RESUMO
All four members of the family of pentopyranosyl-(2'-->4') oligonucleotide systems that contain beta-ribo-, beta-xylo-, alpha-lyxo-, or alpha-arabinopyranosyl units as repeating sugar building blocks are found to be much stronger Watson-Crick base-pairing systems than RNA. The alpha-arabinopyranosyl system is the strongest of all and in fact belongs to the strongest oligonucleotide base-pairing systems known. Whatever the chemical determinants by which nature selected RNA as a genetic system, maximization of base-pairing strengths within the domain of pentose-derived oligonucleotide systems was not the critical selection criterion.
Assuntos
Pareamento de Bases , Oligonucleotídeos/química , Oligorribonucleotídeos/química , Pentoses/química , RNA/química , Arabinose/química , Dicroísmo Circular , Evolução Molecular , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Ribose/química , Temperatura , Termodinâmica , Xilose/químicaRESUMO
[formula: see text] The L-alpha-lyxopyranosyl (4'-->3') oligonucleotide system shows cooperative base-pairing in spite of containing only five instead of the usual six covalent bonds per repetitive backbone unit. In contrast, corresponding D-beta-ribofuranosyl (4'-->3') oligonucleotides do not show adenine-thymine pairing under comparable conditions. The difference in pairing behavior relates to the conformation of the two systems' vicinal 3',4'-phosphodiester substituents, which is diaxial in the lyxopyranosyl system and 3'-axial-4'-equatorial in the ribopyranosyl system.
Assuntos
Pareamento de Bases , Oligonucleotídeos/química , Conformação de Ácido Nucleico , TemperaturaRESUMO
The C-5 substituted uridine derivatives UDP-5-propylamine (7) and UDP-GlcNAc-5-propylamine (8) were synthesized in good yields by Heck alkylation of the 5-mercuriuridines, followed by hydrogenation. The products were characterized by 1H and 13C NMR spectroscopy, electrospray mass spectrometry and UV spectrophotometry. The amines are of interest for the preparation of affinity probes for glycosyltransferases. The benzoylbenzamides of 7 and 8 show strong competitive inhibition of N-acetylglucosaminyltransferase I and II with Ki values ranging from 30 to 100 microM (without irradiation) and may be useful as active site-directed photoaffinity labels. A conjugate of 8 and Sepharose was used for affinity chromatographic purification of N-acetylglucosaminyltransferases I and II. The results indicate that this affinity gel is a stable alternative to the commonly used but unstable UDP-GlcNAc-5-Hg-thiopropyl conjugate.
Assuntos
Marcadores de Afinidade/síntese química , Cromatografia de Afinidade/métodos , N-Acetilglucosaminiltransferases/metabolismo , Propilaminas/síntese química , Uridina/análogos & derivados , Marcadores de Afinidade/metabolismo , Animais , Benzamidas/química , Benzamidas/farmacologia , Sítios de Ligação , Ligação Competitiva , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Cinética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/isolamento & purificação , Propilaminas/metabolismo , Coelhos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrofotometria , Raios Ultravioleta , Uridina/síntese químicaRESUMO
In the synthesis of modified derivatives of octyl O-(alpha-D-mannopyranosyl)-(1-->3)-O-[(alpha-D-mannopyranosyl)-(1-->6)]- beta-D-mannopyranoside, 4.,5-epoxypentyl, a 4-diazirinopentyl, and a 5-(iodoacetamido)pentyl group were attached to the 3''-OH of the trisaccharide. The diazirino derivative may be especially suitable for photolabeling of the active site of N-acetylglucosaminyltransferase I (GlcNAcT-I). In addition, the 2'-OH group of the above-mentioned trisaccharide was reduced to a 2'-deoxy group and substituted 2'-O-methyl group.
Assuntos
Glicoproteínas/química , N-Acetilglucosaminiltransferases/metabolismo , Oligossacarídeos/síntese química , Sítios de Ligação/fisiologia , Sequência de Carboidratos , Dados de Sequência Molecular , Estrutura Molecular , Especificidade por SubstratoRESUMO
8-(Methoxycarbonyl)octyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (10) was synthesized in 54% yield by regioselective diglycosylation of unprotected mannoside 4, employing the trichloroacetimidate donor 1, followed by debenzoylation. Derivatives of compounds 4 and 10 were used to prepare conjugates containing fluorochromes for the study of carbohydrate-lectin interactions, as well as conjugates with phospholipids for the preparation of liposomes.
