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)

Barley (1----3,1----4)-beta-glucan 4-glucanohydrolase (EC 3.2.1.73) isoenzyme EII carries 4% by weight carbohydrate and is more stable at elevated temperatures than isoenzyme EI, which has no associated carbohydrate. The relationship between carbohydrate content and thermostability has been investigated by treatment of the two isoenzymes with N-glycopeptidase F (EC 3.5.1.52). Removal of carbohydrate from isoenzyme EII results in a decrease in the enzyme's thermostability, but treatment of isoenzyme EI with the N-glycopeptidase F has no effect. In addition, removal of a single N-glycosylation site in isoenzyme EII (Asn190-Ala-Ser) by site-directed mutagenesis of the corresponding cDNA led to a reduction in thermostability, while the introduction of this site into isoenzyme EI enhanced stability. We conclude that N-glycosylation of Asn190 enhances the stability of isoenzyme EII at elevated temperatures, but that other factors related to their primary structures also contribute to the differences in thermostabilities of the barley (1----3,1----4)-beta-glucanases.
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PMID:Differences in the thermostabilities of barley (1----3,1----4)-beta-glucanases are only partly determined by N-glycosylation. 151 97

Limited proteolysis of human alpha 2-macroglobulin (alpha 2M) by a novel bacterial proteinase resulted in the isolation of a soluble 20-kDa domain. The isolated fragment contained the receptor recognition site, expressed on alpha 2M complexes, as it competed effectively with alpha 2M-trypsin for binding to the receptor on skin fibroblasts. The fragment also reacted with two monoclonal antibodies which define epitopes that are part of the receptor recognition site. Characterization of the 20-kDa domain showed it to contain an intact disulfide bridge, while its susceptibility to N-glycanase and reaction with concanavalin A indicated the presence of N-linked carbohydrate. The NH2-terminal sequence (Glu-Glu-Phe-Pro-Phe-Ala-Leu-Gly-Val-Glu-Thr-Leu-Pro-Glu-Thr-Cys-Asp-Glu -Pro) proved this fragment to constitute the COOH terminus of human alpha 2M. Proteolysis occurred at Lys1313-Glu which together with the observation that tosyllysine chloromethyl ketone was an effective inhibitor of the bacterial proteinase, would indicate the latter to hydrolyze preferentially peptide bonds carboxyl-terminal to lysine residues.
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PMID:The receptor-binding domain of human alpha 2-macroglobulin. Isolation after limited proteolysis with a bacterial proteinase. 242 72

A detailed study of the oligosaccharide specificity of the almond enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A, was undertaken by comparing the rate of release of intact oligosaccharide chains from defined glycopeptides of all significant classes. The oligosaccharide of a trisialo-triantennary pentaglycopeptide from fetuin was released at the highest rate. A procedure was developed for the isolation of this glycopeptide in high yield from 5 g fetuin. Sequence analysis established the structure as Leu-Ala-Asn(CHO)-Cys-Ser. The Cys(Cm) and the Cys(Ae) derivatives of the glycopeptide were reacted with 4-(dimethylamino)-azobenzene-4'-sulfonyl (dabsyl) chloride to yield a monosubstituted and a disubstituted glycopeptide respectively. This chromophore confers high sensitivity at 436 nm on a pentapeptide backbone having minimal bonds for protease cleavage. A procedure was developed wherein these dabsyl derivatives were used in a high-performance liquid chromatography assay. The dabsyl-pentapeptide was retarded significantly from the dabsyl-glycopeptide and provided a sensitive method (1-2 nmol) of detection of peptide-N4-(N-acetylglucosaminyl)asparagine amidase activity. Enzyme was detected in crude extracts of all eight seed sources surveyed. The enzyme from Pisum sativum was partially purified and its properties were compared with the corresponding enzyme from almonds.
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PMID:Detection and quantification of peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidases. 243 26

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

Membrane preparations of cells expressing the cloned rat hypothalamus melanocortin receptor, MC3, have been photoaffinity labelled using a radiolabelled photoreactive analogue of alpha-MSH, [125I-Tyr2,Nle4,D-Phe7,ATB-Lys11]alpha-MSH. SDS-PAGE followed by autoradiography showed a single band at 53-56 kDa for the native receptor or 35 kDa after deglycosylated with PNGase F, consistent with the predicted cDNA sequence. Receptor binding studies with alpha-MSH, gamma-MSH and [Nle4,D-Phe7]alpha-MSH established that alpha-MSH and gamma-MSH had similar affinities while [Nle4,D-Phe7]alpha-MSH bound 100 times more strongly. These results suggest that the receptor recognises the conserved 'core sequence' (-Met-Glu/Gly-His-Phe-Arg-Trp-) of MSH/ACTH peptides. The binding affinities of alanine-substituted analogues of alpha-MSH were determined to investigate the role of individual residues in ligand-receptor interactions. While in the terminal regions only the replacement of Tyr2 reduced the affinity of the peptide, replacement of Met4, Phe7, Arg8 and Trp9 within the peptide core led to a significant loss of affinity. Glu5 appeared unimportant for receptor recognition.
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PMID:The melanocortin (MC3) receptor from rat hypothalamus: photoaffinity labelling and binding of alanine-substituted alpha-MSH analogues. 806 18

