Characterization of Large Oligosaccharide-Lipids Synthesized in Vitro by Microsomes from Saccharomyces cerevisiae”

Conditions are described for optimizing the synthesis of large oligosaccharide-lipids in microsomal preparations from Saccharomyces cerevisiae. On incubating microsomes with GDP-[”C]Man, the major product obtained was Man9GlcNAc~-P-P-dolichol, but when both GDP-[“CIMan and UDP-[’HIGlc were present in the incubation mixture about half of the MansGlcNAce was elongated to Glc~Man~GlcNAc~-P-P-dolichol. Unlike particulate fractions from mammalian systems, little glucosylation of the yeast microsomal oligosaccharide- lipid was obtained when the concentration of UDP-Glc was less than 10 p ~ , but the synthesis of this product could be maximized by raising the concentration of UDP-Glc to 50 p ~ . Analysis of the yeast MansGlcNAc2 species confirmed that 8 of the 9 mannose residues could be released with a-mannosidase, while the remaining mannosyl residue was in the core trisacchar- ide, Man/3l + 4GlcNAc/31 + 4GlcNAc. Treatment of Glc3Man9GlcNAcz with a-mannosidase released 5 of 9 mannose residues and yielded GlcsMmGlcNAcz. This product appeared to be identical with that obtained in parallel experiments with double labeled oligosaccha-ride-lipid synthesized in oviduct microsomes.

tor protein (I). The oligosaccharide portion of this lipid intermediate in animal systems is now known to be a heterosaccharide containing the common core sequence Manbl + 4GlcNAcPl + 4GlcNAc (Max$GlcNAcz) extended with 8 mannose and as many as 3 peripheral glucose residues (2)(3)(4).
When GDP-[14C]Man is employed as the substrate for in vitro synthesis, more than 80% of the label incorporated into yeast microsomal proteins can be released with 0.1 N NaOH indicating that it is associated with 0-glycosylated serine/ threonine residues (6). Man-P-Dol has been identified as the glycolipid donor for the attachment of the initial mannose residue in these linkages, whereas chain extension is promoted by GDP-Man (6). In addition to the formation of O-mannosylglycoproteins, yeast also synthesize glycoproteins containing asparagine-oligosaccharides of the "high mannose" type. These glycoproteins contain the common core trisaccharide, ManpGlcNAcz, which is elongated by a-linked mannose residues ranging in number from about 12, in the case of carboxypeptidase Y (12,13), to over 50, in the case of invertase (14,15).
By analogy with the mammalian systems described recently (2)(3)(4), the synthesis of N-glycosidic oligosaccharides in yeast would be expected to proceed via large oligosaccharide-lipid intermediates. Small amounts of glycolipid exhibiting characteristics of OS-P-P-Dol from higher cells have been isolated from yeast microsomal preparations (16)(17)(18)(19), and in one case this material served as an oligosaccharide donor for the in vitro glycosylation of endogenous microsomal proteins (18). It has been shown recently that log phase yeast incorporate a small amount of labeled glucose into acid-soluble oligosaccharides, which by paper chromatography appear similar to those obtained from the OS-P-P-Dol of mammalian cells (20). The largest of these species, tentatively identified as Glc3M~GlcNAcz, appears to be. associated with yeast gbcoproteins after a brief pulse with labeled glucose (20).
Despite these advances, a thorough characterization of the oligosaccharide moiety of yeast OS-P-P-Dol, and of the Nglycosylation reaction itself, have been hampered by the extremely low levels of OS-P-P-Dol obtained from yeast. In this communication we describe conditions which yield sufficient quantities of yeast OS-P-P-Dol from in vitro microsomal Man or M, mannose; Glc or G, glucose; GlcNAc or N, N-acetylglucosamine; Endo-H, Streptomyces plicatus endo-P-N-acetylglucosaminidase H; CHO, Chinese hamster ovary.
