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 application of high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective enzymatic deglycosylation treatments is demonstrated in the analysis of glycosylation patterns in recombinant Desmodus salivary plasminogen activator, a heterogeneous glycoprotein. The sample was initially digested with a proteolytic enzyme (endoproteinase Lys-C) and then further treated with either PNGase F to remove N-linked carbohydrates or a combination of neuraminidase and O-glycosidase to remove sialic acid and O-linked carbohydrates. By comparison of the LC-ESI-MS peptide maps for the fully glycosylated and deglycosylated samples, it was possible to unambiguously identify the sites of N-linked glycosylation as well a number of N-linked glycopeptides. The O-link glycopeptides, which are present at low level ( < 1%), were not detected prior to the deglycosylation, nor could changes in peptide elution in the map following deglycosylation be correlated with potential O-linked glycosylation sites.
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PMID:Application of high-performance liquid chromatograph-electrospray ionization mass spectrometry and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry in combination with selective enzymatic modifications in the characterization of glycosylation patterns in single-chain plasminogen activator. 864 33

During fertilization, free-swimming mouse sperm bind to mZP3 (approximately 83 000 Mr), one of three zona pellucida glycoproteins, and once bound undergo the acrosome reaction, a type of cellular exocytosis [Wassarman, P. M., & Litscher, E. S. (1995) Curr. Top. Dev. Biol. 30, 1-19]. Sperm recognize and bind to specific serine/threonine-linked oligosaccharides located at the mZP3 combining site for sperm. Here, we examined certain characteristics of gp55, a approximately 55 000 Mr glycopeptide derived from the carboxy-terminal half of mZP3 polypeptide to which sperm bind [Rosiere, T. K., & Wassarman, P. M. (1992) Dev. Biol. 154, 309-317]. gp55 is heterogeneous with respect to Mr (approximately 47 000-62 000 Mr) and has a relatively low pI (approximately 4.3-4.5) compared to the polypeptide portion of the glycopeptide (pI approximately 6.5). gp55 inhibits binding of sperm to eggs (i.e., exhibits sperm receptor activity) and induces sperm to undergo the acrosome reaction in vitro at about the same concentrations required for intact mZP3 (approximately 50-200 nM). Each of three different size-fractions of gp55, separated by SDS-PAGE, also exhibits bioactivity in vitro. Removal of asparagine-linked (N-linked) oligosaccharides from gp55, by extensive digestion with N-glycanase, reduces its Mr to approximately 21 000 and increases it pI to approximately 5.3, but does not significantly affect its ability to inhibit binding of sperm to eggs or to induce sperm to undergo the acrosome reaction. Similarly, digestion of gp55 with either endo-beta-galactosidase or neuraminidase alters its Mr and/or pI, but does not significantly affect either of its bioactivities. These observations are consistent with the proposal that neither N-linked oligosaccharides nor sialic acid is an essential element of the mZP3 combining site for sperm. They also indicate that a relatively small mZP3 glycopeptide is able to induce sperm to undergo the acrosome reaction (i.e., cellular exocytosis) in vitro.
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PMID:Characterization of mouse ZP3-derived glycopeptide, gp55, that exhibits sperm receptor and acrosome reaction-inducing activity in vitro. 867 30

125I-Glucagon was directly cross-linked to its receptor sites on the MDCK plasma membranes using a UV irradiation procedure. Analysis of the affinity labeled membranes by SDS-PAGE and autoradiography, demonstrated the presence of a single band at 74 kDa. The incorporation of radiolabeled glucagon into this band was abolished by the presence of excess unlabeled hormones, thus indicating a specificity of labeling. Also this band was observed in affinity labeled dog kidney plasma membranes. The size of the MDCK and the dog kidney glucagon receptors were consistently larger than that of the dog liver receptor as judged by electrophoretic mobility. Treatments with neuraminidase, endoglycosidase F, or N-glycanase failed to convert the renal form into the hepatic form of the receptor. Proteolytic mapping of the MDCK and the dog liver glucagon receptors revealed that major domains of both proteins are remarkably similar, yet transient variations in the size of the fragments could be detected after short duration digestions. Overall the data presents evidence that the dog renal receptor represents a structurally unique isoform of the glucagon receptor.
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PMID:Canine kidney glucagon receptor: evidence for a structurally-different, tissue-specific variant of the glucagon receptor. 867 61

