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)

The contribution of N-linked carbohydrate to the complement-inhibitory function of the human erythrocyte membrane glycoprotein, CD59, was investigated. Amino acid sequence analysis of tryptic peptides labeled with [3H]borohydride revealed an N-linked carbohydrate moiety at the Asn18 residue. No O-linked carbohydrate was detected, as judged by the failure of asialo-CD59 to bind peanut agglutinin and by its resistance to digestion by O-glycanase. The apparent molecular mass of CD59 was reduced from 18-20 to 14 kDa upon complete digestion with N-glycanase, with no detectable proteolysis. N-glycanase digestion of CD59 was associated with an 88 +/- 4% loss of the complement-inhibitory activity of the protein, as assessed by its capacity to protect chicken erythrocytes from lysis by the human C5b-9 proteins. By contrast, no change in function was observed after digestion of CD59 with neuraminidase, under conditions that removed greater than 60% of [3H]sialic acid residues. Despite loss of functional activity after N-glycanase digestion, we detected no change in the capacity of the deglycosylated CD59 to incorporate into erythrocyte membranes or to bind specifically and with species selectivity to the C8 and C9 components of the membrane attack complex. In order to alter the branched-chain structure of the N-linked carbohydrate of CD59 without enzymatic digestion, Chinese hamster ovary (CHO) cells transfected with cDNA for human CD59 were grown in the alpha-mannosidase inhibitor, 1-deoxymannojirimycin, resulting in conversion of approximately 70% of the membrane glycoprotein to a high mannose. When grown in the presence of 1-deoxymannojirimycin, the C5b-9-inhibitory activity of CD59 expressed on the surface of the transfected CHO cells was reduced by an amount comparable to that observed for the N-glycanase digested protein. Taken together, these data suggest that normal glycosylation of Asn18 in CD59 is required for the normal expression of its complement-inhibitory activity on membrane surfaces, although these N-linked sugar residues do not contribute to CD59's affinity for the C8 and C9 components of the C5b-9 complex.
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PMID:Contribution of the N-linked carbohydrate of erythrocyte antigen CD59 to its complement-inhibitory activity. 137 27

Carcinoembryonic antigen, an apical membrane glycoprotein expressed in normal human colonic epithelial cells, colonic polyps, tumor, and tissue culture cell lines originating from colonic adenocarcinomas, is generally considered to have a molecular weight of 180,000. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis associated with immunoprecipitation or immunoblotting with both monoclonal (Mab 517 and Mab 601) and polyclonal antibodies, we observed that carcinoembryonic antigen was actually expressed as two discrete apparent molecular weight forms in normal tissues: a broad band averaging at Mr 200,000 and a sharp band at Mr 130,000. This constituted the phenotype of the normal colon. In cancer cells we detected a single band at Mr 170,000 or lower. This variation was mainly the consequence of a modification of the glycosylation pattern of the molecule since deglycosylation by N-glycanase or biosynthesis in the presence of tunicamycin always produced a single molecular weight form, whether or not the source of tissue was normal or cancerous. By close inspection of benign, moderately transformed, and carcinomatous human colonic polyps we noticed that this shift in the molecular weight of carcinoembryonic antigen preceded the detection of other cancer markers such as nonspecific cross-reacting antigen at Mr 95,000 or the histological modifications leading to malignant diagnosis. Carcinoembryonic antigen constitutes, therefore, an important model with which to study the modifications of the glycosylation pattern induced during cancer biogenesis.
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PMID:Carcinoembryonic antigen has a different molecular weight in normal colon and in cancer cells due to N-glycosylation differences. 171 52

