Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

The effect of carbohydrate removal on the properties of the lysosomal enzyme alpha-L-fucosidase has been investigated by comparatively characterizing N-glycanase-treated and mock-treated control fucosidases. N-Glycanase treatment removed approx. 90% of the carbohydrate from purified native human liver fucosidase as determined by carbohydrate assay after gel filtration on Sephadex G-50, and by Western blotting with a lectin-digoxigenin conjugate and densitometric scanning. Removal of carbohydrate from fucosidase does not affect its catalytic activity, its Km value for synthetic substrate, its recognition and rate of hydrolysis of three natural substrates, or its gross conformation as determined by circular dichroism. However, loss of carbohydrate led to significantly decreased activity at acidic pH values (3.1-4.7), a 0.6 pH unit shift to a more neutral optimum and decreased thermostability. The decreased activity at acidic pH values and the more neutral pH optimum of deglycosylated fucosidase suggest that the presence of carbohydrate is physiologically significant in allowing fucosidase to perform its catabolic function more efficiently in the acidic milieu of the lysosome.
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PMID:The effect of carbohydrate removal on the properties of human liver alpha-L-fucosidase. 154 Jun 52

Zona pellucida (ZP), the extracellular glycocalyx surrounding the mammalian oocyte, is believed to mediate species-specific sperm-egg interaction. Despite numerous studies on characterization of ZP glycoconjugates in several species, little or no information is available on the number and chemical nature of the various components of the rat ZP. In this study we have attempted the biochemical characterization of the rat ZP using endo- and/or exo-glycohydrolases. Intact eggs from superovulated rats were radioiodinated by the chloramine-T method, and the labeled ZP components were resolved on SDS-PAGE under nonreducing conditions. These studies show that the rat ZP consists of three components with apparent molecular masses of 205 kDa (ZP1), 119 kDa (ZP2), and 115 kDa (ZP3). Unlike mouse ZP2 and ZP3, which resolve as distinct components on SDS-PAGE, rat ZP2 and ZP3 show substantial overlap in their molecular sizes and isoelectric points. Treatment of the rat ZP components with exo- (neuraminidase and alpha-L-fucosidase) and/or endo- (endoglycosidase H, endoglycosidase F, N-glycanase, and O-glycanase) glycohydrolases indicated the following: 1) Both rat ZP2 and ZP3 contain N-linked oligosaccharide (OS) units as indicated by their sensitivity to endoglycosidase F and N-glycanase. 2) Treatment with N-glycanase caused a reduction in size of the rat ZP2 and ZP3 components by nearly 50% and 60%, respectively, indicating that the two ZP components are highly glycosylated. 3) Rat ZP3 was sensitive to O-glycanase, suggesting that this ZP component contains O-linked OS unit(s). 4) No evidence was obtained for the presence of fucosyl or sialyl residue(s) on the O-linked OS unit(s) of rat ZP3.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Qualitative characterization of oligosaccharide chains present on the rat zona pellucida glycoconjugates. 159 46

The 18 kDa and 32 kDa lectin binding proteins of Chlamydia trachomatis were characterized as glycoproteins by treatments with glycosidases. The proteins of the serovar L2 whole cell lysate were separated by SDS-PAGE and transferred to nitrocellulose paper. After treatment with an enzyme, the proteins were reacted with a biotinylated lectin. Each of the endoglycosidases tested affected the binding of the lectin to the protein. PNGase F inhibited the binding of Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), and Ulex europaeus agglutinin I (UEAI) to both the 18 kDa and 32 kDa proteins. Endoglycosidase F and H inhibited the binding of these lectins to the 32 kDa protein completely and to the 18 kDa protein partially. In the exoglycosidase treatments, alpha-L-fucosidase prevented binding of only UEAI to the two proteins while beta-galactosidase inhibited the binding of SBA. Mannosidase abolished the binding of all the lectins tested. Neuraminidase had no effect. The proteins isolated by electroelution from the excised gels after SDS-PAGE were digested with an endoglycosidase. PNGase F-treated proteins showed a lower molecular weight mobility in which the lectin binding ability was destroyed. Endo-alpha-N-acetylgalactosaminidase had no effect. The polysaccharide stain of isolated proteins with p-phenylenediamine showed a positive reaction. Radiolabeling with [3H]glucosamine did not reveal the 18 kDa and 32 kDa proteins in autoradiography but [3H]galactose did.
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PMID:The characterization of lectin-binding proteins of Chlamydia trachomatis as glycoproteins. 172 47

