Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.22.32 (bromelain)
 1,025 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Asparagine-linked oligosaccharides of stem bromelain glycopeptides were quantitatively released by digestion with the almond glycopeptidase which cleaves beta-aspartylglycosylamine linkage in glycopeptides with oligopeptide moieties. The primary structures of the two oligosaccharide components, (Man)3(Xyl)1(Fuc)1(GlcNAc)2 and (Man)2-(Xyl)1(Fuc)1(GlcNAc)2 were elucidated as Man alpha 1 leads to 6Man alpha 1 leads to 6[Xyl beta 1 leads to 2]Man beta 1 leads to 4GlcNAc beta 1 leads 4[Fuc alpha 1 leads to 3]GlcNAc and Man alpha 1 leads to 6[Xyl beta 1 leads to 2]Man beta 1 leads to 4 GlcNAc beta 1 leads to 4[Fuc alpha 1 leads to 3] GlcNAc, respectively.
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PMID:Complete structure of the carbohydrate moiety of stem bromelain. An application of the almond glycopeptidase for structural studies of glycopeptides. 50 Jun 6

Hydrazinolysis of porcine thyroglobulin glycopeptides and of pineapple stem bromelain [EC 3.4.22.4] permitted the isolation of almost intact carbohydrate chains of these glycoproteins. On the basis of permethylation analyses of the released oligosaccharides after reduction with NaBH4, the core structures of Unit A-type and Unit B-type carbohydrate chains of porcine thyroglobulin were deduced to be Manalpha1 leads to 6[Manalpha1 leads to 3]Manbeta1 leads to 4GlcNAcbeta1 leads to 4[Ralpha1 leads to 6]GlcNAc leads to Asn (Unit A-type, R=H; Unit B-type, R=Fuc), and that of bromelain was found to be Manalpha1 leads to 6[R'1 leads to 2]Manbeta1 leads to 4GlcNAcbeta1 leads to 4[R1 leads to 3]GlcNAc leads to Asn (R'=Xylbeta and R=Fucalpha, or R'=Fucalpha and R=Xylbeta). From these results, it appears that the hydrazinolysis method is applicable to wide variety of glycoproteins which have an N-glycosylamine linkage between the carbohydrate and peptide moieties, regardless of the type of linkage to the most proximal N-acetylglucosamine residue which is bound to asparagine.
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PMID:Studies on the hydrazinolysis of glycoproteins. Core structures of oligosaccharides obtained from Porcine thyroglobulin and pineapple stem bromelain. 103 16

1H- and 13C-NMR assignments for the carbohydrate part of the glycopeptide alpha-D-Man-(1----6)-[beta-D-Xyl-(1----2)]-beta-D-Man-(1----4)-beta-D- GlcNAc-(1----4)-[alpha-L-Fuc-(1----3)]-beta-D-GlcNAc-(1----N)-Asn approximately, derived from the proteolytic enzyme bromelain (EC 3.4.22.4), have been obtained using homo- and heteronuclear correlation spectroscopy, two-dimensional homonuclear Hartmann-Hahn and nuclear Overhauser enhancement experiments. A conformational model for the carbohydrate chain, deduced from the NMR data and consistent with hard-sphere exo-anomeric calculations shows that the rotamer population about the C-5--C-6 bond of beta-Man is restricted to the P omega = 180 rotamer, mainly.
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PMID:Conformational studies on the N-linked carbohydrate chain of bromelain. 236 40

The carbohydrates of BHA, a solubilized hemagglutinin of influenza virus by bromelain digestion, were quantitatively released as oligosaccharides by hydrazinolysis. The oligosaccharide mixture was separated into a neutral and two acidic fractions by paper electrophoresis. Both acidic fractions were resistant to sialidase digestion but were slowly converted to the neutral fraction by incubation with sulfatases. The neutral fraction which comprised about 80% in molar ratio of total oligosaccharides was separated into 13 oligosaccharides by paper chromatography and by Con A-Sepharose column chromatography. Structural studies of these oligosaccharides by sequential exoglycosidase digestion and by methylation analysis revealed that BHA contains a series of high mannose type and bi-, tri-, and tetraantennary complex type sugar chains. Occurrence of Gal beta l leads to 3GlcNAc outer chain in two and bisectional N-acetylglucosamine in one of the biantennary sugar chains is an interesting characteristic of the sugar chains of BHA.
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PMID:Carbohydrates of influenza virus hemagglutinin: structures of the whole neutral sugar chains. 683 Jul 58

