Characterization of phosphorylated oligomannosides from Hansenula wingei mannoprotein

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Abstract
The acidic acetolysis fragments from H. wingei mannoprotein (Yen, P. H., and Ballou, C. E., 1974) were isolated and separated into a mannopentaose monophosphate (Man5P) and a mannotriose monophosphate (Man3P). Based on selective enzymatic and partial acid hydrolysis and 1H and 31P NMR studies, Man5P had the structure P .fwdarw. 6.alpha.Man1 .fwdarw. 3.alpha.Man1 .fwdarw. 2.alpha.Man1 .fwdarw. 2.alpha.Man1 .fwdarw. 2Man (where Man = D-mannopyranose). The 31P NMR spectrum of the Man3P had a mixture of a phosphate monoester and a phosphate diester, which was separated by ion-exchange chromatography. The monoester had the structure P .fwdarw. 6.alpha.Man1 .fwdarw. 3.alpha.Man1 .fwdarw. 3Man, whereas the diester had the properties of a cyclic phosphate. Native H. wingei mannoprotein contains phosphodiester linkages, but the starting mannoprotein preparation was isolated under alkaline conditions that hydrolyze such bonds; there was no phosphodiester signal in the 31P NMR spectrum. The cyclic phosphate apparently is an artifact formed during the acetolysis reaction. Because acetolysis of H. wingei mannoprotein yields only phosphorylated mannotriose and mannopentaose, whereas the mannoprotein contains mannotetraose side chains as well, the phosphorylation process must be a very specific event in the biosynthesis of the glycoprotein.