THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U. S. A. Vol. 258. No. 4, Issue of February 25, pp. 2562-2567, 1983

Glycoprotein Biosynthesis in Yeast PROTEIN CONFORMATION AFFECTS PROCESSING OF HIGH MANNOSE OLIGOSACCHARIDES ON CARBOXYPEPTIDASE Y AND INVERTASE*

(Received for publication, June 4, 1982)

Robert B. Trimble, Frank Maley, and Frederick K. Chu From the Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201

Carboxypeptidase Y and invertase from baker’s yeast, Saccharomyces cerevisiae, have two classes of N- linked high mannose oligosaccharides which may be distinguished on the basis of their susceptibility to hydrolysis by endo-8-N-acetylglucosaminidase H (Endo H). Thus, three of the four oligosaccharides on carbox- ypeptidase Y and seven of nine of those on invertase are readily released by Endo H when these proteins are in their native state (accessible), while the fourth chain on carboxypeptidase Y and the remaining two on in- vertase are hydrolyzed by Endo H only when these proteins are denatured (inaccessible). Analysis of the three accessible oligosaccharides from carboxypepti- dase Y revealed these to be mostly Manll_lnGlcNAc in size and to account for 80% of the mannose and essen- tially all of the phosphate associated with this enzyme. By contrast, the fourth chain from carboxypeptidase Y ranged in size from Man8-lzGlcNAc and contained no phosphate. Comparison of peptide maps with the pri- mary sequence of carboxypeptidase Y (Svendsen, I., Martin, B. M., Viswanatha, T., and Johansen, J. T. (1982) Carlsberg Res. Cornnun 47, 15-27) allowed as- signment of the resistant fourth oligosaccharide to the N-glycosylation sequon located at Asn8.l.

A similar analysis of the accessible oligosaccharide pool from invertase showed that all of the phosphate and over 85% of the mannose was in species larger than ManzoGlcNAc, but the oligosaccharides released after denaturation ranged in size from Man8-12GlcNAc and were devoid of phosphate. The smaller size and lack of peripheral modification found on the oligosaccharides that are initially resistant to Endo H is most easily explained by the hypothesis that as carboxypeptidase Y and invertase fold into their mature configuration, certain glycosylated domains become inaccessible to the mannosyl transferases which catalyze chain exten- sion and phosphomannose addition.

The acquisition of carbohydrate by all eucaryotic orga- nisms, including yeast, starts with transfer of the oligosaccha- ride from the common biosynthetic intermediate, Glcs- MansGlcNAcz-P-P-Doll, and culminates in mature N-linked glycans via a multistep processing pathway with the most

* This work was supported in part by United States Public Health Service Grant GM23900 from the National Institute of General Med- ical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: Dol, dolichol; Endo H, endo-P-N- acetylglucosaminidase H from Streptomyces plicatus; SDS, sodium dodecyl sulfate; ConA, concanavalin A.

extensive modifications occurring in the glycoproteins of mammalian cells. In this case both high mannose and complex oligosaccharides are derived from the glucosylated oligosac- charide precursor following its transfer to nascent proteins (1- 4) . The processing of asparaginyl oligosaccharides in yeast is somewhat different from that in higher eucaryotes, but certain parallels exist. N-Linked glycans in yeast are of the high mannose type exclusively, although peripheral mannose phos- phate residues, similar to those found on mammalian lyso- somal acid hydrolases (5), are found on carboxypeptidase Y ( 6 ) , a yeast vacuolar enzyme. Oligomannosyl chains of yeast proteins are longer than those in animal cells as illustrated by invertase and carboxypeptidase Y with the former possessing 9 chains per 60,000-dalton peptide ranging in size from 8 to over 50 mannose residues (7-lo), and the latter with 4 oligo- mannosyl chains per 50,000-dalton peptide, each averaging 15 sugar residues (1 1).