The three tryptic glycopeptides of cationic peanut peroxidase (C. PRX) and the sole one of anionic peanut peroxidase (A. PRX) were individually coupled to bovine serum albumin to raise antisera. The three categories of antibodies directed towards three N-glycans of C. PRX (anti-GLa, anti-GLb and anti-GLc) were isolated from antisera with glycan-conjugated ECH Sepharose 4B affinity columns and the distribution of epitopes on the N-glycans was investigated. The reactivity of anti-GLa, anti-GLb and anti-GLc is inhibited 25-40% by 1 M fucose, compared with a slight inhibition by N-acetylglycosamine and xylose. Mannose and galactose showed no inhibition to anti-GLa and only a slight inhibition to anti-GLb and anti-GLc. All of anti-GLa, anti-GLb and anti-GLc recognize A. PRX and horseradish peroxidase but do not recognize fetuin. Also, their reactivity is inhibited by bromelain by more than 70%. The three categories of antibodies present high homogeneity and appear to be directed mainly towards the core structure [Xyl] (Man)3 [Fuc] (GlcNAc)2. An effective and simple method to screen antibodies with carbohydrate specificities is described herein.
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PMID:Immunogenicity of the N-glycans of peanut peroxidase. 752 14

The initial velocities of hydrolysis of nineteen glycopeptides by peptide: N-glycosidase F and A were determined. Substrates were prepared from bovine fetuin, hen ovalbumin, pineapple stem bromelain, bovine fibrin and taka-amylase. From these glycopeptides, several variants with regard to peptide and carbohydrate structure were prepared and derivatized with dabsyl chloride, dansyl chloride or activated resorufin. Tyrosine containing glycopeptides were also used without an additional chromophore. Enzymatic hydrolysis of glycopeptides was quantified by narrow bore, reversed phase HPLC with turnaround cycle times of down to 6 min, but usually 15 min. KM values ranging from 30 to 64 microM and from 4 to 36 microM were found for N-glycosidase F and A, respectively. Relative velocities of hydrolysis of the different substrates by each enzyme varied considerably. Little, if any, similarity of the performance of N-glycosidase F and A with the different substrates was observed. The minimal carbohydrate structure released by peptide: N-glycosidase F was a di-N-acetylchitobiose. N-glycosidase A could release even a single N-acetylglucosamine, albeit 3000 times slower than a di-N-acetylchitobiose or larger glycans. In general the structure of the intact glycan had little effect on activity, and with both enzymes the rate of hydrolysis appeared to be primarily governed by peptide structure and length. However, N-glycosidase F did not release glycans alpha 1,3-fucosylated at the asparagine linked N-acetylglucosamine irrespective of the presence of xylose in the substrate.
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PMID:Kinetic comparison of peptide: N-glycosidases F and A reveals several differences in substrate specificity. 754 Sep 2

The reactivity of sera from honeybee venom allergic patients with the N-glycan of phospholipase A2 was investigated using neoglycoproteins with an enzyme-linked immunosorbent assay. Of 122 sera with appreciable levels of IgE antibodies directed against bee venom as measured by radioallergosorbent test, 34 sera exhibited significant amounts of glycan-reactive IgE. These sera cross-reacted with the N-glycan from the plant glycoprotein bromelain. The interaction of IgE with the N-glycan from phospholipase could be inhibited with glycopeptides from bromelain which shares the alpha 1,3-fucosylation of the asparagine-bound N-acetylglucosamine with bee venom phospholipase. Since defucosylated bromelain glycopeptides or glycopeptides containing a Man3GlcNAc2 oligosaccharide were not recognized by most of these sera, we conclude that alpha 1,3-fucosylation of the innermost N-acetylglucosamine residue of N-glycoproteins forms an IgE-reactive determinant. This structural element is frequent in glycoproteins from plants, and it occurs also in insects. It is suspected to be one of the major causes of the broad allergenic cross-reactivity among various allergens from insects and plants.
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PMID:Fucose alpha 1,3-linked to the core region of glycoprotein N-glycans creates an important epitope for IgE from honeybee venom allergic individuals. 769 94

Crude bromelain extracts from pineapple stems (Ananas comosus) were fractionated by two-step FPLC-cation-exchange chromatography. At least eight basic proteolytically active components were detected. The two main components F4 and F5 together with the most active proteinase fraction F9 were characterized by SDS-PAGE, mass spectroscopy, multizonal cathodal electrophoresis, partial amino acid sequence, and monosaccharide composition analysis. F9 amounts to about 2% of the total protein and has a 15 times higher specific activity against the substrate L-pyroglutamyl-l-phenylanalyl-l-leucine-p-nitroanilide (PFLNA) than the main component F4. The molecular masses of F4, F5, and F9 were determined to 24,397, 24,472, and 23,427, respectively, by mass spectroscopy. Partial N-terminal amino acid sequence analysis (20 amino acids) revealed that F9 differs from the determined sequence of F4 and F5 by an exchange at position 10 (tyrosine-->serine) and position 20 (asparagine-->glycine). F4 and F5 contained fucose, N-acetylglucosamine, xylose, and mannose in ratio of 1.0:2.0:1.0:2.0, but only 50% of the proteins seem to be glycosylated, whereas F9 was found to be unglycosylated. Polyclonal antibodies (IgG) against F9 detected F4 and F5 with tenfold reduced reactivity. The pH optimum of F4 and F5 was between pH 4.0 and 4.5 and for F9 close to neutral pH. The kinetic parameters for PFLNA hydrolysis were similar for F4 (Km 2.30 mM, kcat 0.87 sec-1 and F5 (Km 2.42 mM, kcat 0.68 sec-1), and differed greatly from F9 (Km 0.40 mM, kcat 3.94 sec-1).
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PMID:Isolation and partial characterization of basic proteinases from stem bromelain. 777 62

