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)

A rat alpha-amidating enzyme (alpha-AE) cDNA has been expressed in mouse C127 cells using a bovine papilloma virus vector in which transcription was regulated by the mouse metallothionein 1 promoter. The cDNA encoding the full length alpha-AE protein was modified to terminate translation at a site preceding the transmembrane and cytoplasmic domains, thereby enabling functional enzyme to be secreted into the medium. Purification of recombinant alpha-AE to homogeneity indicated that the enzyme was synthesized and secreted as two proteins of 75-77 kDA. The observed heterogeneity was due to inefficient glycosylation at Asn660, as demonstrated by glycopeptidase F digestion. Using the synthetic peptide, dansyl-Tyr-Val-Gly, the specific activity of the recombinant enzyme at pH 7.0 was found to be 1.4 mumol/min/mg and the Km of the enzyme was determined to be 3 microM. The purified recombinant enzyme has maximal activity at pH 4.5-5.5; however, a rapid inactivation of the enzyme occurs in acidic solutions in vitro. This inactivation is diminished when activity is measured at pH 7.0-10.0. The availability of large amounts of readily purified, active recombinant alpha-AE should allow detailed probing of reaction mechanism, copper coordination chemistry, and turn-over-based inactivation events.
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PMID:Purification and characterization of functional recombinant alpha-amidating enzyme secreted from mammalian cells. 221 57

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

In this report we examine the primary sequence of a variant glycophorin obtained from erythrocytes of an individual who exhibits an unusual MNSs blood group phenotype. We show that this protein is a hybrid molecule constructed from sequences of alpha- and delta-glycophorins (glycophorins A and B) in a alpha-delta arrangement. Serological typing revealed that the donor's phenotype was M+N+S+s+U+; yet his erythrocytes reacted with some but not all examples of anti-S antisera. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a variant glycophorin band, and immunoblotting and reaction with N-glycanase suggested that its amino terminus resembled that of M-alpha-glycophorin but that its carboxyl terminus did not. A preparation highly enriched in the variant was obtained and used to generate peptide fragments for sequencing. The sequence revealed that the variant was a hybrid molecule whose amino terminus corresponded to M-alpha-glycophorin and whose carboxyl terminus corresponded to S-delta-glycophorin. CNBr cleavage of the variant glycophorin yielded four peptides. The sequence of the amino-terminal CNBr peptide (residues 1-8) was identical to the amino-terminal octapeptide of M-alpha-glycophorin. The proceeding peptide (residues 9-61) contained a segment identical to residues 9-58 of alpha glycophorin, but its carboxyl-terminal sequence had the Gly-Glu-Met sequence from S-delta-glycophorin (residues 27-29). The other two peptides, insoluble in aqueous solutions, contained highly hydrophobic sequences, identical to residues 30-52 and 53-68 of delta-glycophorin. Sequences of overlapping peptides generated by trypsin and V8 protease confirmed the hybrid nature of the variant glycophorin: residues 1-58 were identical to residues 1-58 of M-alpha-glycophorin, and residues 59-100 were entirely identical to residues 27-68 of S-delta-glycophorin. The variant glycophorin is expected to have 4 additional residues at its carboxyl terminus that correspond to the carboxyl-terminal residues 69-72 of delta-glycophorin. The amino acid sequence arrangement of the variant alpha-delta-glycophorin is an exact reciprocal of that found in another hybrid glycophorin, Sta, that is a delta-alpha hybrid. We propose that the two hybrid glycophorins represent the two possible products resulting from a reciprocal recombination event.
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PMID:Amino acid sequence of an alpha-delta-glycophorin hybrid. A structure reciprocal to Sta delta-alpha-glycophorin hybrid. 279 68

We have purified recombinant human interleukin 4 (huIL-4), formerly named B-cell stimulatory factor-1, from supernatants of COS-7 monkey kidney and L-929 cells transfected with the cDNA for huIL-4. The purified protein exhibited a specific activity of 2.6 X 10(7) units/mg in a T-cell proliferation assay and consisted of multiple components on sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibiting Mr values of 15,000, 18,000, and 19,000. All forms of huIL-4 eluted on gel filtration chromatography with an apparent Mr of 22,000. Gas-phase microsequencing identified 26 and 8 amino acid residues at the N and C termini, respectively, all of which were consistent with the cDNA sequence. The site of processing of the signal sequence was found to occur between Gly-24 and His-25. Incubation with N-glycanase converted the 18- and 19-kDa variants to a 15-kDa form. Treatment with endo-beta-N-acetylglucosaminidase H reduced the molecular mass of the 18-kDa variant to 15 kDa, but did not have any apparent effects on the mass of the 19-kDa species. The removal of oligosaccharide by any of these treatments did not affect bioactivity in the T-cell proliferation assay. Neither O-glycanase nor endo-beta-N-acetylglucosaminidase D affected the molecular weight of any of these species. These data suggest that differences in carbohydrate structure account, at least in part, for the observed microheterogeneity.
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PMID:Isolation and characterization of multiple variants of recombinant human interleukin 4 expressed in mammalian cells. 326 May 92

