Poly-N-acetyllactosamine synthesis in branched N-glycans is controlled by complemental branch specificity of I-extension enzyme and beta1,4-galactosyltransferase I.
Poly-N-acetyllactosamine is a unique carbohydrate that can carry various functional oligosaccharides, such as sialyl Lewis X. It has been shown that the amount of poly-N-acetyllactosamine is increased in N-glycans, when they contain Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->4GlcNAcbeta1 -->2)Manalpha1-->6 branched structure. To determine how this increased synthesis of poly-N-acetyllactosamines takes place, the branched acceptor was incubated with a mixture of i-extension enzyme (iGnT) and beta1, 4galactosyltransferase I (beta4Gal-TI). First, N-acetyllactosamine repeats were more readily added to the branched acceptor than the summation of poly-N-acetyllactosamines formed individually on each unbranched acceptor. Surprisingly, poly-N-acetyllactosamine was more efficiently formed on Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chain than in Galbeta1-->4GlcNAcbeta1-->6Manalpha-->R, due to preferential action of iGnT on Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chain. On the other hand, galactosylation was much more efficient on beta1,6-linked GlcNAc than beta1,2-linked GlcNAc, preferentially forming Galbeta1-->4GlcNAcbeta1-->6(GlcNAcbeta1-->2)Manalph a1-->6Manbeta -->R. Starting with this preformed acceptor, N-acetyllactosamine repeats were added almost equally to Galbeta1-->4GlcNAcbeta1-->6Manalpha-->R and Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chains. Taken together, these results indicate that the complemental branch specificity of iGnT and beta4Gal-TI leads to efficient and equal addition of N-acetyllactosamine repeats on both side chains of GlcNAcbeta1-->6(GlcNAcbeta1-->2)Manalpha1-->6Manbet a-->R structure, which is consistent with the structures found in nature. The results also suggest that the addition of Galbeta1-->4GlcNAcbeta1-->6 side chain on Galbeta1-->4GlcNAcbeta1-->2Man-->R side chain converts the acceptor to one that is much more favorable for iGnT and beta4Gal-TI.
Ujita
M
M
The Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, California 92037, USA. minoru@burnham-inst.org
McAuliffe
J
J
Hindsgaul
O
O
Sasaki
K
K
Fukuda
M N
MN
Fukuda
M
M
D000017:ABO Blood-Group System; D000943:Antigens, Differentiation; D000951:Antigens, Neoplasm; D002240:Carbohydrate Sequence; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism; D007983:Lewis Blood Group Antigens; D016256:Lewis X Antigen; D008954:Models, Biological; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D000017:ABO Blood-Group System; D000943:Antigens, Differentiation; D000951:Antigens, Neoplasm; D007983:Lewis Blood Group Antigens; D016256:Lewis X Antigen; D009844:Oligosaccharides; D011134:Polysaccharides; D011994:Recombinant Proteins; C037199:poly-N-acetyllactosamine; C106501:N-acetylglucosaminyltransferase IGnT; D005700:Galactosyltransferases; D017351:N-Acetylglucosaminyltransferases; C111678:beta-1,4-galactosyltransferase IV