Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have analyzed the functional domain structure of rat mammary glucosidase I, an enzyme involved in N-linked glycoprotein processing, using biochemical and immunological approaches. The enzyme contains a high mannose type sugar chain that can be cleaved by endo-beta-N-acetyl-D-glucosaminidase H without significantly affecting the catalytic activity. Based on trypsin digestion pattern and the data on membrane topography, glucosidase I constitutes a single polypeptide chain of 85 kDa with two contiguous domains: a membrane-bound domain that anchors the protein to the endoplasmic reticulum and a luminal domain. A catalytically active 39-kDa domain could be released from membranes by limited proteolysis of saponin-permeabilized membranes with trypsin. This domain appeared to contain the active site of the enzyme and had the ability to bind to glucosidase I-specific affinity gel. Phase partitioning with Triton X-114 indicated the amphiphilic nature of the native enzyme, consistent with its location as an integral membrane protein, whereas the 39-kDa fragment partitioned in the aqueous phase, a characteristic of soluble polypeptide. These results indicate that glucosidase I is a transmembrane protein with a luminally oriented catalytic domain. Such an orientation of the catalytic domain may facilitate the sequential processing of asparagine-linked oligosaccharide, soon after its transfer en bloc by the oligosaccharyl transferase complex in the lumen of endoplasmic reticulum.
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PMID:Glucosidase I, a transmembrane endoplasmic reticular glycoprotein with a luminal catalytic domain. 188 88

Trimming glucosidase I has been purified about 400-fold from pig liver crude microsomes by fractional salt/detergent extraction, affinity chromatography and poly(ethylene glycol) precipitation. The purified enzyme has an apparent molecular mass of 85 kDa, and is an N-glycoprotein as shown by its binding to concanavalin A-Sepharose and its susceptibility to endo-beta-N-acetylglucosaminidase (endo H). The native form of glucosidase I is unusually resistant to non-specific proteolysis. The enzyme can, however, be cleaved at high, that is equimolar, concentrations of trypsin into a defined and enzymatically active mixture of protein fragments with molecular mass of 69 kDa, 45 kDa and 29 kDa, indicating that it is composed of distinct protein domains. The two larger tryptic fragments can be converted by endo H to 66 kDa and 42 kDa polypeptides, suggesting that glucosidase I contains one N-linked high-mannose sugar chain. Purified pig liver glucosidase I hydrolyzes specifically the terminal alpha 1-2-linked glucose residue from natural Glc3-Man9-GlcNAc2, but is inactive towards Glc2-Man9-GlcNAc2 or nitrophenyl-/methyl-umbelliferyl-alpha-glucosides. The enzyme displays a pH optimum close to 6.4, does not require metal ions for activity and is strongly inhibited by 1-deoxynojirimycin (Ki approximately 2.1 microM), N,N-dimethyl-1-deoxynojirimycin (Ki approximately 0.5 microM) and N-(5-carboxypentyl)-1-deoxynojirimycin (Ki approximately 0.45 microM), thus closely resembling calf liver and yeast glucosidase I. Polyclonal antibodies raised against denatured pig liver glucosidase I, were found to recognize specifically the 85 kDa enzyme protein in Western blots of crude pig liver microsomes. This antibody also detected proteins of similar size in crude microsomal preparations from calf and human liver, calf kidney and intestine, indicating that the enzymes from these cells have in common one or more antigenic determinants. The antibody failed to cross-react with the enzyme from chicken liver, yeast and Volvox carteri under similar experimental conditions, pointing to a lack of sufficient similarity to convey cross-reactivity.
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PMID:Purification and characterization of trimming glucosidase I from pig liver. 267 80

Glucosidase I, the first enzyme in the N-linked oligosaccharide processing pathway, cleaves the distal alpha 1,2-linked glucose residue from the Glc3-Man9-GlcNAc2 oligosaccharide precursor highly specifically. A human hippocampus cDNA library was screened against oligonucleotide probes, generated by PCR using primers derived from the amino acid sequences of tryptic peptides of pig liver glucosidase I. Two independent lambda clones were isolated which allowed the construction of a full-length glucosidase I cDNA of 2881 bp. This cDNA construct encodes, in a single open reading frame, a polypeptide of 834 amino acids corresponding to a molecular mass of 92 kDa. The 92-kDa protein contains a single N-glycosylation site of the Asn-Xaa-Thr/Ser type at Asn655, as well as a strongly hydrophobic sequence close to its N-terminus (amino acids 38-58) which, most likely, functions as a transmembrane anchor. The amino acid sequences of all tryptic peptides of the pig liver enzyme were found, with little deviation, within the coding sequence. This demonstrates the authenticity of the cDNA construct and the close evolutionary relationship between the enzymes from human hippocampus and pig liver. In contrast, the nucleotide and amino acid sequence revealed no homology with other processing enzymes cloned so far. Transfection of COS 1 cells with the glucosidase I cDNA construct resulted in overexpression (about fourfold) of enzymic activity, which was inhibited strongly by 1-deoxynojirimycin or N,N-dimethyl-deoxynojirimycin. The expressed enzyme, with a molecular mass of 95 kDa, is degraded by endoglycosidase H to a 93-kDa form, indicating that it carries a high-mannose oligosaccharide chain at Asn655. The presence of this glycan is in line with the localization of glucosidase I in the lumen of the endoplasmic reticulum, shown by immunofluorescence microscopy. The hydrophobicity profile as well as the removal by trypsin of an approximately 4-kDa polypeptide from the membrane-associated glucosidase I in intact microsomal structures, supports the view that the enzyme is a type-II transmembrane glycoprotein, which contains a short cytosolic tail of approximately 37 amino acids, followed by a single transmembrane domain and a large C-terminal catalytic domain located on the luminal side of the endoplasmic reticulum membrane.
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PMID:Cloning and expression of glucosidase I from human hippocampus. 763 46