2Portions of this paper (including "Experimental Procedures," Table I

RESULTS
Size of the Oligosaccharides Present in the OS-P-P-Dol Fraction-To establish the size of the oligosaccharides in yeast OS-P-P-Dol synthesized in uitro, 2,500 counts/min of each of the four samples in Table I were subjected to mild acid hydrolysis which is known to release oligosaccharides of the type Glc.Man,GlcNAcz (33,34). The water-soluble products were chromatographed on a Bio-Gel P-4 column which Yeast Oligosaccharide-Lipids had been calibrated with heterosaccharides of known composition ( Fig. 1, inset). As indicated in Fig. 1, the radioactivity liberated from yeast OS-P-P-Dol chromatographed as a mixture of oligosaccharides, the relative proportions of which were a function of the growth phase of the yeast and the presence of UDP-Glc. Some species were as large as Glc3MangGlcNAc~ obtained from oviduct OS-P-P-Dol, while others were as small as MansGlcNAcz isolated from ovalbumin. In the absence of UDP-Glc, the OS-P-P-Dol obtained from both microsomal preparations contained a major oligosaccharide which eluted with a sue consistent with MangGlcNAcz ( Fig. 1, A and B; tube 55). The mid-log phase OS-P-P-Dol always contained more of the smaller species migrating in the Man5.7GlcNAcz region of the profile and, even in the absence of UDP-Glc, often revealed shoulders of radioactivity in tubes 50 and 53 where Glc3Man9GlcNAcz and GlclMangGlcNAcz eluted, respectively. Preincubation of the microsomes at 27°C for 15 min prior to addition of the nucleotide sugars substantially reduced the species larger than MangGlcNAcz without otherwise altering the elution profile (data not shown).
On addition of 100 p~ UDP-Glc to the yeast particulate system, marked changes in the Bio-Gel P-4 oligosaccharide elution profiles were noted (Fig. 1). Thus, with mid-log microsomes (Fig. lA), nearly 45% of the oligosaccharides chromatographed the same as oviduct Glc3MangGlcNAc~. In contrast, when UDP-Glc was incubated with late-log preparations, less than 20% of the oligosaccharides migrated with the fully glucosylated oviduct marker (Fig. 1B), and in some cases, high levels of UDP-Glc failed to stimulate any synthesis of Glc~Man~GlcNAcz-P-P-Dol (data not shown). Sugar Composition of the OS-P-P-Dol Fraction-The sue of the oligosaccharide chains released from yeast OS-P-P-Dol ( Fig. 1) suggests that GDP-Man promotes the synthesis of MangGlcNAcz-P-P-Dol primarily, and that GDP-Man plus UDP-Glc yield oligosaccharide chains with as many as 3 glucose residues. In order to ascertain the actual sugar composition of the yeast oligosaccharides eluting with sues of Glc3Man9GlcNAcz and MangGlcNAcz, OS-P-P-Dol was synthesized by incubating mid-log microsomes for 5 min at 27°C in a scaled up reaction containing 4 ~L M GDP-[14C]Man and 25 ~L M UDP-[3H]Glc. A portion of the isolated OS-P-P-Dol (80,000 3H and 70,000 I4C cpm) was subjected to mild acid hydrolysis, and the water-soluble oligosaccharides were applied to the Bio-Gel P-4 column. As Fig. 2A depicts, nearly 80% of the 3H migrated in Peak I (centered at tube 50), the same region where the oviduct Glc3MangGlcNAcz marker eluted. The remaining 20% of the 3H was in a peak at tube 53 consistent with a GlclMangGlcNAcp species. Nearly 50% of the 14C was present in Peak I1 (centered at tube 55) where MangGlcNAcz would be expected to migrate. It should be noted that with 25 p~ UDP-Glc ( Fig. 2A), only 25% of the 14C eluted with Glc3MansGlcNAcz, which was about half that obtained in the previous experiment with 100 p~ UDP-Glc (Fig. 1A). The relationship between UDP-Glc level and the proportion of the oligosaccharide chains glucosylated is examined more thoroughly in a subsequent section.