Assuntos
Dissacarídeos/síntese química , Glicoconjugados/síntese química , Manosídeos/síntese química , Sequência de Carboidratos , Dados de Sequência MolecularRESUMO
Recombinant rabbit UDP-GlcNAc: alpha-Man-(1-->3R) beta-(1-->2)-N-acetylglucosaminyl-transferase I (EC 2.4.1.101, GlcNAc-T I) produced in the Sf9 insect cell/baculovirus expression system has been used to convert compounds of the form 3-R-alpha-Man(1-->6)(alpha-Man(1-->3)) beta-Man-O-octyl to 3-R-alpha-Man(1-->6)(beta-GlcNAc(1-->2)alpha-Man(1-->3)) beta-Man-O-octyl where R is OH (14), O-methyl (17), O-pentyl (18), O-(4,4-azo)pentyl (19), O-(5-iodoacetamido)pentyl (20) and O-(5-amino)pentyl (21); 2-deoxy-alpha-Man(1-->6)(beta-GlcNAc(1-->2) alpha-Man(1-->3)) beta-Man-O-octyl (16), 4-O-methyl-alpha-Man(1-->6) (beta-GlcNAc(1-->2) alpha-Man(1-->3)) beta-Man-O-octyl (22), 6-O-methyl-alpha-Man(1-->6)(beta-GlcNAc(1-->2) alpha Man(1-->3)) beta-Man-O-octyl (23) and alpha-Man(1-->6)[beta-GlcNAc(1-->2)(4-O-methyl) alpha-Man(1-->3)] beta-Man-O-octyl (15) were also synthesized by this procedure. The yields ranged from 80 to 99%. Products were characterized by high resolution 1H and 13C nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. Compounds 14, 15, 17, 22, and 23 are excellent substrates for UDP-GlcNAc: alpha-Man(1-->6R) beta-(1-->2)-N-acetylglucosaminyltransferase II and the other compounds are inhibitors of this enzyme.
Assuntos
N-Acetilglucosaminiltransferases/metabolismo , Oligossacarídeos/síntese química , Trissacarídeos/metabolismo , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Linhagem Celular , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mariposas , N-Acetilglucosaminiltransferases/isolamento & purificação , Oligossacarídeos/biossíntese , Oligossacarídeos/química , Rotação Ocular , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Especificidade por Substrato , Transfecção , Trissacarídeos/químicaRESUMO
Recombinant human UDP-GlcNAc: alpha-Man-(1-->6)R beta-(1-->2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143, GlcNAc-T II) was produced in the Sf9 insect cell/baculovirus expression system as a fusion protein with a (His)6 tag and partially purified by affinity chromatography on a metal chelating column. The partially purified enzyme was used to catalyze the transfer of GlcNAc from UDP-GlcNAc to R-alpha-Man(1-->6)(beta-GlcNAc(1-->2)alpha-Man(1-->3))beta-Man-O-octyl to form beta-GlcNAc(1-->2)R-alpha-Man(1-->6)(beta-GlcNAc(1-->2)alpha- Man(1-->3))beta-Man-O-octyl where there is either no modification of the alpha-Man(1-->6) residue (7), or where R is 3-deoxy (8), 4-deoxy (9) or 6-deoxy (10). The yields ranged from 64-80%. Products were characterized by 1H and 13C nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. Compounds 7-10 are pentasaccharide analogues of the biantennary N-glycan substrates of N-acetylglucosaminyltransferases III, IV and V.