Fucosidosis is an inherited lysosomal storage disease due to a deficiency of alpha-L-fucosidase activity. Exponentially growing lymphoid cell cultures from a fucosidosis patient (JH) had 16-fold lower extracellular alpha-L-fucosidase protein and 72-fold lower intracellular alpha-L-fucosidase protein with negligible catalytic activity as compared with the mean of 19 control cultures. The percentage of total alpha-L-fucosidase protein released extracellularly by JH cells was 71% as compared with 35% +/- 9% for control cells. During a 1.5 h pulse with 35S-methionine, alpha-L-fucosidase was synthesized by JH cells as an intracellular doublet with Mr of 58,000 and 56,000 and by control cells as an intracellular form with Mr = 58,000. During a subsequent 21 h chase with unlabeled methionine, JH alpha-L-fucosidase was entirely secreted. In contrast, only 25%-30% of control enzyme was secreted with the remainder retained intracellularly. Thus, JH lymphoid cells synthesized a reduced amount of alpha-L-fucosidase that was catalytically inefficient and was hypersecreted. Treatment of JH alpha-L-fucosidase with N-glycanase produced polypeptide chains with Mr of 52,000 and 54,000. Previously, treatment of control alpha-L-fucosidase with N-glycancase produced a single polypeptide chain with Mr of 52,000 (Biochem Genet 1988; 26: 401-20). The doublet polypeptide chains of alpha-L-fucosidase in JH cultures may represent expression of two distinct allelic forms of mutant alpha-L-fucosidase.
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PMID:Defective expression of alpha-L-fucosidase by lymphoid cells of a fucosidosis patient. 187 10

We have previously published a two-dimensional (2-D) mapping technique for N-linked oligosaccharides using pyridylaminated derivatives (PA-oligosaccharides) (N. Tomiya et al. Anal. Biochem. 171, 73-90, 1988). We now report an extension of this method to GalNAc-containing N-linked oligosaccharides. The new 2-D map was prepared from the elution data of 40 different GalNAc-containing oligosaccharides, 16 of which were obtained directly from human urinary kallidinogenase by digestion with glycopeptidase A. The other 24 oligosaccharides were derived by subsequent digestion of the 16 original oligosaccharides with beta-galactosidase or alpha-fucosidase. Each of the 40 oligosaccharide derivatives was separated by high-performance liquid chromatography using ODS-silica and amide-silica columns. The 2-D map constructed by plotting elution position of each oligosaccharide (expressed in terms of glucose units) can be useful as such in delineating the structure of an unknown oligosaccharide by direct placement of its elution positions in the 2-D map. Multiple regression analysis of the data as performed previously yielded parameters related to the contribution of each component monosaccharide unit to the elution profile. The best results were obtained when the GalNAc-containing PA-oligosaccharides were classified into an F-series (those containing Fuc alpha 6GlcNAc-PA) and a Z-series (all others), based on our previous classification method. These calculated values are useful in predicting oligosaccharide structure from known elution values as well as to predict elution volumn from a known structure. The structure of a minor GalNAc-containing oligosaccharide in human urinary kallidinogenase was elucidated using these newly calculated values.
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PMID:Two-dimensional elution map of GalNAc-containing N-linked oligosaccharides. 843 1

