testis. As some glycohormones contain N,N'-diacetylgalactosediamine structures in their N-glycans, we examined the ability of beta 4GalNAc-T3 to synthesize N,N'-diacetylgalactosediamine structures in N-glycans on a model protein. When fetal calf fetuin treated with neuraminidase and beta1,4-galactosidase was utilized as an acceptor protein, beta 4GalNAc-T3 transferred GalNAc to it. Furthermore, the majority of the signal from GalNAc disappeared on treatment with glycopeptidase F. These results suggest that beta 4GalNAc-T3 could transfer GalNAc residues, producing N,N'-diacetylgalactosediamine structures at least in N-glycans and probably in both N- and O-glycans.
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PMID:Molecular cloning and characterization of a novel human beta 1,4-N-acetylgalactosaminyltransferase, beta 4GalNAc-T3, responsible for the synthesis of N,N'-diacetyllactosediamine, galNAc beta 1-4GlcNAc. 1296 86

Tomato spotted wilt virus (TSWV, Genus: Tospovirus, Family: Bunyaviridae) is a major constraint to the production of several different crops of agronomic and horticultural importance worldwide. The amino acid sequence of the two envelope membrane glycoproteins, designated as G(N) (N-terminal) and G(C) (C-terminal), of TSWV contain several tripeptide sequences, Asn-Xaa-Ser/Thr, suggesting that the proteins are N-glycosylated. In this study, the lectin-binding properties of the viral glycoproteins and their sensitivities to glycosidases were examined to obtain information on the nature of potential oligosaccharide moieties present on G(N) and G(C). The viral proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed by affinoblotting using a battery of biotinylated lectins with specificity to different oligosaccharide structures. G(C) showed strong binding with five mannose-binding lectins, four N-acetyllactosamine-binding lectins and one fucose-binding lectin. G(N) was resolved into two molecular masses and only the slow migrating form showed binding, albeit to a lesser extent than G(C), with three of the five mannose-binding lectins. The N-acetyllactosamine- and fucose-specific lectins did not bind to either molecular mass form of G(N). None of the galactose-, N-acetylgalactosamine-, or sialic acid-binding lectins tested showed binding specificity to G(C) or G(N). Treatment of the denatured virions with endoglycosidase H and peptide:N-glycosidase F (PNGase F) resulted in a significant decrease in the binding of G(C) to high mannose- and N-acetyllactosamine-specific lectins. However, no such differences in lectin binding were apparent with G(N). These results indicate the presence of N-linked oligosaccharides of high mannose- and complex-type on G(C) and possibly high mannose-type on G(N). Differences in the extent of binding of the two envelope glycoproteins to different lectins suggest that G(C) is likely to be more heavily N-glycosylated than G(N). No evidence was observed for the presence of O-linked oligosaccharides on G(N) or G(C).
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PMID:The two envelope membrane glycoproteins of Tomato spotted wilt virus show differences in lectin-binding properties and sensitivities to glycosidases. 1496 92

Sex-specific gene expression by Schistosoma mansoni worms has been demonstrated at the transcriptome as well as the proteome levels. In view of the important role of glycans in the biology of schistosomes and the interaction with their human host, we have investigated the sex-specific protein glycosylation. Mass spectrometric profiling and structural characterization of PNGase F-released N-glycans revealed the following gender-specific glycosylation patterns: Complex-type N-glycans of females mainly carried Gal(beta1-4)GlcNAc (LacNAc) and Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc (Lewis x) antennae structures, whereas GalNAc(beta1-4)GlcNAc- (LacdiNAc; LDN) and GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc (LDN-F) were prevalent in N-glycans from males. LDN(-F) motifs were found to occur as repeats on the antennae of large N-glycans that contained up to seven LDN(-F) units. The female complex-type glycans were mostly di-antennary and tri-antennary, whereas male structures were predominantly of the mono-antennary and di-antennary type. Oligomannosidic N-glycans were expressed at similar levels in females and males. The localization of the sex-biased glycan motifs was studied by immunofluorescence microscopy using defined anti-glycan monoclonal antibodies (mAbs). The Lewis x element was strongly expressed in the gut of both males and females, but with respect to tegument localization, the females expressed this structure, while Lewis x seemed to be almost completely absent from the male tegument. The expression of LDN-F was predominantly detected in the parenchyma of both male and female worms as well as in the tegument of the male ventral cavity facing the female. LDN was detected in the tegument of male and female worms at similar levels. The sex-specific expression and differential localization of these antigenic glycan motifs in schistosomes may play a role in male-female interactions during conjugal biology and may lead to a differential immune reaction of the host to the two sexes.
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PMID:Gender-specific expression of complex-type N-glycans in schistosomes. 1682 88