Assuntos
N-Acetilglucosaminiltransferases/metabolismo , Oligossacarídeos/biossíntese , Polissacarídeos/metabolismo , Configuração de Carboidratos , Sequência de Carboidratos , Glicosilação , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/isolamento & purificação , Oligossacarídeos/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por SubstratoAssuntos
N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/metabolismo , Oligossacarídeos/síntese química , Acetilglucosamina/análogos & derivados , Acetilglucosamina/química , Sequência de Carboidratos , Glicopeptídeos/química , Glicosilação , Espectroscopia de Ressonância Magnética , Manose/análogos & derivados , Manose/química , Dados de Sequência Molecular , Estrutura Molecular , Oligossacarídeos/metabolismo , Ramnose/análogos & derivados , Ramnose/química , Especificidade por SubstratoRESUMO
AIM: We have investigated the value of flexion osteotomy for femoral head necrosis with regard to the survival rate. METHOD: We examined 40 patients who were treated by flexion osteotomy for femoral head necrosis at the Hannover Medical School in the years 1969 to 1995. The mean follow-up period was 14.1 years. RESULTS: The mean survival time of the flexion osteotomies was 9.6 years. In 23 of the 40 patients, it failed within 10 years after surgery, which implies a 10-year survival rate of 42.5 %. For these 23 patients, the implantation of a total hip arthroplasty (THA) became necessary after an average time of 6.6 years. CONCLUSION: Due to the poorly predictable success of the flexion osteotomy and the comparatively poor patient satisfaction, the indication for flexion osteotomy in such cases should be reconsidered.
Assuntos
Necrose da Cabeça do Fêmur/diagnóstico por imagem , Necrose da Cabeça do Fêmur/cirurgia , Osteotomia/métodos , Adolescente , Adulto , Criança , Intervalo Livre de Doença , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Radiografia , Falha de Tratamento , Resultado do TratamentoRESUMO
A series of phosphinate transition-state analogues of the L-alanine adding enzyme (MurC) of bacterial peptidoglycan biosynthesis was prepared and tested as inhibitors of the Escherichia coli enzyme. Compound 4 was identified as a potent inhibitor of MurC from Escherichia coli with an IC(50) of 49nM.
Assuntos
Parede Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Peptídeo Sintases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Escherichia coli/efeitos dos fármacos , Peptídeo Sintases/metabolismo , Peptidoglicano/metabolismoRESUMO
The bacterial UDP-N-acetylmuramyl-L-alanine ligase (MurC) from Escherichia coli, an essential, cytoplasmic peptidoglycan biosynthetic enzyme, catalyzes the ATP-dependent ligation of L-alanine (Ala) and UDP-N-acetylmuramic acid (UNAM) to form UDP-N-acetylmuramyl-L-alanine (UNAM-Ala). The phosphinate inhibitor 1 was designed and prepared as a multisubstrate/transition state analogue. The compound exhibits mixed-type inhibition with respect to all three enzyme substrates (ATP, UNAM, Ala), suggesting that this compound forms dead-end complexes with multiple enzyme states. Results from isothermal titration calorimetry (ITC) studies supported these findings as exothermic binding was observed under conditions with free enzyme (K(d) = 1.80-2.79 microM, 95% CI), enzyme saturated with ATP (K(d) = 0.097-0.108 microM, 95% CI), and enzyme saturated with the reaction product ADP (K(d) = 0.371-0.751 microM, 95% CI). Titrations run under conditions of saturating UNAM or the product UNAM-Ala did not show heat effects consistent with competitive compound binding to the active site. The potent binding affinity observed in the presence of ATP is consistent with the inhibitor design and the proposed Ordered Ter-Ter mechanism for this enzyme; however, the additional binding pathways suggest that the inhibitor can also serve as a product analogue.
Assuntos
Escherichia coli/enzimologia , Peptídeo Sintases/antagonistas & inibidores , Ácidos Fosfínicos/metabolismo , Sequência de Bases , Calorimetria , Primers do DNA , Peptídeo Sintases/metabolismo , Ácidos Fosfínicos/farmacologiaRESUMO
UDP-GlcNAc:Man alpha 1-3R beta 1-2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) catalyses the conversion of [Man alpha 1-6(Man alpha 1-3)Man alpha 1-6][Man alpha 1-3]Man beta-O-R to [Man alpha 1-6(Man alpha 1-3)Man alpha 1-6] [GlcNAc beta 1-2Man alpha 1-3]Man beta-O-R (R = 1-4GlcNAc beta 1-4GlcNAc- Asn-X) and thereby controls the conversion of oligomannose to complex and hybrid asparagine-linked glycans (N-glycans). GlcNAc-T I also catalyses the conversion of Man alpha 1-6(Man alpha 1-3)Man beta-O-octyl to Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta-O-octyl. We have therefore tested a series of synthetic analogues of Man"alpha 1-6(Man'alpha 1-3)Man beta-O-octyl as substrates and inhibitors for rat liver GlcNAc-T I. The 2"-deoxy and the 3"-, 4"- and 6"-O-methyl derivatives are all good substrates confirming previous observations that the hydroxyl groups of the Man"alpha 1-6 residue do not play major roles in the binding of substrate to enzyme. In contrasts, all four hydroxyl groups on the Man'alpha 1-3 residue are essential since the corresponding deoxy derivatives either do not bind (2'- and 3'-deoxy) or bind very poorly (4'- and 6'-deoxy) to the enzyme. The 2'- and 3'-O-methyl derivatives also do not bind to the enzyme. However, the 4'-O-methyl derivative is a substrate (KM = 2.6 mM) and the 6'-O-methyl compound is a competitive inhibitor (Ki = 0.76 mM). We have therefore synthesized various 4'- and 6'-O-alkyl derivatives, some with reactive groups attached to an O-pentyl spacer, and tested these compounds as reversible and irreversible inhibitors of GlcNAc-T I. The 6'-O-(5-iodoacetamido-pentyl) compound is a specific time dependent inhibitor of the enzyme. Four other 6'-O-alkyl compounds showed competitive inhibition while the remaining compounds showed little or no binding indicating that the electronic properties of the attached O-pentyl groups influence binding.
Assuntos
N-Acetilglucosaminiltransferases/química , Animais , Sequência de Carboidratos , Dados de Sequência Molecular , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Ratos , Especificidade por SubstratoRESUMO
Cells from patients with acute myeloid leukaemia (AML) contain an abnormally high UDP-GlcNAc: Gal beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase (core 2 beta 6-Gn-T) activity. Upon UV irradiation at 350 nm, the substrate Gal beta 1-3GalNAc alpha-p-nitrophenyl acted as an effective inhibitor for this enzyme but not for several other transferases. Preincubation with Gal beta 1-3GalNAc alpha-benzyl but not GalNAc alpha-benzyl protected core 2 beta 6-Gn-T from inhibition indicating that the inhibitor is specific for the substrate binding site of core 2 beta 6-Gn-T. A number of other nitrophenyl-sugar derivatives similarly acted as inhibitors for core 2 beta 6-Gn-T. GalNAc alpha-pnp at higher concentrations also inactivated UDP-Gal: GalNAc-R beta 3-galactosyltransferase from rat liver and AML cells and inhibition could be reduced by substrate protection. These results suggest that pnp-sugar derivatives may prove useful as specific inhibitors of glycosyltransferases and as affinity labels.
Assuntos
Acetilgalactosamina/análogos & derivados , Dissacarídeos/metabolismo , Dissacarídeos/farmacologia , Leucemia Mieloide/enzimologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Acetilgalactosamina/metabolismo , Acetilgalactosamina/farmacologia , Doença Aguda , Sequência de Carboidratos , Glicosiltransferases/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Raios UltravioletaRESUMO
The (L)-alpha-lyxopyranosyl-(4'-->3')-oligonucleotide system-a member of a pentopyranosyl oligonucleotide family containing a shortened backbone-is capable of cooperative base-pairing and of cross-pairing with DNA and RNA. In contrast, corresponding (D)-beta-ribopyransoyl-(4'-->3')-oligonucleotides do not show base-pairing under similar conditions. We conclude that oligonucleotide systems can violate the 'six-bonds-per-backbone-unit' rule by having five bonds instead, if their vicinally bound phosphodiester bridges can assume an antiperiplanar conformation. An additional structural feature that seems relevant to the cross-pairing capability of the (L)-alpha-lyxopyranosyl-(4'-->3')-oligonucleotide system is its (small) backbone/basepair axes inclination. An inclination which is similar to that in B-DNA seems to be a prerequisite for an oligonucleotide system's capability to cross-pair with DNA.
