The centrally acting beta1,6N-acetylglucosaminyltransferase (GlcNAc to gal). Functional expression, purification, and acceptor specificity of a human enzyme involved in midchain branching of linear poly-N-acetyllactosamines

• Published in: The Journal of biological chemistry Vol. 273; no. 42; pp. 27633 - 27639
• Main Authors: Mattila, P, Salminen, H, Hirvas, L, Niittymäki, J, Salo, H, Niemelä, R, Fukuda, M, Renkonen, O, Renkonen, R
• Format: Journal Article
• Language: English
• Published: United States 16-10-1998
• Subjects: Amino Sugars - biosynthesis; Amino Sugars - chemistry; Baculoviridae - genetics; Carbohydrate Conformation; Carbohydrate Sequence; Catalytic Domain; Glutathione Transferase - genetics; Humans; Models, Biological; Molecular Sequence Data; N-Acetylglucosaminyltransferases - genetics; N-Acetylglucosaminyltransferases - isolation & purification; N-Acetylglucosaminyltransferases - metabolism; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments; Polysaccharides - biosynthesis; Polysaccharides - chemistry; Recombinant Fusion Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Tumor Cells, Cultured - enzymology
• ISSN: 0021-9258

In the present experiments the cDNA coding for a truncated form of the beta1,6N-acetylglucosaminyltransferase responsible for the conversion of linear to branched polylactosamines in human PA1 cells was expressed in Sf9 insect cells. The catalytic ectodomain of the enzyme was fused to glutathione S-transferase, allowing effective one-step purification of the glycosylated 67-74-kDa fusion protein. Typically a yield of 750 microg of the purified protein/liter of suspension culture was obtained. The purified recombinant protein catalyzed the transfer of GlcNAc from UDP-GlcNAc to the linear tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc, converting the acceptor to the branched pentasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 GlcNAc as shown by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, degradative experiments, and 1H NMR spectroscopy of the product. By contrast, the recombinant enzyme did not catalyze any reaction when incubated with UDP-GlcNAc and the trisaccharide GlcNAcbeta1-3Galbeta1-4GlcNAc. Accordingly, we call the recombinant beta1,6-GlcNAc transferase cIGnT6 to emphasize its action at central rather than peridistal galactose residues of linear polylactosamines in the biosynthesis of blood group I antigens. Taken together this in vitro expression of I-branching enzyme, in combination with the previously cloned enzymes, beta1,4galactosyltransferase and beta1, 3N-acetylglucosaminyltransferase, should allow the general synthesis of polylactosamines based totally on the use of recombinant enzymes.