We previously showed that mouse ST8Sia II (STX) exhibits polysialic acid (PSA) synthase activity in vivo as well as in vitro (Kojima, N., Yoshida, Y., and Tsuji, S. (1995) FEBS Lett. 373, 119-122, 1995). In this paper, we reported that the neural cell adhesion molecule (NCAM) was specifically polysialylated by a single enzyme, ST8Sia II. PSA-expressing Neuro2a cells (N2a-STX) were established by stable transfection of the mouse ST8Sia II gene. Only the 140- and 180-kDa isoforms of NCAM in N2a-STX cells were specifically polysialylated in vivo, although other membrane proteins of N2a-STX were polysialylated in vitro. A recombinant soluble mouse ST8Sia II synthesized PSA on a recombinant soluble NCAM fused with the Fc region of human IgG1 (NCAM-Fc) as well as fetuin. However, NCAM-Fc served as a 1500-fold better acceptor for ST8Sia II than fetuin. Treatment of NCAM-Fc with Charonia lampas alpha-fucosidase, which is able to cleave alpha1,6-linked fucose, clearly reduced the polysialylation of NCAM-Fc by ST8Sia II. PSA was not synthesized on the N-glycanase-treated NCAM-Fc polypeptide or the free N-glycans of NCAM-Fc. When fetuin and its glycopeptide and N-glycans of fetuin were used as substrates for ST8Sia II, PSA was found to be synthesized on native fetuin and its glycopeptide but not on free N-glycans. These results strongly suggested that core alpha1, 6-fucose on N-glycans as well as the antennary structures of N-glycans and the polypeptide regions are required for the polysialylation by ST8Sia II. Furthermore, oligo and single alpha2, 8-sialylated glycoproteins were no longer polysialylated by mouse ST8Sia II. Therefore, the single enzyme, ST8Sia II, directly transferred all alpha2,8-sialic acid residues on the alpha2,3-linked sialic acids of N-glycans of specific NCAM isoforms to yield PSA-NCAM. Polysialylation did not require any initiator alpha2, 8-sialyltransferase but did depend on the carbohydrate and protein structures of NCAM.
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PMID:Characterization of mouse ST8Sia II (STX) as a neural cell adhesion molecule-specific polysialic acid synthase. Requirement of core alpha1,6-linked fucose and a polypeptide chain for polysialylation. 870 35

Human seminal plasma alpha-L-fucosidase (EC 3.2.1.51) has been purified 7100-fold to very high purity and specific activity (83,000 nmol/min/mg protein) by affinity chromatography on agarose-epsilon-aminocaproyl-fucopyranosylamine. The purified alpha-L-fucosidase appeared to contain a single subunit of 56-57 kDa (as determined by SDS-PAGE and Western analysis). Lectin blotting and N-glycanase treatment studies indicated that this subunit is N-glycosylated and contains sialic acid residues. Human seminal plasma alpha-L-fucosidase was shown to contain three multimeric forms of 110, 236 and 314 kDa respectively (as determined by Sephadex G-200 chromatography) and therefore probably exists in dimeric, tetrameric and hexameric forms. Kinetic analysis with the 4-methylumbelliferyl-alpha-L-fucopyranoside (4MU-Fuc) substrate indicated a broad acidic optimum (pH 4.0-4.5) with a second neutral optimum (pH 6.4-7.4) with 60-80% of maximal activity. Apparent K(M) and V(max) values for the 4MU-Fuc substrate were determined to be 0.06 mmol/l and 92 micromol/min/mg protein respectively, using Lineweaver-Burk double reciprocal plots. Isoelectric focusing and neuraminidase treatment studies provided further evidence that the purified seminal plasma alpha-L-fucosidase is a sialoglycoprotein with several isoforms between pI values 5-7. The acidic isoforms between pI values 5-6 appear to be related chemically to the more neutral isoforms by sialic acid residues since neuraminidase treatment converted the former into the latter isoforms.
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PMID:Purification and characterization of human seminal plasma alpha-L-fucosidase. 1187 Feb 29

