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.
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.
Mattila
P
P
Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, SF-00014 Helsinki, Finland. pirkko.mattila@helsinki.fi
Salminen
H
H
Hirvas
L
L
Niittymäki
J
J
Salo
H
H
Niemelä
R
R
Fukuda
M
M
Renkonen
O
O
Renkonen
R
R
D000606:Amino Sugars / Q000096:biosynthesis* / Q000737:chemistry*; D016367:Baculoviridae / Q000235:genetics; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D020134:Catalytic Domain; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry*; D011993:Recombinant Fusion Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured / Q000201:enzymology
D000606:Amino Sugars; D010446:Peptide Fragments; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; C066929:polylactosamine; D017351:N-Acetylglucosaminyltransferases; D005982:Glutathione Transferase