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

The electrophoretic analysis of purified Ole e I, the major allergen from Olea europaea pollen, reveals the presence of two main variants, glycosylated (20.0 kDa) and non-glycosylated (18.5 kDa) components. The glycosylated variant has been identified as a concanavalin A-binding glycoprotein. Its carbohydrate moiety has a molecular mass of about 1.3 kDa (5% weight of the glycosylated allergen), based on mass spectrometry analysis. Enzymatic treatment of native Ole e I with the specific glycosidase PNGase F accounts for an oligosaccharide N-linked to the polypeptide chain. This treatment does not sensibly modify the secondary structure of the protein but diminishes the affinity of the allergen for specific IgE antibodies. Tryptic digestion of Ole e I reveals the presence of a single carbohydrate-containing peptide. This peptide was recognized by the sera of hypersensitive individuals. The amino acid sequence of this peptide is Phe-Lys-Leu-Asn-Thr-Val-Asn-Gly-Thr-Thr-Arg, asparagine at the seventh being the carbohydrate attaching site. The obtained data are discussed in terms of the potential role of the sugar moiety in the allergenic activity of Ole e I.
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PMID:Glycosylation site of the major allergen from olive tree pollen. Allergenic implications of the carbohydrate moiety. 830 97

Recombinant human uterine tissue plasminogen activator (tPA) glycosylation mutants carrying an additional N-glycosylation site in the epidermal-growth-factor-like domain due to the replacement of either Tyr67 by Asn (YN-tPA) or Gly60 by Ser (GS-tPA) were expressed in mouse epithelial cells (C127) in the presence of [6-3H]glucosamine. Glycopeptides comprising individual glycosylation sites were isolated and oligosaccharides attached were liberated by treatment with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Oligosaccharide alditols obtained after reduction were either directly characterized by high-pH anion-exchange chromatography (high-mannose and hybrid-type glycans) or preparatively subfractionated after enzymic desialylation and separation from sulphated asialooligosaccharides (complex-type sugar chains). Individual (sub)fractions of glucans were studied by methylation analysis, liquid secondary-ion mass spectrometry and, in part, by exoglycosidase digestion, whereas corresponding deglycosylated peptides were identified by amino acid analysis and N-terminal amino acid sequencing. The results revealed that Asn117 of YN-tPA carried exclusively high-mannose-type glycans with five to nine mannose residues similar to wild-type tPA expressed in this cell line [Pfeiffer, G., Schmidt, M., Strube, K.-H. & Geyer, R. (1989) Eur. J. Biochem. 186, 273-286]. In contrast, Asn117 of GS-tPA carried only small amounts (about 25%) of high-mannose and hybrid-type species and predominantly complex-type sugar chains (about 75%) which were partially incomplete and mostly devoid of fucose. Newly introduced N-glycosylation sites at Asn67 (YN-tPA) or Asn58 (GS-tPA) as well as those at Asn184 and Asn448 were solely substituted by complex-type glycans. Each carbohydrate attachment site displayed a peculiar oligosaccharide pattern with regard to branching and substitution by Gal alpha 3-residues, sulphate groups, intersecting GlcNAc and lactosamine repeats. Our study clearly demonstrates that creation of a new glycosylation site at Asn58 influenced the oligosaccharide processing and, hence, the glycosylation pattern at Asn117, whereas introduction of a new site at Asn67 did not. The relative amounts of complex-type glycans at Asn117 of GS-tPA correlated with the degree of carbohydrate substitution of Asn58. Therefore, it can be concluded that the presence of a sugar chain at the position and not the Gly to Ser mutation itself is responsible for the observed alteration of GS-tPA glycosylation.
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PMID:Glycosylation of two recombinant human uterine tissue plasminogen activator variants carrying an additional N-glycosylation site in the epidermal-growth-factor-like domain. 830