Pneumocystis carinii is a common cause of life-threatening pneumonia in immunodeficient patients. Pulmonary surfactant protein A (SP-A), an alveolar glycoprotein containing collagen-like and carbohydrate recognition domains (CRD), binds P. carinii and enhances adherence to alveolar macrophages. In this study, we examined the structural basis of the interaction between SP-A and the major surface glycoprotein of P. carinii (MSG). Rat SP-A bound to purified rat P. carinii-derived MSG in a saturable and calcium-dependent manner, which was partially reversible by coincubation with excess monosaccharides, or pretreatment of MSG with N-glycanase. Mutant recombinant SP-As with neutral amino acid substitutions for the predicted calcium- and carbohydrate-coordinating residues of the CRD were synthesized in insect cells using baculovirus vectors and tested for binding to MSG. Substitutions of negatively charged (Glu195, Glu202, and Asp215) and polar residues (Asn214) of the CRD with alanine but not substitution of the Arg197 with glycine reduced the binding of SP-A to mannose-Sepharose beads and to MSG. Deletion of the N-linked oligosaccharides from SP-A by mutagenesis of the consensus sequences for glycosylation had no effect on binding. We conclude that the CRD mediates the binding of SP-A to oligosaccharides attached to MSG.
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PMID:The carbohydrate recognition domain of surfactant protein A mediates binding to the major surface glycoprotein of Pneumocystis carinii. 920 57

Glycoamidases (peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, EC 3.5.1.52; also known as peptide: N-glycanases (PNGases) release N-linked oligosaccharides from glycopeptides and/or glycoproteins by hydrolyzing the glycosylated beta-amide bond of the asparagine side chain. The most widely used glycoamidases are those from Flavobacterium meningosepticum (glycoamidase F or PNGase F) and almond emulsin (glycoamidase A or PNGase A). To study the substrate structure requirement of these enzymes systematically, we synthesized >30 glycopeptides containing cellobiose, lactose, GlcNAc, and di-N,N'-acetylchitobiose (CTB). The length of the peptide was varied from one to five amino acids, and glycosylamines were linked to either Asn or Gln located at different positions in the peptide, including NH2 and COOH termini. Neither enzyme could cleave cellobiose and lactose glycopeptides, indicating that the 2-acetamido group on the Asn-linked GlcNAc is important in the recognition by both glycoamidases A and F. GlcNAc peptides could be cleaved by both enzymes, albeit not as effectively as CTB glycopeptides. Neither enzyme requires the Asn-Xaa-(Ser/Thr) sequence (required for N-glycosylation) for activity. Glycoamidase A could even hydrolyze a Gln-bound CTB glycopeptide, whereas the action of glycoamidase F on this substrate is minimal. While glycoamidase A could act on CTB dipeptides, glycoamidase F preferred a tripeptide or longer. The Km and Vmax values of glycoamidase A for t-butoxycarbonyl-(CTB)-Asn-Ala-Ser-OMe were 2.1 mM and 0.66 micromol/min/mg, respectively. A natural glycodipeptide, Man9-GlcNAc2-Asn-Phe, was also completely hydrolyzed by glycoamidase A.
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PMID:Detailed studies on substrate structure requirements of glycoamidases A and F. 934 Nov 45