Detergent and salt extraction studies, as well as cytochemical localization with fluorescein isothiocyanate-bovine serum albumin-L-fucose, have provided further evidence for the plasma membrane association of a novel human sperm, alpha-L-fucosidase. This alpha-L-fucosidase has been solubilized and purified 8600-fold to high specific activity (35 000 U/mg protein) by affinity chromatography on agarose-C(24)-fucosylamine. To our knowledge, this is the first report concerning the purification and characterization of a mammalian plasma membrane-associated alpha-L-fucosidase. Both SDS-PAGE and Western blot analysis indicated the alpha-L-fucosidase is highly purified and contains a single subunit with a molecular mass of 51 kDa. N-glycanase studies indicated the subunit contains N-glycans, and lectin blot analysis detected the presence of mannose, but no terminal galactose or sialic acid residues. Isoelectric focusing indicated the presence of two major alpha-L-fucosidase isoforms (pIs 6.5 and 6.7) and a possible minor isoform (pI 6.3). Treatment of alpha-L-fucosidase with neuraminidase did not change its isoform profile, providing further evidence for the enzyme's lack of sialic acid residues. Kinetic analysis with 4-methylumbelliferyl alpha-L-fucopyranoside indicated that sperm alpha-L-fucosidase has a pH optimum near 7, an apparent K(m) of 0.08 mM, and a V(max) of 6.8 micro mol/min/mg protein. The unusual properties of human sperm alpha-L-fucosidase argue in support of a potentially important, but presently unknown, role for this enzyme in human reproduction.
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PMID:Purification and characterization of plasma membrane-associated human sperm alpha-L-fucosidase. 1260 17

Mammalian alpha-fucosidase (EC 3.2.1.51) is a lysosomal enzyme that catalyzes the removal of fucose residues from glycosphingolipids and its absence in humans results in a rare metabolic disorder called fucosidosis. Among the invertebrates in the molluscs (Unio) two forms of the enzyme have been reported, a 68 kDa non-glycosylated form and a 56 kDa glycosylated form. The glycosylated form has been purified from the seminal fluid of Unio [Biochem. Biophys. Res. Commun. 234 (1997) 54]. In the present study, the 56 kDa glycosylated form has been purified to homogeneity from the whole body tissue of Unio using a series of chromatographic steps. The purified enzyme migrated as a single protein species in 10% SDS-PAGE. Antibodies to the purified enzyme were raised in a rabbit in order to study its biochemical and immunological properties. The purified enzyme is a glycoprotein that exhibits strong binding to Con A-Sepharose gel and can be deglycosylated by PNGase F enzyme suggesting it to be N-glycosylated. The enzyme has been shown to specifically interact with the mannose 6-phosphate receptor protein (MPR 300) purified from goat and Unio. This specific interaction is discussed in view of its possible in vivo binding partners.
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PMID:Biochemical and immunological characterization of a glycosylated alpha-fucosidase from the invertebrate Unio: interaction of the enzyme with its in vivo binding partners. 1535 48

A novel dopachrome conversion enzyme (DCE) is present in insects and involved in their melanization pathway. DCE shares no sequence homology with any noninsect species from bacteria to humans. Several DCE sequences have been available, but enzyme structure and catalytic mechanism are unclear. This study concerns DCE PTMs, especially glycosylation. A mosquito DCE was purified and its monosaccharide composition, N-glycosylation site, and oligosaccharide structures were determined. Results showed that N-acetyl D-glucosamine and D-mannose are the major monosaccharides and L-fucose, D-xylose, and D-arabinose are the minor ones in mosquito DCE. Glycosylation site and oligosaccharide structures were elucidated from MS and MS/MS spectra of trypsin-digested DCE glycopeptides. A single N-glycosylation site (Asn285 -Glu-Thr) was identified in DCE and was proven to be fully glycosylated. Man3GlcNAc2, Man3(Fuc)1-2GlcNAc2, and their truncated structures were the dominant oligosaccharides. In addition, high mannose-type structures (Man4-7(Fuc)GlcNAc2) were also identified. Removal of DCE N-oligosaccharides with peptide N-glycosidase (PNGase F) decreased its activity and thermal stability. However, partial DCE deglycosylation with alpha-mannosidase or alpha-fucosidase somewhat stimulated its activity and improved its thermal stability. During mass spectrometric analysis of DCE glycopeptides, their CID patterns were highly intriguing, in that some glycopeptides underwent both C-terminal rearrangement and formation of dimeric structures during CID. Results of this study provide an interesting example in terms of potential complexity of the glycopeptide CID fragmentation pattern.
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PMID:Proteomic analysis of N-glycosylation in mosquito dopachrome conversion enzyme. 1762 77


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