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)

In this study we report that bone and platelet osteonectin are structurally and functionally heterogeneous in terms of glycosylation and collagen binding capacity. The relative sensitivity of bone and platelet osteonectin to specific glycosidases was used to evaluate potential differences in glycosylation. Although native bone and platelet osteonectin are electrophoretically nonidentical, N-glycanase treatment yielded products with the same apparent molecular weight. Bone osteonectin was also susceptible to cleavage by endo H but not to neuraminidase, while platelet osteonectin was susceptible to neuraminidase but not to endo H. In lectin blotting experiments of bone and platelet osteonectin, concanavalin A bound specifically to bone osteonectin but not to platelet osteonectin. However, Lens culinaris agglutinin bound to platelet osteonectin but not to bone osteonectin. These data suggest that bone and platelet osteonectin differ in their oligosaccharide side chain structures, with bone osteonectin possessing a high mannose-type and platelet osteonectin, a complex-type structure. Solid-phase binding techniques were used to functionally evaluate bone and platelet osteonectin in terms of collagen binding. Although bone osteonectin bound specifically to types I, III, and V collagen, platelet osteonectin had no apparent affinity for these collagen types suggesting that the two proteins are also functionally distinct.
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PMID:The collagen binding specificity of bone and platelet osteonectin is related to differences in glycosylation. 203 56

In this study we demonstrate that the binding region of recombinant truncated human bone osteonectin (tHON) for type V collagen resides between amino acids 1 and 146. After removal of oligosaccharide chain structures from tHON, bovine bone osteonectin (BBON) and human platelet osteonectin (HPON) by N-glycanase, their ability to bind to type V collagen is increased, and HPON affinity to collagen V is the same as that of BBON. These data suggest that glycosylation of osteonectin has a direct or regulatory effect on osteonectin binding to collagen V and that the increase in tHON binding upon removal of carbohydrate is the result of a loss of a down-regulation site or direct interference of the carbohydrate at the binding site. To determine the specific role of each N-glycosylation site in tHON, Asn71 and Asn99 were mutated to Gln (N71Q, N99Q) and Thr73 and Thr101 mutated to Ala (T73A, T101A) to selectively inhibit oligosaccharide attachment. The binding affinity of N99Q and T101Q to collagen V is markedly increased over wild-type tHON, whereas N71Q and T73A are the same as wild-type tHON. The doubled mutant (N71,99Q) binds identically to collagen V as N99Q and T101A. These data suggest that only the position 99 glycosylation site (Asn99-X-Thr101) in tHON is important in the reduction of binding of osteonectin to collagen V. Consistent with the binding data is the observation that both the N71Q and T73A mutant proteins migrate on SDS-polyacrylamide gel electrophoresis gels identically to wild-type tHON, suggesting that there is little or no N-glycosylation of residue 71 in wild-type osteonectin.
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PMID:Role of N-linked glycosylation in human osteonectin. Effect of carbohydrate removal by N-glycanase and site-directed mutagenesis on structure and binding of type V collagen. 755 69

Extracellular and cell surface proteins are generally modified with N-linked glycans and glycopeptide enrichment is an attractive tool to analyze these proteins. The role of N-linked glycoproteins in cardiovascular disease, particularly ischemia and reperfusion injury, is poorly understood. Observation of glycopeptides by mass spectrometry is challenging due to the presence of abundant, nonglycosylated analytes, and robust methods for purification are essential. We employed digestion with multiple proteases to increase glycoproteome coverage coupled with parallel glycopeptide enrichments using hydrazide capture, titanium dioxide, and hydrophilic interaction liquid chromatography with and without an ion-pairing agent. Glycosylated peptides were treated with PNGase F and analyzed by liquid chromatography-MS/MS. This allowed the identification of 1556 nonredundant N-linked glycosylation sites, representing 972 protein groups from ex vivo rat left ventricular myocardium. False positive "glycosylations" were observed on 44 peptides containing a deamidated Asn-Asp in the N-linked sequon by analysis of samples without PNGase F treatment. We used quantitation via isobaric tags for relative and absolute quantitation (iTRAQ) and validation with dimethyl labeling to analyze changes in glycoproteins from tissue following prolonged ischemia and reperfusion (40 mins ischemia and 20 mins reperfusion) indicative of myocardial infarction. The iTRAQ approach revealed 80 of 437 glycopeptides with altered abundance, while dimethyl labeling confirmed 46 of these and revealed an additional 62 significant changes. These were mainly from predicted extracellular matrix and basement membrane proteins that are implicated in cardiac remodeling. Analysis of N-glycans released from myocardial proteins suggest that the observed changes were not due to significant alterations in N-glycan structures. Altered proteins included the collagen-laminin-integrin complexes and collagen assembly enzymes, cadherins, mast cell proteases, proliferation-associated secreted protein acidic and rich in cysteine, and microfibril-associated proteins. The data suggest that cardiac remodeling is initiated earlier during reperfusion than previously hypothesized.
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PMID:Quantitative N-linked glycoproteomics of myocardial ischemia and reperfusion injury reveals early remodeling in the extracellular environment. 2144 15