4-Methylumbelliferyl glycosides of N-acetyl 4-thiochito-oligosaccharides as fluorogenic substrates for chitodextrinase from Vibrio furnissii

The degradation of chitin involves a diverse array of enzymes,some with overlapping substrate specificities. In order to distinguishbetween different types of enzymes, specific substrates areneeded. Toward this end, two new fluorogenic substrates containingthio-glycosidic linkages, 4-methylumbelliferyl N,N'-diacetyl-4-thio-ß-chitobioside(Mu-TCB) and N,N',N'-triacetyl-4,4'-dithio-ß- chitotrioside(Mu-TCT) are described. The substitution of the glycosidic oxygens(except the one that links oligosaccharide with the fluorogenicaglycon) with a sulfur atom resulted in resistance of thesecompounds to N-acetyl-hexosaminidases while they were specificsubstrates for the newly discovered chitodextrinase from Vibriofurnissii (Keyhani,N.O. and Roseman,S. (1996) J. Biol. Chem.,271, 33414–33424) and some bacterial chitinases. The enzymekinetics of these 4-S-linked substrates, Mu-TCB and MuTCT, aswell as the O-linked 4-methylumbelliferyl N,N'diacetyl-ß-chitobioside(Mu-CT) and N,N',N'-triacetyl-ß-chitotrioside (Mu-CT)with the chitodextrinase were studied and compared. The usefulnessof the substrates for screening for chitodextrinase and/or chitinaseactivity was demonstrated. chitodextrinase chitinase fluorometric assay 4-methylumbelliferyl glycoside thiochitooligosaccharide     

Isolation, characterization and inactivation of the mouse Mgat3 gene: the bisecting N-acetylglucosamine in asparagine-linked oligosaccharides appears dispensable for viability and reproduction

The biosynthesis of complex asparagine (N)-linked oligosaccharidesin vertebrates proceeds with the linkage of N-acetylglucosamine(GlcNAc) to the core mannose residues. UDP-N-acetylglucosamine:ß-D-mannosideß1–4 N-acetylglucosaminyltransferase III (GlcNAc-TIII,EC2.4.1.144) catalyzes the addition of GlcNAc to the mannosethat is itself ß1–4 linked to underlying N-acetylglucosamine.GlcNAc-TIII thereby produces what is known as a ‘bisecting’GlcNAc linkage which is found on various hybrid and complexN-glycans. GlcNAc-TIII can also play a regulatory role in N-glycanbiosynthesis as addition of the bisecting GlcNAc eliminatesthe potential for     

Characterization of asparagine-linked oligosaccharides on a mouse submandibular mucin

The asparagine-linked oligosaccharides from an adult femalemouse submandibular gland mucin were released by treatment withpeptide-N⁴-(N-acetyl-ß-glucosaminyl)asparagine amidaseF or endo-ß-N-acetylglucosaminidase H. Endo-ß-N-acetylglucosaminidaseH appeared to be more effective at releasing the asparagine-linkedoligosaccharides from this mucin than was peptide-N⁴-(N-acetyl-ß-glucosaminyl)-asparagineamidase F. After quantitative reductive labelling with the fluorophore,8-aminonaphthalene-1, 3, 6-sulphonic acid, the oligosaccharideswere separated by polyacrylamide gel electrophoresis and isolated.The individual oligosaccharides were sequenced by a batteryof recombinant exoglycosidases. Approximately 50% of the oligosaccharideswere of the high-mannose type. The five-mannose member of thisfamily was the most prevalent. The second group of oligosaccharideswere of the non-bisected hybrid type. No complex asparagine-linkedoligosaccharides were detected. The hybrids exhibited both biantennaryand triantennary branching patterns. The triantennary hybridwas the most common hybrid at >30% of all oligosaccharides.With 98% of the hybrid oligosaccharides sialylated and all lackinga bisecting N-acetylglucosamine, these oligosaccharides as agroup have been only rarely observed in other glycoproteins.The fully sialylated triantennary hybrid may be unique. asparagine-linked oligosaccharides biantennary salivary mucin sialylated hybrid triantennary     

Purification and characterization of novel glycosidases from the bacterial genus Xanthomonas

