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)

Active human tissue plasminogen activator variant kringle-2-serine protease (K2 + SP domains; referred to as MB1004) was synthesized as a secreted protein in Escherichia coli, isolated, and characterized. MB1004 is a relatively large and complex protein, approximately 38 kDa in size and containing nine disulfide bonds. MB1004 without a pro region was secreted into the periplasm of E. coli by fusing the protein to the PhoA leader peptide expressed from the tac promoter. Approximately 1% (20 micrograms/L broth) of the secreted MB1004 was purified from E. coli homogenates as a soluble, active enzyme by using a combination of lysine and Erythrina inhibitor affinity chromatography. Purified MB1004 was monomeric and single-chain, and the N-terminus was identical with the predicted amino acid sequence. The specific activity of purified MB1004 from E. coli was compared against the equivalent recombinant material purified from mammalian cells that was naturally glycosylated (MB1004G) or deglycosylated after treatment with N-glycanase (MB1004N). Results from four different in vitro assays showed that MB1004 and MB1004N had similar activities. Both exhibited 4-12-fold higher specific activity than MB1004G in plasminogen activation assays. These results suggest that an inaccurate picture of specific activity can be obtained if the effects of glycosylation are not considered. By utilization of secretion in E. coli, nonglycosylated MB1004 was purified without in vitro refolding and was shown to be suitable for structure-function studies.
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PMID:Secretion of active kringle-2-serine protease in Escherichia coli. 212 81

The technique of high-pH anion-exchange chromatography with pulsed amperometric detection has recently been shown to be a powerful method for resolving closely related oligosaccharides [M. R. Hardy and R. R. Townsend, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 3289-3293]. This report describes separations involving a total of nineteen different high-mannose, hybrid and complex-type oligosaccharides isolated after peptide: N-glycosidase F (PNGase F) or endo-beta-N-acetylglucosaminidase H digestion of glycoproteins. Separations were carried out at a constant base concentration (0.1 M NaOH) using linear gradients from 0 to 0.2 M sodium acetate. The applicability of this chromatography for profiling the N-linked oligosaccharides of glycoproteins was demonstrated by generating "oligosaccharide maps" of PNGase F-liberated oligosaccharides from recombinant human tissue plasminogen activator, ribonuclease b, human transferrin, and bovine fetuin. Methods for recovering salt-free oligosaccharides after this chromatography were also investigated. On-line ion suppression with an anionic micromembrane suppressor cartridge was found to be capable of effective desalting up to a total sodium ion concentration of 0.15-0.2 M at a flow-rate of 1 ml/min. After high-pH anion-exchange chromatography with ion suppression, collected oligosaccharides were analyzed by fast-atom bombardment mass spectrometry after conversion to permethyl derivatives or after reductive amination with rho-aminobenzoic acid ethyl ester.
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PMID:Analysis of glycoprotein-derived oligosaccharides by high-pH anion-exchange chromatography. 232 8

Recombinant human uterine tissue plasminogen activator (tPA), in part metabolically labeled with [6-3H]glucosamine or [35S]sulfate, was isolated from mouse epithelial cells (C127). Oligosaccharides present were liberated by treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated by high-performance liquid chromatography. The glycans were characterized by digestion with exoglycosidases, methylation analysis and, in part, by acetolysis and 1H-NMR spectroscopy. Glycopeptides comprising individual glycosylation sites were identified by N-terminal amino acid sequencing. The results demonstrate that recombinant tPA from C127 cells carries at Asn117 oligomannosidic glycans with 5-8 mannose residues as well as small amounts of hybrid-type species. Asn184 is only partially glycosylated and substituted by fucosylated triantennary and small amounts of diantennary N-acetyllactosaminic glycans. Likewise, Asn448 carries predominantly fucosylated triantennary species, in addition to, small amounts of diantennary and tetraantennary oligosaccharides. As a characteristic feature, part of the triantennary glycans at Asn184 and Asn448 contain additional Gal(alpha 1-3) substituents and/or sulfate groups linked to position six of beta-galactosyl residues forming NeuAc(alpha 2-3)[HO3S-6]Gal(beta 1-4) units. Oligosaccharides attached to Asn448 are almost completely substituted by (alpha 2-3)- or (alpha 2-6)-linked sialic acid residues and carry the majority of sulfate groups present. Glycans at Asn184 were found to be less sialylated and sulfated.
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PMID:Carbohydrate structure of recombinant human uterine tissue plasminogen activator expressed in mouse epithelial cells. 251 86

A sensitive and specific strategy has been developed for determining the sites of attachment of Asn-linked carbohydrates in glycoproteins, and defining the compositions and molecular heterogeneity of carbohydrates at each specific attachment site. In this carbohydrate 'fingerprinting' strategy, potential glycopeptides are identified by comparing the high pressure liquid chromatography (HPLC) chromatograms of proteolytic digests of a glycoprotein obtained before and after digestion with a glycosidase, usually peptide:N-glycosidase F (PNGase F). The glycopeptide-containing HPLC fractions are analyzed by fast atom bombardment mass spectrometry (FAB MS) prior to and after digestion with PNGase F to identify the former glycosylation site peptide and its sequence location (Carr and Roberts, (1986) Anal. Biochem. 157, 396-406). Carbohydrates are extracted from these fractions as the peracetates which are then permethylated and analyzed by FAB MS. The spectra exhibit molecular weight-related ions for each of the parent oligosaccharides present in the fraction which provide composition in terms of hexose, deoxyhexose, N-acetylhexosamine and sialic acid. The relative ratios of these peaks reflect the relative abundances of the various carbohydrate homologs present in the mixture. The derivatives formed are directly amenable to methylation analysis for determination of linkage. This strategy enables the structural classes of carbohydrates at specific attachment sites to be determined using only a few nmol of glycoprotein. The carbohydrate fingerprinting strategy has been applied to a number of glycoproteins including tissue plasminogen activator, the results for which are described herein.
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PMID:Structural fingerprinting of Asn-linked carbohydrates from specific attachment sites in glycoproteins by mass spectrometry: application to tissue plasminogen activator. 314 14

This report describes the N-glycosylation mapping of recombinant tissue plasminogen activator (rt-PA) using micellar electrokinetic capillary chromatography. The carbohydrate structures were tentatively assigned by comparison with the anion-exchange fractionated oligosaccharides and by a comparison with previously reported data. The separation was shown to rely mainly on the degree of sialylation of the oligosaccharides, allowing a quantitative determination of the proportion of neutral and mono- to tetrasialylated structures. Significant differences in the oligosaccharide distribution of the two variants of rt-PA, which differ by the presence (type I) or the absence (type II) of oligosaccharides at the Asn-184 site, were observed. The distribution of the oligosaccharides at each of the rt-PA glycosylation sites was then determined. Glycopeptides were prepared by tryptic digestion of rt-PA and isolated using two consecutive chromatographic procedures. The glycopeptides were finally treated with N-glycanase, and the resulting oligosaccharides were analysed by capillary electrophoresis. Oligosaccharide mapping revealed that the Asn-448 and Asn-184 sites carry the same population of complex-type oligosaccharides but that the relative amounts of each oligosaccharide vary markedly. High-pH anion-exchange chromatography performed on the desialylated oligosaccharides at each glycosylation site showed that the degree of microheterogeneity was related not only to the degree of sialylation but also to structural differences in the oligosaccharide sequences. From the results as a whole, we concluded that the Asn-448 site contains a greater proportion of heavily sialylated structures and has a higher degree of microheterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:N-glycosylation site mapping of recombinant tissue plasminogen activator by micellar electrokinetic capillary chromatography. 779 87

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