Since mammalian cells contain the full complement of enzymes required for the processing of Glc3Man9GlcNAcz to complex oligosaccharides (2, 3), an important question arises as to why high mannose structures are retained at all. One plausible explanation is that during processing, folding of the nascent polypeptide renders the oligosaccharide chains in certain domains inaccessible to the smooth endoplasmic retic- ulum and Golgi mannosidases required for the removal of specific outer a1,2-linked residues (2, 3), and, thus, complex glycan synthesis is prevented. In support of this proposal, studies with closely related strains of both influenza A (12) and murine leukemia viruses (13) grown in a number of host cells have led to the conclusion that the conformation of certain viral glycoproteins has an effect on their ultimate glycosylation patterns. The observations that at least some of the a-mannosidase and the glycosyl transferase enzymes act after protein folding is complete (14) and that the complex oligosaccharide chains appear to be more exposed at the surface of the influenza hemagglutinin membrane glycopro- tein trimer than the high mannose chains (15) reinforce this concept.

There is evidence in yeast as well that the accessibility of oligosaccharides for processing may be influenced by protein structure. Thus, while three of four high mannose oligosac- charides of carboxypeptidase Y and seven of nine of those on invertase are readily removed by Endo H under nondenatur- ing conditions, the remaining chains are released by Endo H only after the proteins are unfolded in SDS (8, 11, 16). In the present study we have compared the oligosaccharides released by Endo H under native conditions from these two glycopro- teins with those removed after the proteins are denatured. The results form the basis of a hypothesis that the Endo H- resistant high mannose oligosaccharides on native carboxy- peptidase Y and invertase represent a class of carbohydrate chains which became inaccessible early enough in glycopro-

2562

by guest on February 1, 2020http://w

ww

.jbc.org/D

ownloaded from

Oligosaccharide Accessibility and Processing 2563

tein maturation to escape the mannose elongation and phos- phate addition reactions which generate the large polyman- nosy1 chains found in yeast (17).

MATERIALS AND METHODS

Essentially all of the procedures used in the work have been described in detail by us previously. Briefly, Endo H was purified from cultural filtrates of Streptomyces plicatus (18) and a-mannosi- dase was isolated from jack bean meal (19). Invertase was purified (8) from a commercial preparation obtained from Boehringer Mannheim and carboxypeptidase Y, purified from yeast by the method of Kuhn et al. (20), was kindly provided by Dr. T. H. Plummer, Jr. of this Center.

Release of the “accessible” carbohydrate from invertase (25 mg of protein) and carboxypeptidase Y (15 mg of protein) was carried out under native conditions by incubating the substrate glycoproteins in 5 ml of 50 m~ sodium citrate buffer, pH 5.5, with about 0.5 IU of Endo H for 6 h at 37 “C. These conditions are sufficient to remove three of the four chains from carboxypeptidase Y and seven of nine of those from invertase. However, longer digestions with high levels of Endo H, such as the conditions used in Fig. 1, serve to confum the relative resistance of certain oligosaccharides to hydrolysis. Reaction mixtures were chromatographed on a Sepharose 6B column (1.5 X 150 cm) equilibrated with 10 m~ sodium citrate buffer, pH 5.5. The protein fractions were located by absorbance at 280 nm. Carbohydrate was located in the profiles and quantitated by the phenolsulfuric acid procedure (21), scaled down to a total volume of 1.2 ml, with mannose as the standard. The protein regions from column profiles were pooled, concentrated by ultrafiltration, and denatured by heating at 100 “C for 5 min with a 1.4-fold weight excess of SDS. After chilling in ice for 2 h and centrifuging to precipitate the excess SDS, the remaining carbohydrate, representing the “inaccessible” chains on the glycoproteins, was removed by hydrolysis with an additional aliquot of Endo H as described above. The reaction mixtures were again chromatographed on Sepharose 6B, and the protein and car- bohydrate regions located and pooled as above.