Enzymatic analysis of oligosaccharides using exoglycosidaseshas become a powerful tool for determining the sequence andstructure of sugar chains. The principal limitation to thesemethods has been the lack of highly purified and wellcharacterizedenzymes. Using fluorescently labelled carbohydrate substratesand TLC, we have developed a method to identify glycosidaseswith novel specificities. This screening method led to the discoverythat bacteria of the genus Xanthomonas are a rich source ofexoglycosidases. From Xanthomonas manihotis, eight novel exoglyccosidaseshave been isolated and characterized. A novel ß-N-acetylglucosaminidasehas been purified that, unlike those previously described, willcleave N-acetylglactosamine without cleaving N-acetylgalactosamineresidues. A novel ß-galactosidase has been isolatedthat preferentially hydrolyses ß(1     

Relationship between Flower Induction by Benzoic Acid and its Metabolism in Lemna paucicostata 151

The flower-inducing activities in Lemna paucicostata 151 offour major metabolites of benzoic acid (N-benzoyl aspartate,benzyl 6-O-ß-D-apiofuranosyl-O-ß-D-glucopyranoside,O-benzoyl isocitrate and O-benzoyl malate) were measured, andthe effects on the uptake and metabolism of benzoic acid dueto change in the level of the benzoic acid concentration orto the addition of plant hormones were investigated. N-Benzoylaspartate had weak activity, and O-benzoyl isocitrate and malatehad fairly strong activities, while benzyl 6-O-ß-D-apiofuranosyl-ß-D-glucopyranosideshowed no activity. As the concentration of benzoic acid rose,the ratio of N-benzoyl aspartate increased and that of benzyl6-O-ß-D-apiofuranosyl-O-ß-D-glucopyranosidedecreased. GA₃ and IAA, inhibitors of flower induction by benzoicacid, seemed to promote conversion to N-benzoyl aspartate insteadof to benzyl 6-O-ß-D-apiofuranosyl-ß-D-glucopyranoside.The conversion to N-benzoyl aspartate was considered to be adetoxification process and that to benzyl 6-O-ß-D-apiofuranosyl-ß-D-glucopyranosidemay be directly related to flower induction in Lemna. (Received November 2, 1987; Accepted January 23, 1988)     

Synthesis of O-glycan core 3: Characterization of UDP-GlcNAc: GalNAc-R {beta}3-N-acetyl-glucosaminyltransferase activity from colonic mucosal tissues and lack of the activity in human cancer cell lines

UDP-GlcNAc: GalNAc-R ß3-GlcNAc-transferase (core 3ß3-GlcNAc-T, where GlcNAc is N-acetyl-D-glucosamine,GalNAc is N-acetyl-D-galactosamine and T is transferase) isexpressed in a tissue-specific fashion and is high in normalcolonic tissue, but downregulated in colon cancer. To furtherstudy the control of this enzyme, we examined the activity inpig, rat and human colonic tissues, and several human cancercell lines. The enzyme was difficult to solubilize by detergentsand was extremely unstable in the solubilized form. Using syntheticderivatives of the GalNAc-R substrate, we showed that the specificityof the enzyme in normal rat and human colonic mucosa requiresall the substituents of the GalNAc-sugar ring of substratesfor maximal activity. Core 3 ß3-GlcNAc-T was significantlyinfluenced by the structure of the aglycon group. None of theinactive substrate derivatives could inhibit the activity. N-Iodoacetamido-galactosamine     

Light and heavy lysosomes: characterization of N-acetyl-{beta}-D-hexosaminidase isolated from normal and I-cell disease lymphoblasts

We previously reported that I-cell disease lymphoblasts maintainnormal or near-normal intracellular levels of lysosomal enzymes,even though N-acetylglucosamine-1-phosphotransferase activityis severely depressed or absent (Little et al., Biochem. J.,248, 151–159, 1987). The present study, employing subcellularfractionation on colloidal silica gradients, indicates thatboth light and heavy lysosomes isolated from I-cell diseaseand pseudo-Hurler polydystrophy lymphoblasts possess normalspecific activity levels of N-acetyl-ß-D-hexosaminidase,-D-mannosidase and ß-D-glucuronidase. These currentfindings are in contrast to those of cultured fibroblasts fromthe same patients, where decreased intralysosomal enzyme activitiesare found. Column chromatography on Ricinus communis revealedthat N-acetyl-ß-D-hexosaminidase in both heavy andlight I-cell disease lysosomal fractions from lymphoblasts possessesan increased number of accessible galactose residues (30–50%)as compared to the enzyme from the corresponding normal controls.Endo-ß-N-acetylglucos-aminidase H treatment of N-acetyl-ß-D-hexosaminidasefrom the I-cell lysosomal fractions suggests that the majorityof newly synthesized high-mannose-type oligosaccharide chainsare modified to complex-type carbohydrates prior to being transportedto lysosomes. This result from lymphoblasts differs from previousfindings with fibroblasts, where N-acetyl-ß-D-hexosaminidasefrom I-cell disease and pseudo-Hurler polydystrophy lysosomesexhibited properties associated with predominantly high-mannose-typeoligosaccharide chains. The current results imply that differentcell types may modify the carbohydrate side chains of lysosomalenzymes in a differential manner, and that selected cell typesmay also employ mechanisms other than the mannose-6-phosphatepathway for targeting lysosomal enzymes to lysosomes. I-cell disease lymphoblasts lysosomes mannose-6-phosphate oligosaccharide chains pseudo-Hurler polydystrophy     

