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)

Receptor-mediated recognition and adhesion to laminin, a specific glycoprotein from basement membranes, exert an important role in many biological phenomena. Studying cell surface proteins of B16-F10, a metastatic murine melanoma cell line, we identified a 120-140 kDa glycoprotein (gp120/140) that binds laminin. This glycoprotein was recognized by a polyclonal antibody raised against the human fibronectin receptor beta 1-integrin chain, as well as immunoprecipitated by an anti-alpha 6 chain (monoclonal antibody GoH3), characterizing it as an alpha 6/beta 1-integrin. Its binding to laminin was specific and displayed moderate affinity, as its apparent dissociation constant was 18 nM. To characterize the influence of carbohydrate moieties on the laminin-gp120/140 interaction, metaperiodate oxidation, metabolic inhibition of glycosylation, and enzymatic deglycosylation studies were performed. Our results indicate that gp120/140 Asn-linked oligosaccharides play a part in this interaction. Reciprocally, both metaperiodate and N-glycanase treatment of native laminin reduced its binding to gp120/140, characterizing the latter as a lectin-like molecule. These results point to glycosylation processes as a possible mechanism for variable binding specificity profiles among integrins.
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PMID:Asn-linked oligosaccharide-dependent interaction between laminin and gp120/140. An alpha 6/beta 1 integrin. 199 8

Neuroglandular antigen (NGA) was identified as a human melanoma-associated antigen by a panel of murine monoclonal antibodies of both IgG2a (LS62, LS76, LS159) and IgG1 (LS113, LS140, LS152) subclasses, developed in this laboratory (L. Sikora, A. Pinto, D. Demetrick, W. Dixon, S. Urbanski, and L. M. Jerry, Int. J. Cancer, 39: 138-145, 1987). Monoclonal antibody LS62 was used to immunoprecipitate NGA from radiolabeled cultured melanoma cells, and it behaved as a heterogeneous glycoprotein "smear" on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (Mr 29,000-70,000). Radioactive pulse-chase time course experiments using human melanoma cells cultured in the presence or absence of inhibitors of protein glycosylation showed that the antigen consisted of a core protein with a molecular weight of 22,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This molecule was modified by the addition of at least three N-linked oligosaccharide side chains (as revealed by limited N-glycanase digestion) to give a precursor form with a molecular weight of approximately 34,000. Subsequent processing steps yielded a heterogeneous family of glycoproteins with varying amounts of covalently attached carbohydrate. Much of this heterogeneity in both molecular weight and pI (as revealed by two-dimensional electrophoresis) could be removed by treatment of the antigen with neuraminidase, suggesting heavy sialylation of the glycoprotein. NGA could be detected on the surface of melanoma cells by fluorescence-activated cell sorter analysis, surface radioiodination, and, as previously shown, immunoperoxidase staining. However, there was a larger intracellular pool of the molecule and the antigen was rapidly released into the culture supernatant. The function of NGA remains unknown but its elevated expression in transformed melanocytes have prompted this characterization to understand its biochemical nature and relation to other melanoma-associated antigens.
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PMID:Biosynthesis, glycosylation and intracellular processing of the neuroglandular antigen, a human melanoma-associated antigen. 236 31

The secretion of tissue-type plasminogen activator (t-PA) from melanoma cells (Bowes) was investigated with or without monensin treatment. Monensin inhibited secretion of t-PA from the cells to the medium in a dose-and time-dependent manner. The inhibition was accompanied by an intracellular accumulation of t-PA. Electrophoretic enzymography of the cell homogenate showed the main lytic zone at 72 kDa, which reacted with the IgG of anti-t-PA. Analysis of the cell organelles using ultracentrifugation with a discontinuous sucrose density gradient revealed that the activity and the antigen of t-PA were observed near the discontinuous phase of the sucrose gradient. Analysis of 3H-mannose- and 35S-methionine-labeled t-PA in the cell organelles revealed that the radioactivity of each was increased by monensin treatment, and that such treatment increased the ratio of 3H-mannose-related glycoprotein to 35S-methionine-related protein. The sugar chain of intracellular t-PA was analyzed with endoglycosidase H and N-glycanase, which reduced the molecular weight of t-PA by 4.5-10 kDa, indicating the intracellular presence of a high-mannose type sugar chain and a complex-type sugar chain of t-PA. t-PA secreted from the monensin-treated cells possesses a high-mannose type sugar chain only. Therefore, monensin alters the secretion of t-PA by abnormal glycosylation.
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PMID:Effect of monensin on secretion of t-PA from melanoma (Bowes). 251 82

A weakly metastatic wheat-germ-agglutinin-resistant mutant Wa4-b1 was previously shown to be less adherent to endothelial cell extracellular matrix than the more metastatic parental B-16 melanoma cells. This report describes reduced adhesion and spreading of Wa4-b1 cells on the cell-binding domain of fibronectin (CBD) and laminin (LN). Cell surface receptors which mediate such interactions are members of the integrin family of membrane glycoproteins. An antibody that recognizes the beta 1 integrin subunit inhibited spreading on both the CBD and LN. The integrins of the mutant cells immunoprecipitated by the antibody appeared to be structurally altered, showing a greater electrophoretic mobility. The mobility difference between the parent and the mutant receptors was abolished following removal of the glycan moieties of the receptors enzymatically using glycopeptidase F, or chemically using trifluoromethanesulfonic acid, suggesting that the structural alteration of the mutant receptors is in glycosylation. The altered receptors may be responsible for the observed decrease in cell adhesion and spreading of the mutant cells to the CBD and LN. Such a decrease in Wa4-b1 cell interaction with extracellular matrix components may play a role in their decreased metastatic potential.
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PMID:Reduced cell adhesion to fibronectin and laminin is associated with altered glycosylation of beta 1 integrins in a weakly metastatic glycosylation mutant. 278 45

The molecular mechanisms underlying cell attachment and subsequent cell spreading on laminin are shown to be distinct form one another. Cell spreading is dependent upon the binding of cell surface galactosyltransferase (GalTase) to laminin oligosaccharides, while initial cell attachment to laminin occurs independent of GalTase activity. Anti-GalTase IgG, as well as the GalTase modifier protein, alpha-lactalbumin, both block GalTase activity and inhibited B16-F10 melanoma cell spreading on laminin, but not initial attachment. On the other hand, the addition of UDP galactose, which increases the catalytic turnover of GalTase, slightly increased cell spreading. None of these reagents had any effect on cell spreading on fibronectin. When GalTase substrates within laminin were either blocked by affinity-purified GalTase or eliminated by prior galactosylation, cell attachment appeared normal, but subsequent cell spreading was totally inhibited. The laminin substrate for GalTase was identified as N-linked oligosaccharides primarily on the A chain, and to a lesser extent on B chains. That N-linked oligosaccharides are necessary for cell spreading was shown by the inability of cells to spread on laminin surfaces pretreated with N-glycanase, even though cell attachment was normal. Cell surface GalTase was distinguished from other reported laminin binding proteins, most notably the 68-kD receptor, since they were differentially eluted from laminin affinity columns. These data show that surface GalTase does not participate during initial cell adhesion to laminin, but mediates subsequent cell spreading by binding to its appropriate N-linked oligosaccharide substrate. These results also emphasize that some of laminin's biological properties can be attributed to its oligosaccharide residues.
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PMID:Functionally distinct laminin receptors mediate cell adhesion and spreading: the requirement for surface galactosyltransferase in cell spreading. 297 32

Pigmentation-associated antigen (PAA) or gp75 is a glycoprotein localized to the melanosomes of human melanomas and melanocytes to which a mouse monoclonal antibody (AbTA99) has been produced (T. M. Thomson et al. (1985) J. Invest. Dermatol. 85, 169). Treatment of 3H-labeled immunoprecipitated melanoma PAA with alkaline-borohydride, hydrazinolysis, or N-glycanase released three families of carbohydrate chains (I, II, and III). Peak I consists of a major component (Ia) of sialylated triantennary N-linked chains which are partially substituted with fucose on terminal positions as well as on the chitobiose core and a minor component (Ib) which is a sialylated biantennary N-linked species. Peak II was not well characterized but may be a monoantennary complex chain species. Peak III consists of typical N-linked high mannose units with six to seven mannose residues. Melanocyte PAA carbohydrate chains have the same general features as melanoma PAA except that the biantennary complex chain predominates; this difference resembles that observed between the cell surface glycopeptides of transformed animal cells and their nontransformed counterparts. The glycosylation characteristics of this melanosomal glycoprotein are compared with those of glycoproteins from endoplasmic reticulum, Golgi, and lysosomes, and with tyrosinase. It is suggested that the glycosylation pattern is a reflection of the biosynthetic origin and cellular destination of a particular organelle and its constituents.
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PMID:Glycosylation characteristics of pigmentation-associated antigen (GP75): an intracellular glycoprotein of human melanocytes and malignant melanomas. 353 23

Swainsonine, a known inhibitor of the alpha-mannosidase II involved in processing of asparagine-linked glycoproteins, causes accumulation of hybrid-type oligosaccharide-containing glycoproteins in mammalian cells. Swainsonine augments lymphokine-activated, killer-cell induction at suboptimal doses of interleukin-2; the amount needed to increase LAK activity is 100-1000 fold higher than required to completely inhibit mannosidase II. Human mononuclear lymphocytes, when treated with these relatively high (58 microM) concentrations of swainsonine showed a 3-4 fold increase in D-[3H]mannose incorporation into the glycan as compared to glycans of untreated cells. Analysis indicated accumulation of high-mannose type, free oligosaccharides in the soluble fractions of the cell. Chromatographic analysis of glycan obtained by D-[2-3H]mannose labeling of human mononuclear lymphocytes showed synthesis of a new oligosaccharide, at 58 microM of swainsonine, that contained 36% of the total radioactivity incorporated into the glycan (oligosaccharide pool). This oligosaccharide fraction was resistant to hydrolysis by endoglycosidase H, endoglycosidase F, O and N-glycanase, but was susceptible to cleavage by Jack bean alpha-mannosidase and was bound > 90% to concanavalin A-Sepharose. A similar chromatographic elution profile was obtained from glycans labeled with D-[2-3H]mannose from mouse B16F10 melanoma and baby hamster kidney cells subsequent to swainsonine treatment. Methylation analysis of free oligosaccharides obtained from MNL revealed the presence of a pentamannose. These results indicate the accumulation of a free high-mannose oligosaccharide rather than expected hybrid-type structure on treatment of cells with relatively high concentrations of swainsonine.
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PMID:Accumulation of pentamannose oligosaccharides in human mononuclear leukocytes by action of swainsonine, an inhibitor of glycoprotein processing. 825 42

Shedding by cultured human melanoma cells of a well-characterised cell- surface glycoprotein antigen known as "melanotransferrin" was studied with two monoclonal antibodies, 140.240 and 96.5. By means of [35S]-cysteine metabolically-labelled melanoma cells and immunoprecipitation studies, identification was made, by 140.240 in the spent media of two of six melanoma cell lines, of a new molecule of 100-kDa, aside from the 88-kDa molecule. Only the 88-kDa shed molecule was detected in the remaining four melanoma cell lines with both antibodies. None of nine clonal sublines derived from the two melanoma cell lines were found to shed the 100-kDa or 88-kDa molecule exclusively. Both shed antigens were released spontaneously to the medium from the live melanoma cells rather than as a result of cell death and lysis, since there was no obvious cell death or debris in the spent medium nor in the monolayer cells detected at the time of spent medium collection. Digestion of the isolated 100-kDa and 88-kDa shed molecules with N-glycanase followed by SDS-polyacrylamide gel electrophoresis resulted in the appearance of a single band of the 77-kDa molecule, which is deduced to be the polypeptide precursor of the cell-associated 87-kDa antigen. It is concluded that some melanoma lines shed the variant 100-kDa molecule, in addition to the 88-kDa molecule, and that both shed molecules and their cellular counterpart 87-kDa differ in their degrees of glycosylation.
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PMID:Identification with monoclonal antibody 140.240 of a structural variant of melanotransferrin shed by human melanoma cell lines in vitro. 861 5

N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migration and tumor invasion. N-cadherin is a heavily glycosylated protein. Many studies have demonstrated that malignant transformation of a number of cell types correlates with changes of cell surface N-linked oligosacharides. We have studied the carbohydrate profile of N-cadherin synthesized in human melanoma cell lines and the effect of this protein and complex N-glycans on in vitro migration of melanoma cells from the primary tumor site--WM35 and from different metastatic sites WM239 (skin), WM9 (lymph node), and A375 (solid tumor). N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterization of its carbohydrate moieties was carried out by SDS-PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and on-blot deglycosylation using PNGase F for glycan release. N-glycans were separated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and their structures identified by the computer matching of the resulting masses with those derived from a sequence database. The assay of in vitro chemotaxic cell migration was performed using QCM Cell Invasion Assay (Chemicon). N-cadherin from WM35 (primary tumor site) possessed high-mannose and biantennary complex type glycans with alpha2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines possessed mostly tri- or tetra-antennary complex type glycans. In addition, N-cadherin from WM9 (lymph node metastatic site) and A375 (solid tumor metastatic site) contained heavily alpha-fucosylated complex type chains with alpha2,3 linked sialic acid. Blocking of N-cadherin-mediated intercellular interaction by N-cadherin-specific antibodies significantly (of about 40%) inhibited migration of melanoma cells. Inhibition of synthesis of complex type N-glycans by swainsonine (mannosidase II inhibitor) led to 50% decrease of cell migration. The results indicated differences between N-cadherin glycans from primary and metastatic sites and confirmed influence of N-cadherin and complex -type N-glycans on in vitro migration of melanoma cells.
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PMID:The structure of the oligosaccharides of N-cadherin from human melanoma cell lines. 1531 81

We recently demonstrated that the mechanism of processing of an HLA-A*0201-restricted peptide epitope, Tyr(369)(D), derived from the membrane protein tyrosinase, involves retrotranslocation of glycosylated molecules from the endoplasmic reticulum to the cytosol, removal of an N-linked carbohydrate from Asn(371) by peptide N-glycanase, proteolysis by the proteasome and other proteases, and retransport of the resulting peptides into the endoplasmic reticulum for association with HLA-A*0201. Carbohydrate removal results in deamidation of Asn(371) to aspartic acid. The asparagine-containing homolog of this peptide, Tyr(369)(N), is not presented by tyrosinase-expressing cells, and this has been presumed to be due to quantitative glycosylation of Asn(371). Although examining cytosolic intermediates that accumulated in human melanoma cells treated with proteasome inhibitors, we were surprised to find both molecules that had been deglycosylated by peptide N-glycanase and a large number of molecules that had not been previously glycosylated. The failure of Tyr(369)(N) to be processed and presented from these latter molecules may be partially due to a process of deamidation independent of glycosylation. However, we also established that proteasomes degrade tyrosinase molecules that are still glycosylated, giving rise to a set of discrete intermediates that are not observed when unglycosylated molecules are degraded. We propose that Tyr(369)(N) fails to be presented because unglycosylated tyrosinase is degraded rapidly and relatively nonselectively. In contrast, glycosylation alters the selectivity of tyrosinase processing by the proteasome, enhancing the production or survival of Tyr(369)(D).
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PMID:N-glycosylation enhances presentation of a MHC class I-restricted epitope from tyrosinase. 1934 61


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