Abstract
We have characterized the UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase in human tracheal epithelium using asialo ovine submaxillary mucin as the acceptor. Maximal enzyme activity was obtained at pH 6.0-7.5 and at 20-25 mM MnCl2 and at 2% Triton X-100. Cd2+ could substitute for Mn2+ as the divalent ion cofactor. Spermine, spermidine, putrecine, cadaverine, and poly-L-lysine stimulated the enzyme activity at low (2.5 mM) MnCl2 concentration. The apparent Michaelis constants for N-acetylgalactosamine, asialo ovine submaxillary mucin, and UDP-galactose were 15.5, 1.14, and 1.36 mM, respectively. The enzyme activity was not affected by alpha-lactalbumin. The alpha-N-acetygalactosaminide beta 3 galactosyltransferase was shown to be different from the N-acetylglucosamine galactosyltransferase by acceptor competition studies. The product of galactosyltransferase was identified as Gal beta 1 leads to 3GalNAc alpha Ser (Thr) by (a) isolation of [14C]Gal-GalNAc-H2 after alkaline borohydride treatment of the 14C-labeled product, (b) establishment of the beta-configuration of the newly synthesized glycosidic bond by its complete cleavage by bovine testicular beta-galactosidase, and (c) assignment of the 1 leads to 3 linkage by identification of threosaminitol obtained from the oxidation of the disaccharide with periodic acid followed by reduction with sodium borohydride, hydrolysis in 4 N HCl, and analysis on an amino acid analyzer. The 1 leads to 3 linkage was confirmed by its resistance to jack bean beta-galactosidase and by the presence of a m/e 307 ion fragment and the absence of a m/e 276 ion by gas-liquid chromatography-mass spectrometry analysis. When acid and beta-galactosidase-treated human tracheobronchial mucin was used as the acceptor, 3.3% of the product was found as [14C]Gal-GalNAc-H2. The remainder of the [14C]Gal was found in longer oligosaccharides formed by a different beta-galactosyltransferase. This galactosyltransferase is slightly inhibited by alpha-lactalbumin and stimulated by spermine.
Highlights
From the Cystic Fibrosis Center, Rainbow Babies and ChildrensHospital, and the Departments of Biochemistry and Pediatrics, Case Western Reserve University, Cleveland, Ohio 44106
We have characterized the UDP-ga1actose:a-N-ace- sugar nucleotide substrates to the growing oligosaccharide tylgalactosaminide 83 galactosyltransferase inhuman acceptor [1].Each step is catalyzed by a glycosyltransferase tracheal epithelium using asialo ovine submaxillary which is specific for the glycosyl donor and the mucin as the acceptor
The P-galactosidelinkagein the GalPl -+ 3GalNAcaSer shown to be different from the N-acetylglucosamine (Thr) structure is commonly found in mucous glycoproteins galactosyltransferase by acceptor competition studies. [8,9] including human tracheobronchiaml ucous glycoproteins
Summary
From the Cystic Fibrosis Center, Rainbow Babies and ChildrensHospital, and the Departments of Biochemistry and Pediatrics, Case Western Reserve University, Cleveland, Ohio 44106. We have characterized the UDP-ga1actose:a-N-ace- sugar nucleotide substrates to the growing oligosaccharide tylgalactosaminide 83 galactosyltransferase inhuman acceptor [1].Each step is catalyzed by a glycosyltransferase tracheal epithelium using asialo ovine submaxillary which is specific for the glycosyl donor and the mucin as the acceptor. The P-galactosidelinkagein the GalPl -+ 3GalNAcaSer shown to be different from the N-acetylglucosamine (Thr) structure is commonly found in mucous glycoproteins galactosyltransferase by acceptor competition studies. We report studies of complete cleavage by bovine testicular&galactosidase, the a-N-acetylgalactosaminide D l ”+ 3 galactosyltransferase and (c) assignment of the 1+ 3 linkage by identification in human tracheal epithelium[15], including definitecharacof threosaminitoi obtained from the oxidation of the terization of its product. The 1-+ 3 linkage was confirmed by its resistance to jack bean 8-galac-
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