The oligosaccharide pools from Sepharose 6B were desalted by passage through a Bio-Gel P-2 (100-200 mesh) column (2 X 25 cm) equilibrated with 0.1 N acetic acid and concentrated by rotary evap- oration. High resolution gel filtration for sizing the oligosaccharides was performed on a column (0.9 X 195 cm) of Bio-Gel P-4 (minus 400 mesh), and 0.6-ml fractions were collected (22). The column was calibrated with a series of Endo H-released invertase oligosaccharides purified by repeated chromatography on the Bio-Gel P-4 column. The Man/GlcNAc ratio in the phosphate-free Man+l4 GlcNAc species was confvmed by integrating their ‘H-NMH 360 MHz spectra, kindly provided by Dr. Paul Atkinson at Albert Einstein College of Medicine. The Manlc,-&lcNAc species were assigned from their elution posi- tions (K,, values) on a Bio-Gel P-6 (minus 400 mesh) column (0.9 X 195 cm) which provided a linear relationship between the log of the hexose number, H (where mannose is 1 and GlcNAc is 2.5). and the elution volume.

Phosphate was determined by the method of Ames (23) using tubes (12 X 75 mm), acid washed to reduce the background phosphate level. Oligosaccharides were chromatographed on QAE-Sephadex (Phar- macia) columns (1 x 2.5 cm) equilibrated in 2 mM Tris base as described by Varki and Kornfeld (5). The conditions for stepwise elution of these oligosaccharides with 2-ml aliquots of increasing sodium chloride concentration in 2 mM Tris base were established in preliminary experiments.

SDS-polyacrylamide gel electrophoresis was carried out in a 0.75- mm thick acrylamide slab consisting of a 5 or 10% separating gel and a 3.5% stacking gel with the buffer system of Laemmli (24). The samples shown in Fig. 1 represent 1 to 2 pg of protein, and Endo H disgestions for this experiment were carried out with 0.1 IU of Endo H/mg of substrate protein/ml for the times indicated in the legend.

RESULTS AND DISCUSSION

Demonstration of Oligosaccharides Inaccessible to Endo H on Carboxypeptidase Y a n d Invertase-Previous studies from this laboratory have shown the value of Endo H from S. plicatus in characterizing the carbohydrate and protein com- ponents of carboxypeptidase Y and invertase from baker’s yeast (7, 8, 11). During this work it was observed that both glycoproteins contained two classes of high mannose N-linked

oligosaccharides which could be differentiated on the basis of their sensitivity to hydrolysis by Endo H. One class, repre- senting three of four chains on carboxypeptidase Y and seven of nine on invertase, was readily released from the native proteins on incubation with Endo H for only 1 to 2 h. The other class, representing the remaining carbohydrate, could be released by Endo H only if these glycoproteins were de- natured with SDS. The results of these studies are summa- rized by the SDS-polyacrylamide gel profiles shown in Fig. 1.

After 1 h, Endo H converted native carboxypeptidase Y from the 62,000-dalton species (Fig. lA, lane I) to a series of bands which differed in size from each other by multiples of about 3,000 daltons (lane 2), consistent with the removal of from 0 to 2 oligosaccharides. After 20 h of hydrolysis a single band of about 52,000 daltons was present (lane 3), the species known to retain a single oligosaccharide chain (11). Removal of three oligosaccharides from carboxypeptidase Y reduces its ability to bind ’251-C0nA on SDS-polyacrylamide gels (25) but does not diminish its exopeptidase activity (16). The Endo H digestion conditions employed in Fig. 1 (lane 3) are about 10- fold more rigorous than required for the complete removal of the three accessible chains from native carboxypeptidase Y (compared to the large scale digestions described under “Materials and Methods”) which attests to the resistance of the fourth oligosaccharide to Endo H. Continuing this incu- bation (Fig. 1) for 96 h failed to release the fourth oligosac- charide, but addition of SDS to the reaction mixture resulted in the removal of the remaining chain by the residual Endo H to yield an inactive homogeneous species of about 50,000 daltons (lane 4), which did not bind ’251-ConA (16).