The glycosylation state of lecithin:cholesterol acyltransferase (LCAT) may be important in determining its enzymatic activity. We compared glycosylation structure, enzyme kinetics, and phosphatidylcholine (PC) acyl specificity of human LCAT from four sources: human plasma (pLCAT), media from HepG2 cells (HepG2 LCAT), media from SF21 cells infected with a recombinant baculovirus (bLCAT) and media from stably transfected Chinese hamster ovary (CHO) cells (CHO LCAT). bLCAT was underglycosylated (molecular weight approximately 50 kDa) and resistant to digestion by N-glycanase F, endoglycosidase F, and neuraminidase. CHO and HepG2 LCAT were overglycosylated (approximately 68 kDa and approximately 70-75 kDa) compared to pLCAT (approximately 65 kDa). CHO LCAT, like pLCAT, was sensitive to N-glycanase F and neuraminidase but not to endoglycosidase F. HepG2 LCAT demonstrated resistance to N-glycanase F and endoglycosidase F. Apparent Km values for all four enzymes were similar (1.4-9.2 microM cholesterol) for recombinant high density lipoproteins (rHDL) containing sn-1 16:0, sn-2 18:1 PC (POPC). Apparent Vmax values (nmol cholesteryl ester formed/h per micrograms) were 52.6 for pLCAT, 48.6 for CHO LCAT, 15.3 for bLCAT, and 8.3 for HepG2 LCAT. Changes in PC acyl specificity in the presence and absence of cholesterol were characterized by comparing the ratio of LCAT activity on rHDL containing sn-1 16:0, sn-2 20:4 PC (PAPC) or POPC (PAPC/POPC activity ratio). The ratios for pLCAT, bLCAT, CHO LCAT, and HepG2 LCAT activity were 0.63, 0.49, 0.56, and 0.51 with cholesterol and 0.34, 0.29, 0.36, and 0.99 without cholesterol, respectively. We conclude that LCAT source influences glycosylation structure, which affects the apparent Vmax for cholesteryl ester formation with only minor changes in apparent Km or acyl substrate specificity.
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PMID:Glycosylation structure and enzyme activity of lecithin:cholesterol acyltransferase from human plasma, HepG2 cells, and baculoviral and Chinese hamster ovary cell expression systems. 872 18

Monoclonal antibodies to opioid-binding cell adhesion molecule (OBCAM) were produced against a synthetic OBCAM peptide. Immunoblotting analysis revealed that the antibodies reacted with 58 and/or 51 kDa proteins in P2 membranes from bovine, rat, mouse, guinea pig and rabbit brains. In bovine brain, the 58 and 51 kDa proteins were present in the striatum and cerebral cortex at high levels, but not in the pituitary. OBCAM was also detected in the cerebellum mainly in the 51 kDa form. In other tissues, the proteins were found in the spleen at very low levels, but not at all in the liver or kidney of the rat. OBCAM was effectively solubilized from bovine P2 membranes by bacterial phosphatidylinositol specific-phospholipase C (PI-PLC), indicating that OBCAM is a glycosylphosphatidylinositol (GPI)-anchored protein. PI-PLC treatment, however, had little effect on the opioid binding activity of the residual P2 membranes. The molecular weight of the proteins (58 and 51 kDa) was reduced to 36 kDa following treatment with N-glycanase but not further reduced after subsequent treatment with neuraminidase and O-glycanase, suggesting that OBCAM has N-glycosylated carbohydrate chains and that its two isoforms are different, at least, in the degree of N-glycosylation. Taken together, these results suggest that OBCAM consists of 58/51 kDa GPI-anchored glycoproteins which are highly N-glycosylated and are expressed mainly in the nervous system.
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PMID:Characterization and tissue distribution of opioid-binding cell adhesion molecule (OBCAM) using monoclonal antibodies. 874 Apr 43

A material of Mr 24,000 has been isolated from a cachexia-inducing mouse tumor (MAC16) and shown to initiate protein degradation in isolated gastrocnemius muscle. Biological activity was destroyed by preincubation with peptide N-glycosidase F (PNGase F) and endo-alpha-N-acetylgalactosaminidase (O-glycosidase) but not by neuraminidase or trypsin. Antibody reactivity was destroyed by treatment with periodate, indicating carbohydrate moieties to be the antigenic determinants. Antigenic activity was also reduced by treatment with PNGase F and O-glycosidase and was completely destroyed by treatment with chondroitinase ABC but was unaffected by treatment with either trypsin or chymotrypsin, confirming that the N- and O-linked sulfated oligosaccharide chains were both the antigenic and biological determinants. Biosynthetic labeling of MAC16 cells using a combination of [35S]sulfate and [6-3H]GlcN gave a single component of Mr 24,000 containing both radiolabels. Similar material could not be isolated from a cell line (MAC13) originating from a tumor that does not cause cachexia in vivo. Digestion of 3H/35S material with PNGase F produced two fragments of Mr 14,000 and 10,000 containing both radiolabels, and digestion with O-glycosidase produced three fragments of Mr 14,000, 6,000, and 4, 000, the first two contained both radiolabels and the third contained only 3H. Digestion of the fragment of Mr 14,000 released by PNGase F with O-glycosidase also gave fragments of Mr 6,000 and 4, 000. The products from both digestions were acidic as determined by anion exchange chromatography on DEAE-cellulose. The negative charge on the fragment of Mr 4,000 was removed by treatment with alkaline phosphatase. This suggests that the charge originated from phosphate residues, and this has been confirmed by biosynthetic labeling of MAC16 cells with [32P]orthophosphate, where radiolabel was incorporated into material of Mr 24,000 and into the fragment of Mr 4,000 after treatment with O-glycosidase. To determine the size of the polypeptide core MAC16 cells were biosynthetically labeled with L-[2,5-3H]His which after chemical deglycosylation produced a major component of Mr 4,000. These results suggest a model for the Mr 24, 000 material consisting of a central polypeptide chain of Mr 4,000 and with phosphate residues that may be attached to the polypeptide or a short oligosaccharide chain containing GlcN, one O-linked sulfated oligosaccharide chain containing GlcN, and of Mr 6,000 and one N-linked sulfated oligosaccharide chain of Mr 10,000 also containing GlcN. Neither chain was cleaved into disaccharides with chondroitinase ABC, suggesting that the material is a sulfated glycoprotein.
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PMID:Structural analysis of a tumor-produced sulfated glycoprotein capable of initiating muscle protein degradation. 913 70

Analysis of the carboxymethylated subunit of human cartilage oligomeric matrix protein (COMP) by matrix-assisted laser desorption time-of-flight mass spectrometry indicated a protonated molecular mass of 86949 +/- 149 Da, compared with 83547.0 Da calculated from the sequence. Treatment with N-glycanase caused a reduction in mass of 3571 +/- 219 Da, but there was no loss of mass after treatment with O-glycanase or neuraminidase. Peptides containing two putative sites of N-glycosylation were purified and characterized. Analysis of the masses of these after N-glycanase treatment indicated that one was substituted at Asn-101 with an oligosaccharide of mass 1847. 2 +/- 6.6 Da, and the other was unsubstituted at Asn-124. The remaining site of attachment, at Asn-721, was, therefore, also substituted with an oligosaccharide of mass 1724 +/- 226 Da. Analysis of the total monosaccharide content by chemical methods indicated that there were no additional oligosaccharide substituents. The MALDI-TOF mass spectra of COMP from bovine fetal and adult cartilage were compared, indicating a more heterogeneous pattern of substitution at Asn-101 in the fetal form. Since COMP is distributed throughout the pericellular and territorial environments in developing cartilage but occupies the interterritorial zone in mature cartilage, these changes in glycosylation may allow for different intermolecular interactions.
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PMID:Post-translational modifications in cartilage oligomeric matrix protein. Characterization of the N-linked oligosaccharides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. 916 39