Assuntos
Oligorribonucleotídeos/química , Pareamento de Bases , Cromatografia Líquida de Alta Pressão , Modelos Moleculares , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Oligorribonucleotídeos/síntese química , Oligorribonucleotídeos/metabolismo , Pentoses , Ribose , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , TemperaturaRESUMO
UDP-GlcNAc: Man alpha 3R beta 2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) is the key enzyme in the synthesis of complex and hybrid N-glycans. Rat liver GlcNAc-T I has been purified more than 25,000-fold (M(r) 42,000). The Vmax for the pure enzyme with [Man alpha 6(Man alpha 3)Man alpha 6](Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc beta-Asn as substrate was 4.6 mumol min-1 mg-1. Structural analysis of the enzyme product by proton nuclear magnetic resonance spectroscopy proved that the enzyme adds an N-acetylglucosamine (GlcNAc) residue in beta 1-2 linkage to the Man alpha 3Man beta-terminus of the substrate. Several derivatives of Man alpha 6(Man alpha 3)Man beta-R, a substrate for the enzyme, were synthesized and tested as substrates and inhibitors. An unsubstituted equatorial 4-hydroxyl and an axial 2-hydroxyl on the beta-linked mannose of Man alpha 6(Man alpha 3)Man beta-R are essential for GlcNAc-T I activity. Elimination of the 4-hydroxyl of the alpha 3-linked mannose (Man) of the substrate increases the KM 20-fold. Modifications on the alpha 6-linked mannose or on the core structure affect mainly the KM and to a lesser degree the Vmax, e.g., substitutions of the Man alpha 6 residue at the 2-position by GlcNAc or at the 3- and 6-positions by mannose lower the KM, whereas various other substitutions at the 3-position increase the KM slightly. Man alpha 6(Man alpha 3)4-O-methyl-Man beta 4GlcNAc was found to be a weak inhibitor of GlcNAc-T I.
Assuntos
Glicoproteínas/biossíntese , Fígado/enzimologia , N-Acetilglucosaminiltransferases/metabolismo , Animais , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Espectroscopia de Ressonância Magnética , Manose/metabolismo , Dados de Sequência Molecular , N-Acetilglucosaminiltransferases/isolamento & purificação , Ratos , Especificidade por SubstratoRESUMO
UDP-GlcNAc:Galbeta1-3GalNAc-R (GlcNAc to GalNAc) beta-1, 6-N-acetylglucosaminyltransferase (C2GnT) catalyses the formation of O-glycan core 2. Purification and characterization of C2GnT from natural sources has been hampered by the instability of this enzyme. We have been able to prepare a stable partly purified recombinant human C2GnT by expression of a truncated form of the enzyme in the baculovirus/Spodoptera frugiperda 9 (Sf9) insect cell system. C2GnT activity was secreted into the Sf9 culture medium (15 pmol/min per microl; approx. 0.2 mg/l) and was stable at 4 degrees C either in solution or after lyophilization. Endoglycosidase H and N-glycanase F treatment of the radiolabelled C2GnT indicated the presence of N-glycans at both potential N-glycosylation sites. The elimination of one or both of the two potential N-glycosylation sites or treatment of the virus-infected insect cells with tunicamycin resulted in loss of enzyme activity due in part to protein degradation.
Assuntos
N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/biossíntese , Animais , Linhagem Celular , Primers do DNA , Estabilidade Enzimática , Glicosiltransferases/metabolismo , Humanos , Cinética , Mutagênese Sítio-Dirigida , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/isolamento & purificação , Reação em Cadeia da Polimerase , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Spodoptera , TransfecçãoRESUMO
UDP-GlcNAc:Man alpha 1-6R beta(1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complex N-glycans. We have tested a series of synthetic analogues of the substrate Man"'alpha 1-6(GlcNAc"beta 1-2Man'alpha 1-3)Man beta-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attaches N-acetylglucosamine in beta 1-2 linkage to the 2"'-OH of the Man"'alpha 1-6 residue. The 2"'-deoxy analogue is a competitive inhibitor (Ki = 0.13 mM). The 2"'-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3"'-, 4"'- and 6"'-OH groups are not essential for binding or catalysis since the 3"'-, 4"'- and 6"'-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3"'-position to pentyl and substituted pentyl groups causes competitive inhibition (Ki = 1.0-2.5 mM). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3"'-O-(4,4-azo)pentyl group and a 3"'-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man"'alpha 1-6 residue are essential for binding although the 2"'- and 3"'-OH face the catalytic site of the enzyme. The 4-OH group of the Man beta-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
N-Acetilglucosaminiltransferases/metabolismo , Animais , Metabolismo dos Carboidratos , Sequência de Carboidratos , Carboidratos/síntese química , Fígado/enzimologia , Dados de Sequência Molecular , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Ratos , Especificidade por SubstratoRESUMO
UDP-GlcNAc:GlcNAc beta 1-2Man alpha 1-6R (GlcNAc to Man) beta 1,6- N-acetylglucosaminyltransferase V (GlcNAc-T V) adds a GlcNAc beta 1-6 branch to bi- and triantennary N-glycans. An increase in this activity has been associated with cellular transformation, metastasis and differentiation. We have used synthetic substrate analogues to study the substrate specificity and inhibition of the partially purified enzyme from hamster kidney and of extracts from hen oviduct membranes and acute myeloid leukaemia leukocytes. All compounds with the minimum structure GlcNAc beta 1-2Man alpha 1-6Glc/Man beta-R were good substrates for GlcNAc-T V. The presence of structural elements other than the minimum trisaccharide structure affected GlcNAc-T V activity without being an absolute requirement for activity. Substrates with a biantennary structure were preferred over linear fragments of biantennary structures. Kinetic analysis showed that the 3-hydroxyl of the Man alpha 1-3 residue and the 4-hydroxyl of the Man beta- residue of the Man alpha 1-6(Man alpha 1-3)Man beta-R N-glycan core are not essential for catalysis but influence substrate binding. GlcNAc beta 1-2(4,6-di-O-methyl-)Man alpha 1-6Glc beta-pnp was found to be an inhibitor of GlcNAc-T V from hamster kidney, hen oviduct microsomes and acute and chronic myeloid leukaemia leukocytes.
