Carbohydrate chains on yeast carboxypeptidase Y are phosphorylated.

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Abstract
Carboxypeptidase Y, a vacuolar enzyme from S. cerevisiae, was digested with endo-.beta.-N-acetyl-D-glucosaminidase H to release the 4 oligosaccharide chains that are linked to asparagine in the glycoprotein. The oligosaccharides were fractionated into a neutral and acidic component and the latter proved to be phosphorylated. From its gel filtration pattern, the neutral fraction was a mixture of at least 4 homologs, the smallest of which had a proton NMR spectrum almost identical to that given by an IgM oligosaccharide with 8 mannoses and 1 N-acetylglucosamine [Cohen, R. E. et Ballou, C. E.]. The yeast oligosaccharide has 1 additional mannose unit in an .alpha.1 .fwdarw. 3 or .alpha.1 .fwdarw. 6 linkage; the larger homologs appear to have 2, 3, and 4 more mannose units. One phosphorylated oligosaccharide with a mannose/phosphate ratio of 12.5 was reduced with NaB3H4 and subjected to mild acid hydrolysis. This released mannose and mannobiose that were glycosidically linked to the phosphate group; complete acid hydrolysis yielded D-mannose 6-phosphate. The recovered oligosaccharide phosphomonoester, which contained 11 or 12 mannose units, was digested exhaustively with .alpha.-mannosidase and the product of this reaction was treated with alkaline phosphatase, which yielded radioactive Man3GlcNAcH2. The mannosidase-resistant phosphorylated oligosaccharide has the structure Man .fwdarw. P .fwdarw. 6.alpha.Man .fwdarw. .alpha.Man .fwdarw. 6.beta.Man .fwdarw. 4GlcNAcH2, in which some of the phosphate groups are substituted with mannobiose instead of mannose. A 2nd phosphorylated oligosaccharide with a mannose/phosphate ratio of 6.5 probably contains 2 phosphodiester groups but its structure has not been fully investigated.