We previously described the production of monoclonal antibodies against a preparation of membrane glycoproteins from human brain [Berglund et al. (1987) J. Neurochem. 48, 809-815]. One of the glycoproteins, recognized by monoclonal antibody CF3, was specifically expressed in the brain. We now report the isolation and characterization of this glycoprotein, called glycoprotein 135 (Gp135). Gp135 was purified by means of lentil lectin affinity chromatography and immunoaffinity chromatography, using monoclonal antibody CF3, from a crude membrane extract of human brain cortex. Gp135 was shown to consist of a glycosylated single polypeptide chain with an apparent molecular mass of 135 kDa. The size of the polypeptide moiety was estimated to 115 kDa following N-glycanase digestion. The glycoprotein is anchored in the membrane by a glycosylphosphatidylinositol tail, as shown by phospholipase C digestion and liposome incorporation experiments. Amino acid sequence analysis of the amino terminal, and of an internal peptide obtained by V8 protease digestion of the glycoprotein, revealed a strong similarity to three previously described glycoproteins from chicken (contactin and F11) and mouse (F3) brains. These glycoproteins belong to the immunoglobulin superfamily and are implicated in cell adhesion phenomena in the developing brain. Gp135 may be the human counterpart to one or several of these glycoproteins.
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PMID:Isolation and characterization of a membrane glycoprotein from human brain with sequence similarities to cell adhesion proteins from chicken and mouse. 202 73

The cytoplasmic tubulovesicular and canalicular membranes of gastric parietal cells are intimately involved in hydrochloric acid secretion. To characterise the glycoproteins of these membranes, we examined a panel of lectins for reactivity with parietal cells in paraffin sections of rat, dog and pig stomach. The poly-N-acetyllactosamine-specific lectin from Lycopersicon esculentum (tomato) and from Solanum tuberosum (potato), and the galactose-specific lectin Ricinus communis agglutinin (RCA120), showed strong cytoplasmic binding of parietal cells of all three species, with a pattern indicative of an intracellular membrane network. Binding to parietal cells was confirmed by double-labelling studies with parietal cell auto-antibodies from patients with autoimmune gastritis. Mucous cells and mucin also bound these lectins strongly. Other gastric cell types did not stain with either tomato or potato lectin, but stained weakly with RCA120. Electron-microscopic examination of lectin binding sites using biotinylated tomato lectin or RCA120 and streptavidin-gold, revealed specific binding to the luminal face of parietal cell tubulovesicular and canalicular membranes as well as the contents of mucous cell secretory granules. Tomato lectin and RCA120 reacted by lectin blotting with a major species of apparent molecular weight 60-90 X 10(3) Mr from rat, dog and pig gastric membranes. A tubulovesicular membrane fraction, enriched 10-fold for K(+)-dependent phosphatase activity, was also enriched three-fold for tomato lectin binding as assessed by a solid-phase lectin assay. The 60-90K (K = 10(3) Mr) component, in 125I-labelled detergent extracts of dog tubulovesicular membranes, bound to an affinity support of tomato lectin-Sepharose and was specifically eluted with N,N',N'-triacetylchitotriose. Digestion with N-glycanase collapsed the 60-90K component into a sharp 35K band. We conclude that: (1) a 60-90K membrane glycoprotein localised on the luminal face of tubulovesicles and canaliculi of parietal cells interacts strongly with tomato lectin and RCA120; and (2) the glycoprotein is composed of a 35K core protein glycosylated with N-glycans probably containing poly-N-acetyllactosamine sequences with terminal galactosyl residues. The properties of this 60-90K glycoprotein are identical to a major parietal cell autoantigen recognised by sera of patients with autoimmune gastritis.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Poly-N-acetyllactosamine-specific tomato lectin interacts with gastric parietal cells. Identification of a tomato-lectin binding 60-90 X 10(3) Mr membrane glycoprotein of tubulovesicles. 216 41

The human erythrocyte glucose transporter is a fully integrated membrane glycoprotein having only one N-linked carbohydrate chain on the extracellular part of the molecule. Several authors have suggested the involvement of the carbohydrate moiety in glucose transport, but not definitive results have been published to date. Using transport glycoproteins reconstituted in proteoliposomes, kinetic studies of zero-trans influx were performed before and after N-glycanase treatment of the proteoliposomes: this enzymatic treatment results in a 50% decrease of the Vmax. The orientation of transport glycoproteins in the lipid bilayer of liposomes was investigated and it appears that about half of the reconstituted transporter molecules are oriented properly. Finally, it could be concluded that the release of the carbohydrate moiety from the transport glycoproteins leads to the loss of their transport activity.
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PMID:Glycosylation of the human erythrocyte glucose transporter is essential for glucose transport activity. 226 93