A high serum alpha-fetoprotein (AFP) level was found in a patient with endometrial adenocarcinoma of the uterus, which appeared to be hepatoid on histological examination. The AFP of this unusual patient was purified by immunoaffinity chromatography and characterized. The electrophoretic profiles on sodium dodecyl sulfate-polyacrylamide get electrophoresis both before and after glycopeptidase F treatment were indistinguishable from those of a hepatoma AFP. This indicates that the patient's AFP was also composed of a single polypeptide chain of Mr 67,000 and an N-linked sugar chain of Mr 3,000. Amino acid sequence analyses of this AFP, and of AFP from hepatoma and umbilical cord serum indicated that the N-terminal sequences were essentially the same. The sequence, Arg-Thr-Leu-His-Arg-Asn-Glu-Tyr-Gly-Ile, was slightly different from previous reports, but matched that deduced from the cDNA sequence. AFP isoforms due to microheterogeneity of the sugar chain were analyzed by lectin affinity electrophoresis using a series of lectins. The AFP isoform profiles were distinct from those of proteins derived from cord serum, hepatoma, yolk sac tumor and gastric cancer. The reverse-transcription of RNA from the tumor tissue followed by a polymerase chain reaction using primers with AFP-specific sequences gave a product of the size and nucleotide sequence expected for AFP. mRNAs possessing the requisite sequences for albumin and transferrin syntheses were also detected in the tumor. The expression of these hepatocyte-specific proteins supported the hepatoid nature of this tumor.
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PMID:Biochemical characterization of alpha-fetoprotein and other serum proteins produced by a uterine endometrial adenocarcinoma. 876 25

Point mutations in the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor have been shown to cause constitutive activation which results in precocious puberty in affected males. We introduced one of these mutations, Asp-556 --> Gly, into the rat LH/hCG receptor and demonstrated that the mutant receptor constitutively activated adenylate cyclase in transfected 293 T cells. The cell surface expression of the mutant receptor was lower than that of the wild type receptor. Pulse-chase studies showed that the 73-kDa precursor of both the mutant and wild type receptors was synthesized at comparable efficiencies. However, post-translational processing of the mutant receptor to the mature 92-kDa form, which has N-linked complex type oligosaccharide chains, was impaired. Sensitivity of the mutant receptor to peptide-N-glycanase F and endoglycosidase H, and insensitivity to sialidase indicated that the 73-kDa species represents the high mannose form that has not yet been trafficked through the medial and trans Golgi. Additionally, although the wild type receptor was palmitoylated, the mutant receptor was not. Although the high mannose 73-kDa species is capable of binding LH/hCG, our results show that post-translational processing in the Golgi is required for the mature 92-kDa receptor to reach the cell surface.
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PMID:Post-translational processing in the Golgi plays a critical role in the trafficking of the luteinizing hormone/human chorionic gonadotropin receptor to the cell surface. 903 11

Human protein S (HPS) has three potential N-linked glycosylation sites at Asn458, 468, 489. To study the role of glycosylation at these sites, PCR mutagenesis was used to abolish the consensus sequence of each N-linked glycosylation site (Asn458-->Gln, Ser460-->Gly; Asn468-->Gln, Thr470-->Gly; Asn489-->Gln, Thr491-->Gly) in full-length HPS cDNA. Each resulting construct was expressed in human kidney 293 cells by stable transfection of cDNA/SV40/adeno/pBR322-derived expression vectors, and conditioned medium was collected for recombinant protein purification. SDS-PAGE gels revealed that glycosylation mutants migrate identically and faster than the wild-type rHPS, showing that each of the three potential N-glycosylation sites contain a similar amount of carbohydrate. Mass spectral analysis yielded similar results and a molecular mass of approximately 78,000 for wild-type HPS. To demonstrate that the difference in mobility between wild-type and mutant protein S is due to their carbohydrate content, plasma-derived HPS and recombinant HPS were subjected to N-glycanase digestion and subsequently shown to migrate identically on SDS-PAGE gels. All forms of HPS have similar time courses for cleavage by alpha-thrombin. Functional studies indicate that wild-type rHPS possesses the same cofactor specific activity as plasma-derived HPS, as tested by a standard clotting assay. Asn458 and Ser460 mutant rHPS have only a slightly higher cofactor activity, whereas the other four mutants have similar clotting activities, compared to wild-type rHPS. In a purified component system, glycosylation mutants of protein S showed a slightly enhanced ability to stimulate APC-mediated factor Va inactivation after an initial lag phase. The interaction of rHPS glycosylation mutants with human C4b-binding protein (C4bp) was also studied by solution phase equilibrium binding assay. Two mutants (Asn458, Ser480) have marginally lower dissociated constants (Kd) with C4bp, whereas the others have the same apparent Kd as wild-type rHPS.
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PMID:The effect of N-linked glycosylation on molecular weight, thrombin cleavage, and functional activity of human protein S. 924 50


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