Glucosidase I initiates the processing of the oligosaccharide, Glc3Man9GlcNAc2, in newly assembled glycoproteins by excising the distal alpha 1,2-linked glucosyl residue in the oligosaccharide. Earlier, the enzyme purified from the ER of rat and bovine mammary gland has been found to have M(r) of 85 kDa, as examined by SDS-PAGE along with a domain structure in which a 39 kDa lumenally-oriented region is anchored to the ER through a transmembrane segment and a short cytoplasmic tail. These studies were further extended to include the enzyme from several different tissues of the rat, mouse, guinea pig and bovine mammary glands, sheep liver and pig kidney. Using anti-rat glucosidase I antibody as a probe and several biochemical parameters such as SDS-PAGE analysis, trypsin-catalyzed digestion, ConA-binding, endo H susceptibility and peptide mapping analysis by cleavage of the tryptophanyl peptide linkages within the enzyme, it was found that glucosidase I in all of the tissue sources examined has an M(r) of 85 kDa and is cross-reactive to anti-rat glucosidase antibody. The enzyme is a high mannose glycoprotein, and has domain features in its structure; the enzyme from mouse, rat, guinea pig and bovine mammary glands and sheep liver is sequentially cleaved by trypsin to generate fragments of 69, 55 and 39 kDa. The rate of release of the different fragments differs for different sources, indicating some evolutionary changes in its primary structure. The trypsin-released fragments from pig kidney enzyme are 69, 45 and 29 kDa in size, identical to the same observed earlier for pig liver.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Conserved structural features in glycoprotein processing glucosidase I from several tissues and species. 800 15

A generally applicable, rapid, and sensitive method for profiling and sequencing of glycoprotein-associated N-linked oligosaccharides from protein gels was developed. The method employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation and purification and in-gel deglycosylation using PNGase F for glycan release. Profiles of the neutral glycans from bovine ribonuclease B, chicken ovalbumin, and human immunoglobulin G (IgG), as well as sialic acid-containing sugars (following esterification of the acidic groups) of bovine fetuin and bovine alpha1-acid glycoprotein, were obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and by normal-phase high-performance liquid chromatography following fluorescent labeling. Oligosaccharides were sequenced using specific exoglycosidases, and digestion products were analyzed by MALDI MS. Between 50 and 100 pmol (1.5 to 15 microg) of glycoprotein applied to the gel was sufficient to characterize its oligosaccharide contents. The identity of all glycoproteins investigated could be confirmed after deglycosylation by in-gel trypsin treatment followed by MALDI MS mass mapping and matching the measured molecular weights to a sequence database. The technique was used for the characterization of the glycan moieties of human immunodeficiency virus recombinant gp120 (Chinese hamster ovary cells) and to monitor changes in the glycosylation of this glycoprotein when produced in the presence of a glucosidase I inhibitor. Furthermore, since heavy and light chains of IgG became separated by SDS-PAGE, it could be established that most glycans were associated with the heavy chains.
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PMID:Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography. 923 2

In the current model for Glc3Man9GlcNAc2-P-P-Dol assembly, Man5GlcNAc2-P-P-Dol, Man-P-Dol, and Glc-P-Dol are synthesized on the cytoplasmic face of the ER and diffuse transversely to the lumenal leaflet where the synthesis of the lipid-bound precursor oligosaccharide is completed. To establish the topological sites of Glc-P-Dol synthesis and the lipid-mediated glucosyltransfer reactions involved in Glc3Man9GlcNAc2-P-P-Dol synthesis in ER vesicles from pig brain, the trypsin-sensitivity of Glc-P-Dol synthase activity and the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) was examined in sealed microsomal vesicles. Since ER vesicles from brain do not contain glucose 6-phosphate (Glc 6-P) phosphatase activity, the latency of the lumenally oriented, processing glucosidase I/II activities was used to assess the intactness of the vesicle preparations. Comparative enzymatic studies with sealed ER vesicles from brain and kidney, a tissue that contains Glc 6-P phosphatase, demonstrate the reliability of using the processing glucosidase activities as latency markers for topological studies with microsomal vesicles from non-gluconeogenic tissues lacking Glc 6-P phosphatase. The results obtained from the trypsin-sensitivity assays with sealed microsomal vesicles from brain are consistent with a topological model in which Glc-P-Dol is synthesized on the cytoplasmic face of the ER, and subsequently utilized by the three Glc-P-Dol-mediated GlcTases after "flip-flopping" to the lumenal monolayer.
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PMID:Topological studies on the enzymes catalyzing the biosynthesis of Glc-P-dolichol and the triglucosyl cap of Glc3Man9GlcNAc2-P-P-dolichol in microsomal vesicles from pig brain: use of the processing glucosidases I/II as latency markers. 985 42