Previous studies have shown that plasma membrane compounds are involved in the contact-dependent inhibition of growth of human diploid fibroblasts. The purification of the active plasma membrane glycoprotein is described in this report. The glycoprotein has an apparent molecular mass of 60-70 kD and, due to differential sialylation, isoelectric points between pH 5.5. and 6.2. Treatment with sialidase yielded one spot in two-dimensional gel electrophoresis with an isoelectric point of 6.3. After removal of the N-glycosidically linked oligosaccharide chains, the apparent molecular mass is reduced by approximately 22 kD. Treatment was diluted NaOH, which removes the O-glycosidically linked portion of oligosaccharides, resulted in a reduction of the apparent molecular mass by approximately 5 kD. The addition of 50 ng/ml of this glycoprotein-for which the term "contactinhibin" is proposed-in immobilized form to sparsely seeded human fibroblasts resulted in a reversible 70-80% inhibition of growth. The inhibition was not confined to human fibroblasts as other cells were also inhibited, with the exclusion of transformed cells, which are refractory to contactinhibin. The inhibitory activity was abolished by treatment with beta-galactosidase or glycopeptidase F, indicating that the glycan moiety is the biologically active part of the molecule. Confluent cultures treated with antibodies raised against contactinhibin were released from the contact-dependent inhibition of growth. In addition to enhanced saturation density, these cultures exhibited a crisscross growth pattern and the formation of foci. Immunocytochemical studies showed that contactinhibin was associated with vimentin. Furthermore, contactinhibin was found to be not expressed in a species- or organ-specific manner.
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PMID:Isolation and characterization of a 60-70-kD plasma membrane glycoprotein involved in the contact-dependent inhibition of growth. 227 80

Using affinity chromatography with the monoclonal antibody 271A6, which binds selectively to telencephalic regions of the rabbit brain, we have purified a telencephalon-specific antigen to apparent homogeneity and characterized it as a membrane glycoprotein. The telencephalon-specific membrane protein (named "telencephalin") has a molecular weight of about 500,000 and is composed of four subunits each of mol. wt 130,000. Its digestion with N-glycanase reduced the subunit mol. wt by 23,000, indicating that each subunit has several N-asparagine-linked oligosaccharide chains. Immunohistochemical analysis using polyclonal antibody against the purified telencephalin shows that expression of the entire protein is restricted to the telencephalon. In addition, segment-specific expression of telencephalin was observed in all mammalian species examined (mouse, rat, guinea-pig, rabbit, cat and monkey). The telencephalon is the most rostral segment of the brain, and comprises the cerebral neocortex, paleocortex, hippocampus, septum, striatum and olfactory bulb. The present results indicate that all regions of the mammalian telencephalon express the segment-specific membrane glycoprotein, telencephalin, and suggest that telecephalin is involved in functions specific to the surface membrane of telencephalic neurons.
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PMID:Mammalian telencephalic neurons express a segment-specific membrane glycoprotein, telencephalin. 235 99

Antibodies were affinity purified from crude antiserum by elution from the 24 kDa region of preparative one-dimensional Western blots containing immobilized adult Schistosoma mansoni inner bilayer membrane proteins. They were shown to be specific for a single acidic polypeptide complex, Smgp24, following immunoblotting from two-dimensional polyacrylamide gels. These antibodies were then used to detect the presence of the Smgp24 complex in fractions prepared from lectin affinity chromatography, phase separation in Triton X-114 and chemical and enzymatic carbohydrate modification treatments. The 24 kDa antigen was bound and specifically eluted from both concanavalin A and lentil lectin affinity matrices. In addition, the electrophoretic mobility of the antigen was shifted to approximately 20 kDa after treatment with endoglycosidase F and N-glycanase, but was not appreciably altered following treatment with endoglycosidase H, neuraminidase, or sodium meta-periodate. The 20 kDa species produced by endoglycosidase F or N-glycanase treatment no longer bound to the lectin affinity resins. The Smgp24 complex also partitioned almost quantitatively into the detergent-enriched phase after phase separation in Triton X-114 solutions. These results indicate that the Smgp24 complex is an antigenic integral membrane glycoprotein and may consist of a single polypeptide backbone which is extensively post- or co-translationally modified.
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PMID:Biochemical properties of a 24 kilodalton membrane glycoprotein antigen complex from Schistosoma mansoni. 318 20