The extent of N-glycosylation of yeast external invertase at each of the 14 potential sites was determined by the combination of proteolytic digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS). The average molecular mass of the intact external invertase was determined as 97 kDa by MALDI/TOF-MS. The intact protein was digested with trypsin, Lys-C and Asp-N, followed by high-performance liquid chromatographic separation. The proteolytic digests were analyzed by MALDI/MS screening for the glycopeptides. The glycopeptides were then treated with peptide:N-glycosidase F (PNGase F) and/or endo-beta-N-acetylglucosaminidase (Endo H) and the molecular mass of the deglycosylated peptide was determined by MALDI/MS and matched with the peptide predicted by a computer program. The sequences of some peptides or deglycosylated peptides were identified by the MALDI post-source decay technique. The size of the oligosaccharide, the degree of glycosylation and the distribution of the oligosaccharides at each individual potential glycosylation site were characterized. This information goes for beyond previously published data and sometimes differs from them. During this study, the amino acid sequence originally derived from the DNA sequence of the gene coding for invertase was also verified and it was found that this protein when expressed from SUC2 gene might be created as more than one sequence which differ by a few amino acid substitutions (Asn58<-->Thr, Asn65-->His and Val412<-->Ala).
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PMID:Determination of N-linked glycosylation of yeast external invertase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 1022 60

Coagulation factor V (FV) circulates in two forms, FV1 and FV2, having slightly different molecular masses and phospholipid-binding properties. The aim was to determine whether this heterogeneity is due to the degree of glycosylation of Asn(2181). FVa1 and FVa2 were isolated and digested with endoglycosidase PNGase F. As judged by Western blotting, the FVa2 light chain contained two N-linked carbohydrates, whereas FVa1 contained three. Wild-type FV and three mutants, Asn(2181)Gln, Ser(2183)Thr, and Ser(2183)Ala, were expressed in COS1 cells, activated by thrombin, and analyzed by Western blotting. Wild-type FVa contained the 71 kDa-74 kDa doublet, whereas the Asn(2181)Gln and Ser(2183)Ala mutants contained only the 71 kDa light chain. In contrast, the Ser(2183)Thr mutant gave a 74 kDa light chain. This demonstrated that the third position in the Asn-X-Ser/Thr consensus affects glycosylation efficiency, Thr being associated with a higher degree of glycosylation than Ser. The Ser(2183)Thr mutant FVa was functionally indistinguishable from plasma-purified FVa1, whereas Asn(2181)Gln and Ser(2183)Ala mutants behaved like FVa2. Thus, the carbohydrate at Asn(2181) impaired the interaction between FVa and the phospholipid membrane, an interpretation consistent with a structural analysis of a three-dimensional model of the C2 domain and the position of a proposed phospholipid-binding site. In conclusion, we show that the FV1-FV2 heterogeneity is caused by differential glycosylation of Asn(2181) related to the presence of a Ser rather than a Thr at the third position in the consensus sequence of glycosylation.
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PMID:Partial glycosylation of Asn2181 in human factor V as a cause of molecular and functional heterogeneity. Modulation of glycosylation efficiency by mutagenesis of the consensus sequence for N-linked glycosylation. 1052 Dec 65

The effect of ammonium chloride was determined on a culture of CHO cells transfected with the human erythropoietin (EPO) gene. Cell growth was inhibited above a culture concentration of 5 mM NH(4)Cl with an IC-50 determined to be 33 mM. The specific production of EPO increased with the addition of NH(4)Cl above 5 mM. At 10 mM NH(4)Cl, the final cell density after 4 days in culture was significantly lower but the final yield of EPO was significantly higher. This appeared to be due to continued protein production after cell growth had ceased. The metabolic effects of added NH(4)Cl included higher specific consumption rates of glucose and glutamine and an increased rate of production of alanine, glycine, and glutamate. The EPO analyzed from control cultures had a molecular weight range of 33-39 kDa and an isoelectric point range of 4.06-4.67. Seven distinct isoforms of the molecule were identified by two-dimensional electrophoresis. This molecular heterogeneity was ascribed to variable glycosylation. Complete enzymatic de-glycosylation resulted in a single molecular form with a molecular mass of 18 kDa. Addition of NH(4)Cl to the cultures caused a significant increase in the heterogeneity of the glycoforms as shown by an increased molecular weight and pI range. Enzymatic de-sialylation of the EPO from the ammonia-treated and control cultures resulted in identical electrophoretic patterns. This indicated that the effect of ammonia was in the reduction of terminal sialylation of the glycan structures which accounted for the increased pI. Selective removal of the N-glycan structures by PNGase F resulted in two bands identified as the O-glycan linked structure (19 kDa) and the completely de-glycosylated structure (18 kDa). The proportion of the O-linked glycan structure was reduced, and its pI increased in cultures to which ammonia was added. Thus, the glycosylation pattern altered by the presence of ammonia included a reduction in terminal sialylation of all the glycans and a reduction in the content of the O-linked glycan. The addition of a sialidase inhibitor to the cultures had no effect on the ammonia-induced increase in EPO heterogeneity. Also, the effect of ammonia on glycosylation could not be mimicked using the weak base chloroquine in our system.
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PMID:Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation. 1074 5


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