Hepatic asialoglycoprotein receptor, which may mediate the clearance of circulating thyroglobulin, is known to have a high affinity for GalNAc. Recently, the receptor has been reported to be present also in the thyroid, implicating interaction with thyroglobulin. Here, mammalian thyroglobulins were analyzed for GalNAc termini by Western blotting with GalNAc-recognizing lectins labeled with peroxidase or (125)I. Wistaria floribunda lectin was found to bind human thyroglobulin and, to some extent, bovine, but not porcine thyroglobulin. After desialylation, the lectin bound all of the thyroglobulins tested. The binding was inhibited by competitive inhibitor GalNAc. Peptide N-glycanase treatment of human desialylated thyroglobulin resulted in the complete loss of reactivity with W. floribunda lectin, indicating that the binding sites are exclusively on N-glycans. The binding sites on human desialylated thyroglobulin were partly sensitive to beta-galactosidase, and the remainder was essentially sensitive to beta-N-acetylhexosaminidase. On the other hand, the binding sites of bovine and porcine desialylated thyroglobulins were totally sensitive to beta-galactosidase. Thus the lectin binds beta-Gal termini, as well as beta-GalNAc. GalNAc-specific Dolichos biflorus lectin also bound human thyroglobulin weakly. In contrast to W. floribunda lectin, desialylation diminished binding, suggesting that these two lectins recognize different GalNAc-terminated structures. Again, the binding was inhibited by GalNAc and by treatment with peptide N-glycanase. These results strongly indicate the presence of distinct GalNAc termini of N-glycans on human thyroglobulin.
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PMID:Presence of beta-linked GalNAc residues on N-glycans of human thyroglobulin. 1709 89

The mutant beta1,4-galactosyltransferase (beta4Gal-T1), beta4Gal-T1-Y289L, in contrast to wild-type beta4Gal-T1, can transfer GalNAc from the sugar donor UDP-GalNAc to the acceptor, GlcNAc, with efficiency as good as that of galactose from UDP-Gal. Furthermore, the mutant can also transfer a modified sugar, C2 keto galactose, from its UDP derivative to O-GlcNAc modification on proteins that provided a functional handle for developing a highly sensitive chemoenzymatic method for detecting O-GlcNAc post-translational modification on proteins. We report herein that the modified sugar, C2 keto galactose, can be transferred to free GlcNAc residues on N-linked glycoproteins, such as ovalbumin or asialo-agalacto IgG1. The transfer is strictly dependent on the presence of both the mutant enzyme and the ketone derivative of the galactose. Moreover, the PNGase F treatment of the glycoproteins, which cleaves the N-linked oligosaccharide chain, shows that the modified sugar has been transferred to the N-glycan chains of the glycoproteins and not to the protein portion. The application of the mutant galactosyltransferase, beta4Gal-T1-Y289L, to produce glycoconjugates carrying sugar moieties with reactive groups, is demonstrated. We envision a broad potential for this technology such as the possibilities to link cargo molecules to glycoproteins, such as monoclonal antibodies, via glycan chains, thereby assisting in the glycotargeting of drugs to the site of action or used as biological probes.
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PMID:Direct identification of nonreducing GlcNAc residues on N-glycans of glycoproteins using a novel chemoenzymatic method. 1737 Sep 97


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