The glycoproteins responsible for calcium-dependent oligodendrocyte aggregation were purified and characterized. Using detergent extraction, lentil-lectin-Sepharose 4B affinity chromatography, and preparative gel electrophoresis, 3 proteins were purified to apparent homogeneity, with relative Mrs of 120,000, 140,000, and 180,000. The aggregation assay showed that all 3 proteins had the ability to block antibody-mediated inhibition of oligodendrocyte aggregation. The 120,000 protein was the most active of the three. Antisera were raised in rabbits to these 3 individual proteins. Western blot analyses showed that all three antisera recognized 120,000, 140,000, and 180,000 proteins, which indicated that the proteins were related. Western-blot analyses of cultured oligodendrocytes and purified rat myelin showed only the 120,000 protein. Immunoprecipitation of iodinated membrane proteins of cultured oligodendrocytes also indicated the presence of only the 120,000 Mr protein. Deglycosylation of the 120,000 protein by N-glycanase resulted in a 110,000 protein. The immunoblot pattern suggested some similarities between oligodendrocyte adhesion molecules and the neural cell adhesion molecule (N-CAM). Therefore, the 120,000, 140,000, and 180,000 Mr proteins were compared to N-CAM by Western-blot analysis, immunofluorescence staining, and by immunoprecipitation. The results suggest that oligodendrocytes contain a 120,000 membrane glycoprotein that is related to N-CAM.
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PMID:Oligodendrocyte cell adhesion molecules are related to neural cell adhesion molecule (N-CAM). 377 36

Ammodytoxin A, the presynaptic neurotoxin from Vipera ammodytes ammodytes venom, was found to bind specifically and with high affinity to bovine cortex synaptic membrane preparation. The detected ammodytoxin A high-affinity binding was characterized by equilibrium binding analysis which revealed a single high-affinity binding site with Kd 4.13 nM and Bmax 6.67 pmoles/mg of membrane protein. 125I-ammodytoxin A was covalently cross-linked to its neuronal acceptor using a chemical cross-linking technique. As revealed by subsequent SDS-PAGE analysis and autoradiography, 125I-ammodytoxin A specifically attached to membrane components with apparent mol. wts 53,000-56,000. Besides by the native ammodytoxin A, the binding of radioiodinated ammodytoxin A to the neuronal acceptor was highly attenuated, also by other two iso-neurotoxins from V. a. ammodytes venom, ammodytoxins B and C, and neurotoxin crotoxin B from the venom of the South American rattlesnake (Crotalus durissus terrificus). Vipera berus berus phospholipase A2 was a weaker inhibitor, whereas nontoxic phospholipase A2, ammodytoxin I2 and myotoxic phospholipase A2 homologue, ammodytin L, both from V. a. ammodytes venom as well, were very weak inhibitors. No inhibitory effect on 125I-ammodytoxin A specific binding at all was, however, obtained with alpha-dendrotoxin, beta-bungarotoxin and crotoxin A, respectively. Treatment of synaptic membranes with proteinase K and Staphylococcus aureus V-8 proteinase, a combination of PNGase F and neuroaminidase, heat or acid lowered the 125I-ammodytoxin A specific binding to various extents but never completely abolished it. The ammodytoxin A binding site in bovine synaptic membranes is thus most likely a combination of membrane glycoprotein acceptor and membrane phospholipids. As ammodytoxin A reduced the second negative component of the perineural waveform, measured on mouse triangularis sterni preparation, which is very likely a result of an inhibition of a fraction of the terminal K+ currents, the ammodytoxin A acceptor could well be connected with K+ channels.
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PMID:Ammodytoxin A acceptor in bovine brain synaptic membranes. 757 Jun 29


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