Characterization of an N-acetylglucosamine-6-O-sulfotransferase from human respiratory mucosa active on mucin carbohydrate chains.
A microsomal GlcNAc-6-O-sulfotransferase activity from human bronchial mucosa, able to transfer a sulfate group from adenosine 3'-phosphate 5'-phosphosulfate onto methyl-N-acetylglucosaminides or terminal N-acetylglucosamine residues of carbohydrate chains from human respiratory mucins, has been characterized. The reaction products containing a terminal HO3S-6GlcNAc were identified by high performance anion-exchange chromatography. Using methyl-beta-N-acetylglucosaminide as a substrate, the optimal activity was obtained with 0.1% Triton X-100, 30 mM NaF, 20 mM Mn2+, 5 mM AMP in a 30 mM MOPS (3-(N-morpholino) propanesulfonic acid) buffer at pH 6.7. The apparent Km values for adenosine 3'-phosphate 5'-phosphosulfate and methyl-beta-N-acetylglucosaminide were observed at 9.1 x 10(-6) M and 0.54 x 10(-3) M, respectively. The enzyme had more affinity for carbohydrate chains with a terminal GlcNAc residue than for methyl-beta-N-acetylglucosaminide; it was unable to catalyze the transfer of sulfate to position 6 of the GlcNAc residue contained in a terminal Galbeta1-4GlcNAc sequence. However, oligosaccharides with a nonreducing terminal HO3S-6GlcNAc were substrates for a beta1-4 galactosyltransferase from human bronchial mucosa. These data point out that GlcNAc-6-O-sulfotransferase must act before beta1-4 galactosylation in mucin-type oligosaccharide biosynthesis.
Degroote
S
S
Unité INSERM 377, place de Verdun, F-59045 Lille, France.
Lo-Guidice
J M
JM
Strecker
G
G
Ducourouble
M P
MP
Roussel
P
P
Lamblin
G
G
D000117:Acetylglucosamine / Q000378:metabolism; D001667:Binding, Competitive; D001980:Bronchi / Q000201:enzymology*; D002236:Carbohydrate Conformation; D002851:Chromatography, High Pressure Liquid; D005690:Galactose / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009077:Mucins / Q000378:metabolism*; D009092:Mucous Membrane / Q000201:enzymology; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism; D015238:Sulfotransferases / Q000737:chemistry / Q000378:metabolism*; D000097811:Carbohydrate Sulfotransferases
D000117:Acetylglucosamine; D005690:Galactose; D005700:Galactosyltransferases; D009077:Mucins; D010724:Phosphoadenosine Phosphosulfate; D013431:Sulfates; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases