Isolation of Carboxypeptidase Y and Digestion with Endoglucosaminidase H. From 100 lb ofyeast cake, =500 mg ofpure carboxypeptidase Y was obtained. As noted by Kuhn et al. (12), two peaks of enzyme activity were found at the DEAE-Sephadex A-50 isolation step, here designated CPY-I and CPY-II. These two peaks were separated, and each gave a single peak when it was rechromatographed. The carbohydrate content of CPY-I was 14.4% and that of CPY-II was 14.7%. Hasilik and Tanner (5) have reported 14.1% carbohydrate for the enzyme. Both carboxypeptidase preparations contained organic phosphate, the mannose/phosphate molar ratios being 12.5 for CPYI and 10. 8 for CPY-II. Both fractions ofcarboxypeptidase Y were inactivated with toluenesulfonyl fluoride (14) to prevent autodigestion and digestion of the endoglucosaminidase H during treatment to release the carbohydrate chains. The yield of inactivated CPY-I was 260 mg and that of inactivated CPY-II was 230 mg. Both preparations gave single major bands that were indistinguishable on acrylamide/NaDodSO4 gel electrophoresis (Fig. 1).
Methylation of intact CPY-I, followed by hydrolysis, reduction, and acetylation of the methylated derivative, yielded the alditol acetates of 2,3,4,6-tetra-O-methylmannose (5 mol), 2,4,6-tri-0-methylmannose (3 mol), 3,4,6-tri-0-methylmannose (2 mol), 2,4-di-0-methylmannose (1 mol), and 3,4,-di-0- methylmannose (2 mol). The two di-O-methyl derivatives are those expected for the branching at positions 2 and 3 ofthe 1-*6- linked backbone mannose that is characteristic of yeast glycoprotein core oligosaccharides (13). Also typical are the two tri- 0-methyl ethers that reflect the presence of mannose in 1-*2 and 1--3 linkage. Samples of inactivated CPY-I and CPY-II (150 mg each) were digested with 0.75 unit ofendoglucosaminidase H for 96 hr (19). As shown in Fig. 1, both carboxypeptidase preparations were converted quantitatively to single faster-migrating protein bands that were again indistinguishable. The enzyme digests were then fractionated on a Bio-Gel P-30 (100-200 mesh) column (2 x 90 cm) by elution with water (Fig. 2). The separated protein peak (fractions 23-31) contained very little mannose; 95% of the mannose was distributed in a neutral peak (fractions 78-93) and a broader peak (fractions 32-71) associated with most of the phosphate. The latter was divided into three fractions: fraction a with a mannose/phosphate ratio of6.5, fraction b with a ratio of 8.0, and fraction c with a ratio of 12.5. These results suggest that fraction a has two phosphate groups per oligosaccharide chain, that fraction c has one, and that fraction b is an overlapping mixture of fractions a and c.
Nature of the Neutral Oligosaccharide. The material in fractions 78-93 (fraction CPY-IId; see Fig. 2) was combined, and its neutral nature was confirmed by the absence of phosphate and the failure ofthe carbohydrate to bind when passed through a Dowex 1 (acetate) column. The sample, chromatographed on a Bio-Gel P4 (>400 mesh) column (2 X 190 cm) by elution with water, had at least four components that appeared to differ from each other by increments of about one mannose unit (Fig. 3A). The following characterization was limited to the smallest oligosaccharide of the mixture.
Fractions 114-121 (see Fig. 3A) were combined, and the material was reduced with NaB3H4 (see Materials and Methods). This reduced sample was then chromatographed on the calibrated Bio-Gel P-4 column, from which it eluted in the position ofMan9GIcNAcH2 (Fig. 3B). From this, we conclude that the larger oligosaccharides in Fig. 3A range in size from Man11GlcNAc to Man13GlcNAc.
The MangGlcNAcH2 sample gave an anomeric proton NMR spectrum (Fig. 4) that was almost identical to that of an oligosaccharide Man8GlcNAcH2 obtained from IgM and designated rGP-563-I (13). This latter oligosaccharide has the structure shown in bold type in Fig. 4. The carboxypeptidase oligosaccharide has one more mannose unit, however, and, from the shoulder (g) on peak b, we conclude that about halfofthe chains have an unsubstituted al-46-linked mannose, whereas the excess area under peak a,e,e' would be consistent with about half of the chains having an additional terminal al-*3-linked mannose. The postulated modifications are shown in italics on the structure in Fig. 4.
