Gemmill T R, Trimble R B
Department of Biomedical Sciences, State University of New York at Albany School of Public Health, Albany, New York 12201, USA.
Glycobiology. 1998 Nov;8(11):1087-95. doi: 10.1093/glycob/8.11.1087.
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).
研究了经内切β-N-乙酰葡糖胺酶H从粟酒裂殖酵母释放的大N-连接寡糖，以确定所存在的带负电荷的丙酮酸化半乳糖（Gemmill，T.R.和Trimble，R.B.，1996，《生物化学杂志》，271，25945 - 25949）是如何连接到寡糖链上的。生物素化的人血清淀粉样蛋白P和花生凝集素分别与天然和去丙酮酸化的粟酒裂殖酵母糖蛋白结合，表明丙酮酸化表位可能处于β构型。通过高场1H NMR对完整聚糖以及部分酸水解从中释放的二糖片段进行检测，结果表明丙酮酸化半乳糖种类实际上是通过β1,3连接到第二个半乳糖，并且在标称的Gal57Man64GlcNAc N-聚糖上平均发生五到六次。丙酮酸-2，(4,6)Gal-β1,3Gal表位在化学上类似于在副粘病毒感染的牛肾细胞糖蛋白上发现的乙醛-Galβ1,3Gal基团（Prehm，P.，Scheid，A.和Choppin，P.W.，1979，《生物化学杂志》，254，9669 - 9677）。这些粟酒裂殖酵母聚糖中丙酮酸与β连接的半乳糖之间1:1的化学计量比表明，要么向末端β1,3Gal添加丙酮酸的效率很高，要么丙酮酸化的Gal以整体形式转移到N-连接链中α1,2连接的Gal残基上。与许多产生半乳甘露聚糖的真菌不同，后者向其糖蛋白中添加大量呋喃糖形式的Gal，在粟酒裂殖酵母大的半乳甘露聚糖中所有可检测到的Gal都处于吡喃糖形式，这与高等真核生物中的情况相同。目前的研究表明，粟酒裂殖酵母的外链结构是一个多α1,6Man主链，其2-O位被Gal或丙酮酸化的半乳糖二糖取代，并且几乎不含有α1,2连接或2-O取代的Man。这与酿酒酵母的外链不同，酿酒酵母的外链是被α1,2连接的Man侧链取代的多α1,6Man（Ballou，C.E.，1990，《酶学方法》，185，440 - 470）。
