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The authors made a detailed comparison of the core oligosaccharide units from …

Biology Articles » Biochemistry » Carbohydrate Biochemistry » Carbohydrate chains on yeast carboxypeptidase Y are phosphorylated » Discussion

- Carbohydrate chains on yeast carboxypeptidase Y are phosphorylated

Yeast carboxypeptidase Y contains phosphate in diester form that is linked as mannosylphosphate and mannobiosylphosphate units to position 6 of mannose in some of the oligosaccharide units of the glycoprotein. These observations show a striking parallel between this yeast vacuolar hydrolytic enzyme and the lysosomal enzymes of higher organisms, some of which are also phosphorylated (9). Whether the phosphorylated oligosaccharide chains ofcarboxypeptidase Y specify the localization of this enzyme in the vacuole is unknown, but it is certain that the mere presence of phosphate in a mannoprotein does not determine location; some secreted yeast mannoproteins also carry mannosylphosphate and mannobiosylphosphate groups in the outer chain (22). The novelty in the present report is the discovery ofphosphate in the core oligosaccharide ofan intracellular yeast mannoprotein.

It has been reported (12), and we confirm, that partially purified carboxypeptidase Y is resolved on DEAE-Sephadex into two peaks of enzyme activity. It is possible that this separation is based on different phosphate contents. The molar ratios of mannose/phosphate were 12.5 in CPY-I and 10.8 in CPY-II. As carboxypeptidase contains =50 mannose units per molecule (5), this means that there may be four phosphates in CPY-I and five in CPY-LI. The additional phosphate group in CPY-II would be consistent with its later elution from DEAE-Sephadex. In our isolation, the ratio CPY-I/CPY-II was -0. 8, whereas Kuhn et al. (12) found a ratio of about 0.5. The finding of four or five phosphate groups per enzyme molecule and the fact that one-fourth ofthe four oligosaccharide chains are not phosphorylated suggests that some oligosaccharide chains must carry more than one phosphate group. The elution pattern of the oligosaccharides from the Bio-Gel P-30 column does reveal heterogeneity in the extent of phosphorylation, and the early fraction (CPY-IIa) appears to have about twice as much phosphate as the later one (CPY-IIc). By 31P NMR we have shown that all of the phosphate in carboxypeptidase Y is diesterified and, because strong acid hydrolysis yields mannose 6-phosphate quantitatively, it is clear that the phosphate is esterified to position 6 of mannose units in some of the oligosaccharide chains. In most or all of the phosphodiester units, the other attached group is mannose or mannobiose in an acid-labile glycosylphosphate linkage. The phosphorylated mannose unit ofthe carboxypeptidase Y oligosaccharide CPY-IIc is probably attached as a side chain to the first al-+6-linked mannose unit of the backbone by an al-*2 or al-*3 linkage (Fig. 7). Ifthe phosphorylated oligosaccharides are related structurally to those of the outer chain (22), the linkage is probably al-42. This structure has obvious similarity to that reported by Tabas and Kornfeld (23) for a mouse lymphoma P-glucuronidase in which N-acetylglucosamine phosphate is esterified to the core oligosaccharide.

All of the carbohydrate chains on carboxypeptidase Y are not phosphorylated, and one of the neutral oligosaccharides was shown to have a structure almost identical to that of an IgM chain with eight mannoses (13). The unphosphorylated carboxypeptidase Y oligosaccharide fraction (CPY-IId) is composed of at least four homologs that appear to have 9, 11, 12, and 13 mannoses, respectively. This neutral fraction makes up about one-fourth of the total oligosaccharide component and could all be localized at a particular site in the polypeptide chain, but we have not investigated this feature. Methylation of intact carboxypeptidase Y showed the presence of 2- and 3-0-substituted, as well as 2,6- and 3,6- doubly 0-substituted mannoses. Because the carbohydrate chains are heterogeneous, the observed ratios of these units do not fit any single structure well, but all of the linkages are expected in the yeast core oligosaccharides. The somewhat high proportion of 1-3-linked mannose is consistent with the presence of this linkage in the mannobiose unit previously found attached to phosphate in the mannoprotein outer chain (22).

That the observed heterogeneity of the carboxypeptidase-Y oligosaccharides results from phosphatase and mannosidase digestion during isolation of the enzyme seems unlikely for several reasons. Some of the neutral oligosaccharides are larger than the corresponding part ofthe acidic ones; none ofthe oligosaccharides contains monoesterified phosphate, which would presumably be an intermediate in any degradative pathway; and the smallest neutral oligosaccharide detected contained nine mannose units, whereas smaller oligosaccharides are readily formed by digestion with jack bean a-mannosidase. Yeast mannoproteins show a high species polymorphism (24), and the carbohydrate chains of the external mannoprotein, invertase, are known to be heterogeneous in size (25) and in the structure of the core oligosaccharides (4). As we show in the present study, carboxypeptidase Y, an intracellular mannoprotein that possesses only core oligosaccharides, has a very heterogeneous assortment ofcarbohydrate chains that differ in both size and state of phosphorylation. An important aim in extending this study will be to elucidate the origin and function of this diversity.

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