Nature of the Phosphorylated Component of Carboxypeptidase Y. CPY-I and CPY-II were hydrolyzed in 1 M trifluoroacetic acid at 120°C for 2 hr. The acid was evaporated under a stream of N2, and the residue was assayed enzymically for Dmannose 6-phosphate (15, 16). The results (Table 1) show that the phosphate was almost quantitatively accounted for as mannose 6-phosphate.
Phosphorylated oligosaccharide CPY-IIc [fractions 56-71 (see Fig. 2)] was passed through a Bio-Gel P-4 (>400 mesh) column (2 X 190 cm) and eluted with 0.1 M NH4OAc in 3-ml fractions. The majority of the carbohydrate appeared in a symmetrical peak between fractions 97 and 113. The combined material was analyzed by proton-decoupled 31P NMR, and it gave a single signal at -1.7 ppm (pH 7.0 in 0.1 M Na EDTA), a chemical shift characteristic of phosphate diesters (20). S. cerevisiae mannoproteins often contain diesterified phosphate in which one component of the diester is mannose or mannobiose in glycosyl linkage that is subject to release by mild acid hydrolysis (21). Phosphorylated CPY-IIc oligosaccharide was heated at 100TC for 30 min in 0.01 M HCI, and the products were separated on a Bio-Gel P-2 (>400 mesh) column (0.2 x 114 cm) by elution with 0.1 M NH4OAc (Fig. 5). Peaks corresponding to mannose and mannobiose were obtained, and their identities were confirmed by paper chromatography (not shown). The weight ratio oligosaccharide residue/mannose plus mannobiose was 21, whereas a ratio of about 12 would be expected if all of the phosphate groups were esterified with mannose. Incomplete hydrolysis of the diester bond is the most likely explanation for this result because subsequent phosphatase digestion gave only a 70% yield of neutral oligosaccharide. In confirmation ofthis conclusion, a second mild acid hydrolysis of the recovered oligosaccharide gave sufficient additional mannose to bring the weight ratio oligosaccharide/mannose plus mannobiose to 10.6.
A sample of the acidic CPY-IIc oligosaccharide was reduced with NaB3H4, and the product was subjected to mild acid hydrolysis and treatment with alkaline phosphatase to dephosphorylate the monoester oligosaccharide component. This gave a neutral radioactive oligosaccharide that was separated into two peaks on the calibrated Bio-Gel P-4 column. These two appear to differ by one mannose unit, and their sizes correspond to Man11GlcNAcH2 and Man12GlcNAcH2 (not shown).
Location of the Mannose 6-Phosphate in the Acid-Stable Oligosaccharide. Strong acid hydrolysis of intact carboxypeptidase Y gives mannose 6-phosphate (see above), so this structure must be present in the acidic oligosaccharide recovered after mild acid hydrolysis. The location of the mannose phosphate was determined by subjecting the NaB3H4-reduced oligosaccharide to exhaustive digestion with a-mannosidase. The recovered acidic radioactive product was dephosphorylated with alkaline phosphatase and then fractionated on the calibrated Bio-Gel P-4 column (Fig. 6). It eluted in the position of Man3GlcNAcH2. Assuming the radioactive product was derived from an oligosaccharide such as that in Fig. 4, this suggested three possible structures: aMan-*6aMan--36,Man-> 4GlcNAcH2, aMan--2(or 3)aMan--6,8Man--)4GlcNAcH2, and aMan--2aMan--3f3Man--4GlcNAcH2. To discriminate between these, we used partial acetolysis (17); only the oligosaccharides with 1--6 linkages would be degraded readily in this reaction. As shown in Fig. 6, the acetolysis ofMan3GlcNAcH2 labeled only in the glucosaminitol residue yields a radioactive product having the elution position of ManGlcNAcH2. This is consistent with either of the first two structures above. Because the original phosphorylated oligosaccharide (CPY-TIc) contains 11 or 12 mannoses, however, the backbone probably has four 1-+6-linked mannose units, which would preclude attachment of phosphate to position 6 of the third mannose in the mannosidase- resistant tetrasaccharide. From this consideration, we favor the structure aMan--2 (or 3) aMan- 6,3Man-*4GlcNAcH2 for this oligosaccharide.