Glycosidases from Cotyledons of Pisum sativum L.

-Mannosidase and ß-N-acetylglucosaminidase were purifiedfrom extracts of cotyledons of germinating Pisum sativum L.A 13-fold purification of a-mannosidase free from ß-N-acetylglucosaminidaseactivity was achieved by precipitation in ammonium sulphate,column chromatography on DEAE-cellulose, and treatment with2 M pyridine. ß-N-Acetylglucosaminidase was purified200-fold by the use of (NH₄)₂SO₄, and chromatography on ConcanavalinA¹-Sepharose and Sephacryl-200. This preparation showed no measurablecontamination by -mannosidase activity. Both glycosidases appearto be glycoproteins and demonstrate optimal activity at pH valuesof 4.0–4.5. Both glycosidases appear to have very similarmolecular weights, with -mannosidase being slightly larger thanß-N-acetylglucosaminidase. An extensive search forthe activity of aspartylglycosylamine amido hydrolase in peacotyledons proved unsuccessful.     

Terminal {beta}-linked tyvelose creates unique epitopes in Trichinella spiralis glycan antigens

Indirect evidence that the immunodominant N-glycans of the parasite,Trichinella spiralis are capped by novel ß-linked3,6-dideoxy-D-arabinohexopyranosyl residues (tyvelase, Tyv)was obtained from immunochemical assays employing monoclonalantibodies and synthetic oligosaccharides. Three of four previouslycharacterized monoclonal antibodies generated from the lymphocytesof T.spiralis infected rats bind BSA glycoconjugates bearingthe synthetic epitope ß-D-Tyvp(1     

Proton NMR study of triantennary complex type N-linked glycan chains: assignment of proton chemical shifts of the {beta}-Man residue in a basic unit of the triantennary glycan chain having a GlcNAc{beta}1->6Man{alpha}1->6Man{beta}->sequence

The chemical shifts of ring protons of the ß-Man residuein a triantennary complex type N-linked glycan chain havinga GlcNAcß1     

Glycosylation pattern and processing of envelope gene products encoded by glycosylation mutants of Friend spleen focus-forming virus

The protein encoded by the envelope gene of Friend spleen focus-formingvirus is responsible for the acute leukaemogenicity of thisvirus. In order to correlate glycosylation and intracellularprocessing of this protein with viral pathogenicity, envelopegene products of pathogenic and apathogenic glycosylation mutantswere expressed in Rat-1 cells and metabolically labelled with[6-³H] glucosamine. Following immunoprecipitation, primary andsecondary gene products (gp55, gp65) were separated by preparativepolyacrylamide gel electrophoresis. Oligosaccharides were releasedfrom tryptic glycopeptides by treatment with endo-ß-N-acetylglucosaminidaseH (gp55), peptide-N⁴-(N-acetyl-ß-glucosaminyl)asparagineamidase F (gp65) or by reductive ß-elimination. Resultingglycans were characterized by cochromatography with authenticoligosaccharide standards using different HPLC systems and digestionwith exoglycosidases. The results revealed that the primaryenvelope gene products of pathogenic glycosylation mutants were,in part, further processed in Rat-1 cells similar to wild-typeglycoprotein, resulting in polypeptides carrying complex-typeN-glycans as well as partially sialylated O-linked oligosaccharides.In contrast, corresponding glycoproteins encoded by apathogenicmutants were found to remain at the level of the primary translationproduct exclusively comprising high-mannose-type N-glycans.Hence, intracellular maturation of the envelope gene productsin this model cell line seems to correlate with the in vivopathogenicityof the glycosylation mutants studied. carbohydrate structure glycoprotein murine leukaemia virus oligosaccharide processing SFFV     

Development of {beta}-amylase activity and polymorphism in wheat seedling shoot tissues8