Assuntos
Inibidores Enzimáticos/metabolismo , Leucemia Mieloide Aguda/enzimologia , Leucócitos/efeitos dos fármacos , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Animais , Sequência de Carboidratos , Galinhas , Cricetinae , Feminino , Humanos , Leucemia Mieloide Aguda/sangue , Leucócitos/enzimologia , Dados de Sequência Molecular , Estrutura Molecular , Especificidade por SubstratoRESUMO
UDP-GlcNAc:alpha-6-D-mannoside [GlcNAc to Man alpha 1-6] beta-1,2-N-acetylglucosaminyltransferase II (GlcNAc-T II, EC 2.4.1.143) is a Golgi enzyme catalyzing an essential step in the conversion of oligomannose to complex N-glycans. A 1.2-kb probe from a rat liver cDNA encoding GlcNAc-T II was used to screen a human genomic DNA library in lambda EMBL3. Southern analysis of restriction endonuclease digests of positive phage clones identified two hybridizing fragments (3.0 and 3.5 kb) which were subcloned into pBlueScript. The inserts of the resulting plasmids (pHG30 and pHG36) are over-lapping clones containing 5.5 kb of genomic DNA. The pHG30 insert (3.0 kb) contains a 1341-bp open reading frame encoding a 447-amino-acid protein, 250 bp of G + C-rich 5'-upstream sequence and 1.4 kb of 3'-downstream sequence. The pHG36 insert (3.5 kb) contains 2.75 kb of 5'-upstream sequence and 750 bp of the 5'-end of the open reading frame. The protein sequence showed the domain structure typical of all previously cloned glycosyltransferases, i.e. a short 9-residue putative cytoplasmic N-terminal domain, a 20-residue hydrophobic non-cleavable putative signal-anchor domain and a 418-residue C-terminal catalytic domain. Northern analysis of human tissues showed a major message at 3 kb and minor signals at 2 and 4.5 kb. There is no sequence similarity to any previously cloned glycosyltransferases including human UDP-GlcNAc:alpha-3-D-mannoside [GlcNAc to Man alpha 1-3] beta-1,2-N-acetylglucosaminyltransferase I (GlcNAc-T I) which has 445 amino acids with a 418-residue C-terminal catalytic domain. The human GlcNAc-T I and II genes (MGAT1 and MGAT2) map to chromosome bands 5q35 and 14q21, respectively, by fluorescence in situ hybridization. The entire coding regions of human GlcNAc-T I and II are each on a single exon. There is 92% identity between the amino acid sequences of the catalytic domains of human and rat GlcNAc-T II. Southern analysis of restriction enzyme digests of human genomic DNA indicates that there is only a single copy of the MGAT2 gene. The full-length coding region of GlcNAc-T II has been expressed in the baculovirus/Sf9 insect cell system, the recombinant enzyme has been purified to near homogeneity with a specific activity of about 20 mumol.min-1.mg-1 and the product synthesized by the recombinant enzyme has been identified by high-resolution 1H-NMR spectroscopy and mass spectrometry.