-
Fractions 49 to 51 ( I ) and 54 to 56 ( I I ) in Fig. 2A were pooled separately and rechromatographed on Bio-Gel P-4 to assess the homogeneity of each. Pool I yielded a single symmetrical peak with a constant 14C/3H ratio throughout the profile (Fig. 2B). Hydrolysis of a portion of this material ( Fig.  2 B ) to its component sugars with acid, followed by paper chromatography in Solvent D, verified that the 14C and 3H were in mannose and glucose, respectively. Correcting for the counting efficiency and specific activity of the labels present in this oligosaccharide provided a molar Man/Glc ratio of 2.93. From the estimated size of this species, HexoseltGlcNAcz (Fig. l), and the molar ratio of labeled sugars present, ita composition was calculated to be G l c 3 .~~M a~,~G l c N A c~.
Rechromatography of Pool I1 provided the profile shown in Fig. 2C. A small amount of 3H migrated in the front of the otherwise symmetrical peak, the center of which eluted as anticipated for HexosegGlcNAcz. Fractions 55 to 57 were pooled and a portion of the total was subjected to acid hydrolysis followed by paper chromatography in Solvent D.
All of the 14C migrated with mannose indicating that the composition of this species was MangGlcNAcz.
To characterize the Oligosaccharides obtained from the double labeled OS-P-P-Dol fraction further, rechromatographed Pools I and I1 (Fig. 2, B and C, respectively) were digested with a-mannosidase, and the products were resolved on the Bio-Gel P-4 column. The elution pattern of the GlcaMangGlcNAcz species shown in Fig. 2 0 now revealed two peaks. one (tubes 95 to 97) with 57% of the "C which migrated with mannose on paper chromatography (Solvent D) and the other (tubes 55 to 58) with a size equivalent to Hexose7.8GlcNAc2. The latter species contained 43% of the l4C and all of the 3H, and, although its profile was somewhat broader than observed for the other oligosaccharides chromatographed on this column, the 14C/3H ratio was constant in all fractions. From the corrected counting efficiencies of the radionuclides present, a molar Man/Glc ratio of 1.37 was obtained for the pooled oligosaccharide. Utilizing Hexose7GlcNAcz as the size of this species, its composition based on the molar ratio of sugars present was Glc2.95Man4.06GlcNAc2. Thus a-mannosidase appeared to remove 5 mannose residues from the large glucosylated heterosaccharide (Peak I). Chromatography of the a-mannosidase-digested Mans-GlcNAcz species also provided two peaks (Fig. 2E). In this case 91% of the I4C eluted in the monosaccharide region (tubes 95 to 98), and, on paper chromatography in Solvent D, this was identified as mannose. The other peak (tubes 73 to 75) contained 9% of the 14C and eluted in the position observed for the core trisaccharide, ManflGlcNAc2. The pooled material (tubes 73 to 75), when reduced with sodium borotritide (27), co-migrated with authentic Manfll + 4GlcNAcfl1 +. 4Gl~NAc[~H]-ol on paper chromatography in Solvents C and D. Digestion of the reduced trisaccharide with fl-mannosidase yielded a single tritiated product, identified chromatographically (Solvent D) as GlcNAcPl + 4Gl~NAc[~H]-ol, which on complete acid hydrolysis provided only gl~cosamin[~H]-itol. Thus, the MangGlcNAcz oligosaccharide isolated from the yeast glycolipid fraction contains the expected common core trisaccharide structure (25).