Increasing ß-amylase activity in wheat (Triticum aestlvum,var. Star) seedling shoot tissues was consistently accompaniedby the development of a characteristic polymorphism of the enzyme,as shown by electrophoresis employing amylopectin-containingpolyacrylamide gels. Very young shoot tissue contained one principalform of the enzyme (ß1), whereas two other major forms(ß2, ß3) appeared complementary to thisupon further growth. In vitro incubation experiments indicatedthat the polymorphism arose via a probably proteolytic conversionof ß1 into ß2 and ß3. The conversioninvolved neither an activation of ß-amylase nor asignificant modification of ß-amylase component plvalues. The electrophoretic ß-amylase patterns ofsubcellular leaf compartments suggested that ß1 issynthesized in the cytoplasm of leaf mesophyfi cells and thatthe other forms arise upon transfer of this ‘primary’form into the vacuole. The development of shoot ß-amylaseactivity did not require light, but appeared to be under thenegative control of the chloroplast and was stimulated by mineralnutrients. No clear relationship between ß-amylaseactivity and starch metabolism was evident, since the leaf activitywas largely absent from mesophyll protoplasts, could not beunequivocally demonstrated in the mesophyll chioroplasts, anddeveloped regardless of whether the tissues contained significantamounts of starch or not. Key words: Wheat, leaves, ß-amylase, polymorphism, compartmentation     

Autolysis Promotes the Extension Capacity of Zea mays Coleoptile Cell Walls in Response to Acid pH Solutions

The relationship between autolytic degradation of ß(1–3),(1–4)-D-glucanand acid pH-induced extension of isolated Zea mays cell wallshas been investigated using a constant-load extension technique.Acidic buffer (4.5) was able to induce an additional extension(E_a) on cell walls already extended at pH 6.8 buffer under a20 g-mass load, indicating that the additional extension (E_a)was the parameter that better represented the effect of thedifferent treatments on the mechanical properties of maize coleoptilecell walls. The additional extension in response to acidic pHwas higher when cell walls had been previously autolysed for24 h at pH 5.5. Furthermore, the acid-pH effect was dependenton the presence during the constant load extension of some thermo-labilefactors, suggesting the participation of expansins. Acid pHincreased E_a of native cell walls through an increase in theplastic extension (E_p) in agreement with a one step mechanismleading directly to irreversible (plastic) wall extension assuggested by Cosgrove (1977). The autolytic degradation of ß(1–3),(1–4)-D-glucan was also able to modify the mechanicalproperties of maize coleoptile cell walls increasing its elasticextension (E_e) in response to pH 4.5 buffer but that modificationonly leads to an increase in wall extension when expansins areactive, suggesting a cooperation between ß-glucanturnover and expansin action. (Received August 5, 1998; Accepted March 16, 1999)     

{beta}1,4-Galactosyltransferase activity in B cells detected using a simple ELISA-based assay

Lymphocytic ß1,4-galactosyltransferase (ß1,4-GalTase,EC 2.4.1.38 [EC] ) activity was measured in B cells using a neoglycoprotein,N-acetylglucosamine-phenylisothlocyanate-bovine serum albumin(GlcNAc-pITC-BSA), as an acceptor substrate in a novel enzyme-linkedimmunosorbent assay (ELISA)-based method. This assay provedto be much simpler to use than the lengthy and expensive radiochemicalassays commonly used, and has the additional advantage thatit specifically detects the enzyme mediating transfer via theGalß1,4GlcNAc linkage. A F(ab')₂ antibody againstGalTase was able to specifically inhibit the reaction. Greatersensitivity for ß1,4-GalTase activity was obtainedusing GlcNAc-pITC-BSA as an acceptor substrate rather than ovalbumin.Low levels of ß-galactosidase activity were detectablein lymphocyte cell lysates at acidic pH, although such activitywas not detectable at the neutral pH used in the ß1,4-GalTaseactivity assay. Using this assay with the GlcNAc-pITC-BSA acceptor,similar ß1,4-GalTase activities were observed in CD19⁺B cells from patients with rheumatoid arthritis (RA) to thoseseen in normal control individuals. ELISA ß1,4-galactosyltransferase lymphocyte neoglycoprotein radiochemical     

Human serum contains a chitinase: identification of an enzyme, formerly described as 4-methylumbelliferyl-tetra-N-acetylchitotetraoside hydrolase (MU-TACT hydrolase)