We enzymatically deglycosylated pig lung angiotensin I-converting enzyme (ACE) to study the involvement of its glycanic chains in its physicochemical and catalytic properties. The effects of endoglycosidases F2 and H, and of N-glycanase were assessed by ACE mobility in SDS-PAGE. N-Glycanase only was completely effective with or without previous denaturation, leading to a shift in ACE M(r) from 172 to 135 kDa; endoglycosidase F2 produced the same shift but only without previous denaturation. Deglycosylated ACE had the same kcat as native ACE for the substrate hippuryl-histidyl-leucine, and an identical Stokes radius as measured by size-exclusion high performance liquid chromatography. Neuraminidase had no effect on ACE Stokes radius but slightly decreased its kcat which could be related to variations in ionization of the active site. The isoelectric point of ACE, as, determined by isoelectric focusing, increased from 4.5-4.8 to 5.0-5.3 after either endoglycosidase F2 or neuraminidase digestion, but still with microheterogeneities which thus did not seem to be related to ACE glycans. Deglycosylated ACE did not bind onto agaroselectins in contrast to native ACE which bound strongly to concanavalin A showing interactions involving oligomannosidic or biantennary and sialylated N-acetyl-lactosaminic isoglycans. Finally, tunicamycin, an inhibitor of N-glycosylation, did not modify ACE secretion by endothelial cells. Thus, ACE glycans have no drastic effects on structural and biological properties of the protein, but they may have a functional role on intracellular targeting of both secreted and membrane-bound ACE isoforms, also for the protection of the soluble plasma form against hepatic lectins and the maintenance of its hydrosolubility.
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PMID:Structural and biological roles of glycosylations in pulmonary angiotensin I-converting enzyme. 918 38

An important risk factor for thrombosis is the polymorphism R506Q in factor V that causes resistance of factor Va to proteolytic inactivation by activated protein C (APC). To study the potential influence of the carbohydrate moieties of factor Va on its inactivation by APC, factor V was subjected to mild deglycosylation (neuraminidase plus N-glycanase) under nondenaturing conditions. The APC resistance ratio values (ratio of activated partial thromboplastin time [APTT] clotting times with and without APC) of the treated factor V were increased (2.4 to 3.4) as measured in APTT assays. O-glycanase treatment of factor V did not change the APC resistance ratio. The procoagulant activity of factor V as well as its activation by thrombin was not affected by mild deglycosylation. Treatment of factor V with neuraminidase and N-glycanase mainly altered the electrophoretic mobility of the factor Va heavy chain, whereas treatment with O-glycanase changed the mobility of the connecting region. This suggests that the removal of the N-linked carbohydrates from the heavy chain of factor Va, which is the substrate for APC, is responsible for the increase in susceptibility to inactivation by APC. Thus, variability in carbohydrate could account for some of the known variability in APC resistance ratios, including the presence of borderline or low APC resistance ratios among patients who lack the R506Q mutation.
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PMID:The carbohydrate moiety of factor V modulates inactivation by activated protein C. 919 57

Endothelial cell-specific molecules are potential targets for new therapeutic strategies in the control of inflammatory reactions, immune responses and neoangiogenesis. We describe the production and characterization of MEC 14.7, a monoclonal antibody directed to murine endothelial cells recognizing a glycosylated protein with an apparent molecular mass of about 100 kDa in cultured endothelioma cell lysate and about 80 kDa in lung lysate. MEC 14.7 antigen was selectively expressed by the endothelium in vivo, particularly in small vessels and neoformed capillaries and by developing vascular structures in embryonal bodies. Deglycosylation of the molecule with neuraminidase, O- and N-glycanase showed that the MEC 14.7 epitope is neuraminidase-sensitive. MEC 14.7 antigen was purified from lung lysates by chromatographic techniques, and sequenced internal peptides indicated it was identical with murine CD34. Thus the apparent molecular mass of CD34 is heterogeneous, depending on the glycosylation state in the different cell types. Immunomagnetic isolation and culture of MEC 14.7-positive bone marrow cells showed that this antibody recognizes hematopoietic progenitors (particularly myelomonocytic) and can be used in murine models of bone marrow reconstitution.
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PMID:Characterization of MEC 14.7, a new monoclonal antibody recognizing mouse CD34: a useful reage for identifying and characterizing blood vessels and hematopoietic precursors. 927 Aug 80


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