Effect of UDP-Glc Concentration on the Distribution of Oligosaccharide Species Present in Yeast OS-P-P-Dol-Microsomal preparations from animal cells incorporated glucose into OS-P-P-Dol when UDP-Glc was as low as 0.2 p M (41). Since the yeast system did not incorporate appreciable glucose into this glycolipid unless UDP-Glc was above 10 p , it was of interest to determine the concentration at which UDP-Glc provided an optimal level of glucosylation. Preincubated midlog and late-log microsomes were supplemented with 4 p~ GDP-Man and varying amounts of UDP-Glc up to 100 PM. After 15 min the OS-P-P-Dol was isolated, and the oligosaccharides released by mild acid hydrolysis were applied to the Bio-Gel P-4 column. For each UDP-Glc concentration tested, the relative amount of each oligosaccharide species present in the elution profile was estimated from the amount of radioactivity in the respective peaks and shoulders. The apparent number of mannose residues in each oligosaccharide was used to normalize the various fractions. The results, shown in Fig.  3, provide the percentage of each oligosaccharide species present in the OS-P-P-Dol formed at each UDP-Glc level, and reveal that both mid-log and late-log microsomes required at least 50 ~L M UDP-Glc to yield the maximal amount of Glc3MangGlcNAc2-P-P-Dol. As indicated, there was a direct relationship between the UDP-Glc level and that of the fully glucosylated oligosaccharide in both systems. Thus, as the glucosylated species increased, the MangGlcNAc2 species decreased indicative of a precursor-product relationship. With the exception of a decline in the putative monoglucosylated glycolipid in mid-log microsomes, the proportion of all other species was relatively unaffected by increasing the UDP-Glc concentration (Fig. 3).

DISCUSSION
Incorporation of radioactive sugars from GDP-Man and/or UDP-Glc into large oligosaccharide-lipids by yeast microsomes has been shown to rarely exceed 1% of the sugar nucleotides added (17-19). A major objective of the current work, therefore, was to improve the extent of transfer from these nucleotides to the glycolipid fraction so that the latter's attendant oligosaccharides could be more thoroughly characterized. By modifying the system of Lehle and Tanner (18) to include dithiothreitol and UDP-GlcNAc, a 5-fold increase in the yield of OS-P-P-Dol was obtained. Eliminating MnCh from the reactions and optimizing the concentration of each assay component resulted in a further 3-to 4-fold increase. Thus, the present study represents about a 20-fold improvement in the in vitro synthesis of yeast lipid-linked oligosaccharides. Relevant to previous studies (7, 17-19, 39), the following characteristics of the yeast particulate system were observed: (a) freshly isolated microsomes, or those frozen and stored in liquid Nz, were required for maximal OS-P-P-Dol synthesis; (6) microsomes stored at 0°C or -2OOC lost their synthetic capacity with a half-life of 24 h and neither sucrose nor glycerol were effective in stabilizing this activity; (c) Triton X-100, even at levels below O.l%, diminished OS-P-P-Dol synthesis by over 5-fold and doubled the Man-P-Dol levels relative to the values reported in Table I; (d) exogenously added dolichol phosphate was without effect on the system unless added in combination with Triton X-100, in which case very large amounts of Man-P-Dol were synthesized with little appearance of OS-P-P-Dol. A major finding in these studies was that the size and sugar composition of the oligosaccharides synthesized by yeast microsomes was not only dependent upon the growth state of the yeast but also on the level of UDP-Glc added to the in vitro assays (Figs. 1 and 3). The two predominant oligosaccharide-lipids synthesized by yeast microsomes from UDP-[3H]Glc and GDP-["CIMan were shown by analysis to be GlcWmgGlcNAcz and MangGlcNAcz (Fig. 2). Lesser amounts of oligosaccharides with sizes consistent with GlclMangGlcNAcz, Man.~GlcNAcz, and MansGlcNAcz were also found in the OS-P-P-Dol fraction ( Figs. 1 and 2). The yeast microsomal system appeared to form OS-P-P-Dol in which all of the mannose residues were labeled uniformly since a-mannosidase released 91% of the label from ['4C]MangGlcNAcz (Fig. 2E), compared with a theoretical value of 89% expected for 8 of 9 residues. The remaining 9% of the ['4C]mannose was present in a trisaccharide shown to be the common core sequence, ManpGlcNAcz, which also has been identified on the reducing end of lipid-linked oligosaccharides from oviduct (43), CHO cells (3), and Nil 8 fibroblasts (4). Treatment of the yeast Glc3MangGlcNAcz double labeled oligosaccharide with a-mannosidase released 57% of the ["C]mannose compared with a value of 55.6% expected for 5 of 9 residues. Removing 5 mannose residues from the parent oligosaccharide should produce a Hexose7GlcNAc~ species with a composition of Glc3MamGlcNAcz. Clearly, the r3H]-Glc/[14C]Man ratio present in the oligosaccharides before (Fig. 2B) and after (Fig. 2 0 ) a-mannosidase digestion, coupled with the percentage of mannose removed, confirms this assumption.