The conformational behavior of the N-glycan Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc beta of stem bromelain as part of the intact glycoprotein was investigated and compared with that of the same N-glycan as part of a bromelain-derived glycopeptide. Proton chemical shifts of the glycoprotein N-glycan were determined by 2D HOHAHA and 2D NOESY measurements, making use of the glycopeptide 1H NMR data. During each 2D NMR experiment about 4% of the glycoprotein denatured. Experimental data concerning interproton distances of the intact glycoprotein N-glycan were obtained by NOESY 1H NMR spectroscopy. Several theoretical models for the N-glycan, obtained by molecular dynamics simulations of the glycopeptide, were investigated. Comparison of experimental and theoretical NOESY cross peak intensities was performed with the program CROSREL. In comparison with the glycopeptide, the distribution of populations between two main conformations of the Fuc alpha 1-3GlcNAc linkage was altered. In addition, the omega = 60 degrees (gt) rotamer of the Man alpha 1-6Man linkage seems to be present for a significant period of time, whereas in the glycopeptide the omega = -60 degrees (gg) conformation exists exclusively. Except for the Xyl beta 1-2Man linkage, the mobilities around the glycosidic linkages in the glycoprotein were reduced compared with those in the glycopeptide, especially concerning the Fuc alpha 1-3GlcNAc and Man alpha 1-6Man linkages. These findings might be the result of an interaction of the polypeptide chain with the Fuc alpha/Man alpha side of the N-glycan. A qualitative analysis of the NMR spectra showed a larger degree of mobility in the denatured glycoprotein N-glycan than in the intact glycoprotein.
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PMID:Conformational analysis of the xylose-containing N-glycan of pineapple stem bromelain as part of the intact glycoprotein. 779 34

The binding to concanavalin A (Con A) by pyridylaminated oligosaccharides derived from bromelain (Manalpha1,6(Xylbeta1,2) Manbeta1, 4GlcNAcbeta1,4(Fucalpha1,3)GlcNAc), horseradish peroxidase (Manalpha1,6(Manalpha1,3) (Xylbeta1,2)Manbeta1, 4GlcNAcbeta1,4(Fucalpha1,3) GlcNAc), bee venom phospholipase A2 (Manalpha1,6Manbeta1,4GlcNAcbeta1,4GlcNAc and Manalpha1,6(Manalpha1,3)Manbeta1,4GlcNAcbeta1,4 (Fucalpha1,3)GlcNAc) and zucchini ascorbate oxidase (Manalpha1,6(Manalpha1,3) (Xylbeta1,2)Manbeta1,4 GlcNAcbeta1,4GlcNAc) was compared to the binding by Man3GlcNAc2, Man5GlcNAc2 and the asialo-triantennary complex oligosaccharide from bovine fetuin. While the fetuin oligosaccharide did not bind, bromelain, zucchini, Man2GlcNAc2 and horseradish peroxidase were retarded (in that order). The alpha1,3-fucosylated phospholipase, Man3GlcNAc2 and Man5GlcNAc2 structures were eluted with 15 mM alpha-methylmannoside. It is concluded that core alpha1,3-fucosylation has little or no effect on ConA binding while xylosylation decreases affinity for ConA. In a parallel study comparing the endoglycosidase D (Endo D) sensitivities of Man3GlcNAc2, IgG-derived GlcNAcbeta1, 2Manalpha1,6(GlcNAcbeta1,2Manalpha1,3)Manbeta1,+ ++4GlcNAcbeta1,4(Fucalpha1,6)GlcNAc, the phospholipase Manalpha1,6(Manalpha1,3) Manbeta1, 4GlcNAcbeta1,4(Fucalpha1,3)GlcNAc, and horseradish and zucchini pyridylaminated N-linked oligosaccharides, it was found that only the Man3GlcNAc2 structure was cleaved. The IgG structure was sensitive only when beta-hexosaminidase was also present. Thus, in contrast to core alpha1,6-fucosylated structures, such as those present in mammals, the presence of core alpha1,3-fucose, as found in structures from plants and insects, and/or beta1,2-xylose, as found in plants, causes resistance to Endo D.
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PMID:Concanavalin A binding and endoglycosidase D resistance of beta1,2-xylosylated and alpha1,3-fucosylated plant and insect oligosaccharides. 955 83


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