Since 1988 an endoglucosaminidase, provisionally named MU-TACThydrolase, has been known that hydrolyses the artificial substrate4-methylumbelliferyl-tetra-N-acetyl-chitotetraoside (MU-[GlcNAc]₄,where GlcNAc is N-acetyl-glucosamine). The biological functionof the enzyme was unknown. In this paper evidence is presentedshowing that this endoglucosaminidase from human serum is infact a chitinase that is different from lysozyme. The factssustaining this finding are: (i) the identification of the productsformed from MU-[GlcNAc]₃ as [GlcNAc]₂ and [GlcNAc]₃; (ii) chitinand ethylene glycolchitin can be degraded by the enzyme; (iii)the chitinase inhibitor allosamidin also inhibits the actionof MU-TACT hydrolase from human serum; (iv) no hydrolysis ofthe lysozyme substrate Micrococcus lysodeikticus. The enzymealso occurs in rat liver. It was demonstrated that upon Percolldensity gradient centrifugation the enzyme from this tissuedistributed parallel to the lysosomal marker enzymes ß-N-acetylhexosaminidaseand ß-galactosidase, indicating a lysosomal localizationfor this enzyme. It is proposed that the enzyme functions inthe hydrolysis of chitin, to which mammals are frequently exposedduring infection by pathogens. allosamidin chitinase human serum lysozyme MUTACT hydrolase     

Effect of cycloheximide and cordycepin on auxin-induced elongation and {beta}-glucan degradation of noncellulosic polysaccharides of Avena coleoptile cell wall

The effect of cycloheximide (10^–5 M) and cordycepin (10^–4M) used as protein and RNA synthesis inhibitors, respectively,on auxin action in noncellulosic ß-glucan degradationof Avena coleoptile cell wall was investigated. Both depressedauxin-induced ßglucan degradation of the cell wallas well as auxin-induced elongation and cell wall loosening,suggesting that the process of ß-glucan degradationof the cell wall is closely associated with cell wall looseningand that auxin enhances the activity of an enzyme for ß-glucandegradation through de novo synthesis of RNA and protein butnot through activation of the enzyme in situ. Kinetic studywith the inhibitors showed that RNA metabolism involved in ß-glucandegradation was stimulated by auxin treatment of only 15 minwhile a longer lag phase (about 1 hr) existed for the synthesisof the enzyme. (Received December 16, 1978; )     

Enzymatic evidence for the presence of a critical terminal hexa-arabinoside in the cell walls of Mycobacterium tuberculosis

A species of Cellulomonas was isolated from soil by enrichmentculture and shown to secrete enzymes capable of degrading mycobacterialcell wall arabinogalactan, both the insoluble peptidoglycan-boundand base-solubilized forms. The major degradation product waspurified and characterized as a hexa-arabinofuranoside, [ß-D-Araf-(1     

HL 60 leukaemia cells chemically induced to differentiate retain some surface glycan features of undifferentiated cells not found in normal leukocytes

Human HL 60 myeloid leukaemia ells have the potential to differentiateinto either macrophage-like cells or granulocyte-like cellsunder the stimulus of chemical treatments. Using glycotechnologyprocedures, the glycosylation patterns of differentiated andundifferentiated HL 60 cells were analysed and compared withthose of normal human peripheral monocytes. Both in vitro differentiationsresult in significant morphologic and functional changes, butwe observed that the glycosylation patterns of undifferentiatedand differentiated HL 60 cells exhibit several common glycosidicfeatures that are absent in normal peripheral monocytes: thepresence of (i) bisecting ß-N-acetylglucosamine attachedat the C-4 position of the ß-mannose of polyantennarycomplex-type carbohydrate chains and (ii) complex-type carbohydratechains enriched with non-reducing terminal ß-N-acetylglucosamineresidues. Moreover, the three populations of HL 60 cells expresssmall amounts of biantemary complex-type structures (<6%),whereas normal peripheral monocytes expressed >20% of suchstructures. Thus, the cell glycosylation pattern could reflectthe pathological state of the HL 60 cells. differentiation glycosylation HL 60 cell monocytes     

Processing of asparagine-linked oligosaccharides in insect cells. N-Acetylglucosaminyltransferase I and II activities in cultured lepidopteran cells

The levels of ß1,2-N-acetylglucosaminyltransferase(GlcNAc-T) I and II activities in cultured cells from Bombyxmori(Bm-N), Mamestra brassicae (IZD-Mb-0503) and Spodoptera frugiperda(Sf-9 and Sf-21) were investigated. Apart from initial experimentswith Man