An apparent discrepancy involves the elution properties of Glc3Man.,GlcNAcz from yeast and oviduct, both of which migrated on Bio-Gel P-4 as if they were in actuality 1 hexose residue larger (tube 57). From their composition, these oligosaccharides were expected to elute with a peak at tube 60 on the calibrated column. This anomaly may be due to the configuration of Glc3Man4GlcNAcz, which should be a Linear molecule based on the proposed structure of the parent Glc3Man&1cNAcz compound (3,4). Since the calibration oligosaccharides were all derived from a structure containing two di-substituted mannose branch points (3, 4, 21, 26, M), the extended Hexose7GlcNAcz may have a greater hydrodynamic volume, and hence elute earlier, than a more compact branched oligosaccharide of the Same composition. Verification of this concept should occur when the structure of the Glc3Ma~GlcNAcz species is confirmed.
The size, Glc/Man molar ratio, and a-mannosidase sensitivity of the largest oligosaccharide in yeast OS-P-P-Dol suggests that its structure is similar to, if not identical with, that of the comparable species from mammalian and avian cells. It is not clear why maximal synthesis of the heterosaccharide in yeast microsomes requires a level of UDP-Glc which is 1 to 2 orders of magnitude above that needed in particulate fractions from higher cells (4,41,(45)(46)(47). Although glycogen and glucan synthetases are very active in yeast microsomes, depletion of UDP-Glc cannot be the answer since more than half of that added remains at the end of the incubations. It is equally unclear at present why the late-log microsomes incorporate glucose poorly into OS-P-P-Dol, while synthesizing high levels of MangGlcNAcz-P-P-Dol (Fig. 1). One explanation is that since late-log phase cells, which have a doubling time about 3 times that of mid-log phase cells, require less Glc3MangGlcNAcz-P-P-Dol for protein glycosylation, their levels of UDP-G1c:OS-P-P-Dol glucosyltransferase enzyme(s) are correspondingly lower. Potentially related to these studies in yeast, Schmitt and Elbein (48) recently reported that by blocking protein synthesis in canine kidney cells with puromycin, a rapid inhibition of OS-P-P-Dol synthesis occurred. In this case, the decreased availability of protein acceptor sites appeared, by a feedback mechanism, to prevent accumulation of OS-P-P-Dol. Determination of the levels of yeast glucosylating enzymes at different growth phases should help clarify the site of impaired glucosylation in the late-log phase microsomes.
