All pyruvylated galactose in Schizosaccharomyces pombe N-glycans is present in the terminal disaccharide, 4, 6-O-[(R)-(1- carboxyethylidine)]-Galbeta1,3Galalpha1- |
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Authors: | Gemmill TR; Trimble RB |
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Institution: | Department of Biomedical Sciences, State University of New York at Albany School of Public Health, Albany, New York 12201, USA. |
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Abstract: | The large N-linked oligosaccharides released from Schizosaccharomyces pombe
by endo-beta-N-acetylglucosaminidase H were examined to determine how the
negatively chargedpyruvylated galactoses present (Gemmill,T.R., and
Trimble,R.B., 1996, J. Biol. Chem ., 271, 25945-25949) were attached to the
oligosaccharide chains. Binding of biotinylated human serum amyloid P and
peanut agglutinin to native and depyruvylated S.pombe glycoproteins,
respectively, indicated that the pyruvylated epitope was likely to be in
the beta configuration. Examination by high- field 1H NMR of whole glycans
and a disaccharide fragment released from them on partial acid hydrolysis
showed that the pyruvylated galactose species was in fact beta1,3-linked to
a second galactose, and this occurred an average of five to six times on
nominal Gal57Man64GlcNAc N- glycans. The pyruvate-2,(4,6)Gal-beta1,3Gal
epitope is chemically similar to acetaldehyde-Galbeta1,3Gal groups found on
the glycoproteins from Paramyxovirus-infected bovine kidney cells (Prehm,
P., Scheid,A. and Choppin,P.W. ,1979, J. Biol. Chem ., 254, 9669-9677). The
1:1 stoichiometry between pyruvate and beta-linked galactose in these
S.pombe glycans indicates that either pyruvate addition to terminal
beta1,3Gal is highly efficient or that pyruvylated Gal is transferred en
bloc to alpha1,2-linked Gal residues in theN-linked chains. In
contradiction to many galactomannan-producing fungi, which add substantial
amounts of Gal in the furanose form to their glycoproteins, all detectable
Gal in the large S.pombe galactomannans is in the pyranose form, as found
in higher eukaryotes. The current work shows that the S.pombe outer chain
structure is a poly-alpha1,6Man backbone 2- O-substituted with either Gal
or the pyruvylated galactobiose and contains little alpha1,2-linked or
2-O-substituted Man. This is in contrast to the S. cerevisiae outer chain,
which is poly-alpha1,6Man substituted with alpha1,2-linked Man sidechains
(Ballou,C.E. ,1990, Methods Enzymol , 185, 440-470).
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