Endo-H treatment of the microsomal proteins after a 5-min incubation with UDP-r3H]Glc and GDP-[l4C)Man released a single major oligosaccharide (Fig. 5), which had the same [3H]Glc/['4C]Man ratio as the largest oligosaccharide in the OS-P-P-Dol from that reaction (Fig. 2B). The presence of the smaller oligosaccharides in the OS-P-P-Dol fraction at both 5 and 20 min (Figs. 1 and 2), but not on the proteins until 20 min, suggests that the glucosylated oligosaccharide is transferred preferentially. The absence of GlcMangGlcNAcz oligosaccharide in the glycolipids after a 20-min incubation without UDP-Glc (Fig. 1) and its apparent release by Endo-H from the microsomal proteins (Fig. 4) is not necessarily contradictory. In the presence of GDP-Man alone, log-phase microsomes always synthesized some Glc3MangGlcNAcn-P-P-Dol suggesting the presence of an endogenous pool of UDP-Glc. Support for this belief was obtained on preincubating the microsomes in the absence of UDP-Glc, which resulted in a greatly reduced synthesis of Glc3MangGlcNAcZ-P-P-Dol. Consistent with this explanation was the finding that the addition of UDP-Glc promoted a substantial increase in the largest oligosaccharide in both the glycolipid (Fig. 1) and protein (Fig. 4)

fractions.
A recent study by Parodi (20), performed with yeast in vivo, led to the same conclusions as those reported here with the in vitro microsomal system. Acid extraction of yeast yielded oligosaccharides which co-migrated with those isolated from the oligosaccharide-lipid synthesized by mammalian microsomes in vitro. The largest of these, most probably GlcaMangGlcNAcz, was also found in yeast proteins after a brief pulse with ['4C]glucose. On chasing with cold glucose, the labeled oligosaccharides fist became shorter as a result of glucose removal and then larger due to mannose addition. Yeast OS-P-P-Dol in whole cells (20) and in the particulate fraction ( Figs. 1 and 2) provide strikingly similar oligosaccharide profiles in that Glc3MangGlcNAcz and MangGlcNAcz are the predominant species. This pattern is somewhat different than that in most higher eukaryotic cells examined to date, where the glucosylated species is present almost exclusively (49-54). Although small amounts of MansGlcNAcz and MansGlcNAcn have been identified as intermediates in the biosynthesis of the large glucosylated OS-P-P-Dol in CHO cells (55) and chick fibroblasts (X), respectively, MangGlcNAcz appears to be the major precursor in yeast (Fig.   3). While smaller oligosaccharides are also found in the yeast OS-P-P-Dol fractions (Figs. 1, 2, and 3), their relationship to the glycoprotein biosynthetic pathway remains to be elucidated.
The requirement for glucose in the initial glycosylation of proteins (41,42) and its subsequent processing as well as that of mannose from the newly transferred oligosaccharides in animal, viral, and avian systems is now well documented (49, 51, 56-62). Since yeast do not produce the "complex" oligosaccharides found in animal cells there is no apparent reason for yeast to trim Glc3Man9GlcNAcz to MansGlcNAca as occurs during complex carbohydrate synthesis in higher cells (49,51,  58,62). Nevertheless, after a 20-min incubation in vitro, yeast microsomal proteins contained a species consistent with Man7GlcNAc which may have arisen by the processing of a larger oligosaccharide (Fig. 4). It is not known whether the yeast oligosaccharide chains are reduced in size to species smaller than MangGlcNAc2 after glucose removal (20), nor is it known how much of the original species transferred to protein is conserved in the "inner core" structure (63) of yeast mannoproteins, but processing reactions may prepare the oligosaccharide for its subsequent elongation with 50 or more mannose residues (15). Studies currently in progress should identify the intermediates formed during OS-P-P-Dol biosynthesis as well as the processing reactions that may be required for the completion of mature yeast glycoproteins. In recent experiments (a), Glc3MangGlcNAc2-P-P-Dol has been found to be a 20-fold more active substrate than MangGlcNAcn-P-P-Dol for oligosaccharide transfer to endogenous protein acceptors in a solubilized yeast microsomal system. Our results, and also those of Parodi (20), thus support the thesis that the glucosylated lipid-linked oligosaccharide is the natural donor for the initial step in protein N-glycosylation in yeast, as it is in animal and avian systems (41, 42, 47, 51, 5 4 , 58, 60).