Incubation of native invertase for 1 h with Endo H reduced its size, without loss of activity, from over 90,000 daltons (Fig. lB, lane I) to a population of 68,000-, 66,000-, and 64,000- dalton species (Fig. lB, lane 2), all of which bound “‘I-ConA on SDS-acrylamide gels (25). Incubation for 20 h (lane 3) reduced their respective sizes to 66,000, 64,000, and 62,000 daltons, with the smallest unable to bind “‘I-ConA (25). This species was shown previously to retain 9 N-acetylglucosamine

A M r 1 2 3 4

Q 94 K-

67K-

rn 43K-

B Mr I 2 3 4

67 K

FIG. 1. SDS-acrylamide gel electrophoresis of the products of Endo H hydrolysis of yeast carboxypeptidase Y (A, 10% gel), and external invertase (B, 5% gel). The incubation conditions were: lane 1, -Endo H; lane 2, +Endo H for 1 h; lane 3, +Endo H for 20 h; lane 4, A, 0.25% SDS + Endo H for 20 h and B, boiled in 0.25% SDS for 1 min then +Endo H for 20 h. The positional markers for 10% gel are phosphorylase b (94,000). bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), and trypsin inhibitor (20,100). Those for 5% gel are Escherichia coli RNA polymerase subunits p’ (165,000), /? (155,000), u (90,000), and bovine serum albu- min (67,000).

by guest on February 1, 2020http://w

ww

.jbc.org/D

ownloaded from

2564 Oligosaccharide Accessibility and Processing

residues (8). Addition of SDS to this incubation mixture did not promote release of the remaining carbohydrate by the residual Endo H (not shown), but after heating in SDS and supplementing with additional Endo H all of the resistant oligosaccharides were liberated, providing only the homoge- neous 62,000-dalton mannose-free species (Fig. lB, lane 4). Consistent with recent studies on external invertase from the yeast mutant X2180-mnn2 (9), the 64,000-, 66,000-, and 68,000- dalton bands seen in Fig. 1B (lanes 2 and 3) represent subunits which retain 1, 2, or 3 short oligomannosyl chains, respec- tively.

The presence of oligosaccharides on carboxypeptidase Y and invertase which cannot be released by Endo H prior to protein denaturation (Fig. 1) provides strong evidence that protein conformation rather than the carbohydrate structure itself confers this resistance. Thus, N-linked high mannose oligosaccharides which are inaccessible to Endo H in mature glycoproteins may represent a general class of carbohydrate chains that became inaccessible to processing glycosidases and sugar transferases at an early stage in their synthesis. In the case of carboxypeptidase Y and invertase, carbohydrate chains masked from processing enzymes by protein folding should be smaller and have a lower degree of peripheral modification than those accessible to the mixed mannosyl transferases associated with chain elongation and phospho- mannose addition (17,26). To test this prediction directly, the oligosaccharides accessible to Endo H on native carboxypep- tidase Y and invertase were isolated and compared with those hydrolyzed by Endo H subsequent to protein denaturation.

Characterization of Accessible and Inaccessible Oligosac- charides from Carboxypeptidase Y-The three oligosaccha- ride chains released from native carboxypeptidase Y by Endo H, which correspond to those liberated when the 52,000-dalton product was generated (Fig. l A , lane 3), were chromato- graphed on a calibrated Bio-Gel P-4 (minus 400 mesh) column. The profile, shown in Fig. 2 A , reveals species which ranged in size from Mang-zoGlcNAc, with most of the hexose eluting at a size equivalent to Man14-18GlcNAc. Based on the work of Hashimoto et al. (6), the presence of phosphate on carboxy- peptidase Y was expected, and, in agreement with their data our analyses provided an average value of about four phos- phates per enzyme molecule. The release by Endo H of about 80% of the mannose from native carboxypeptidase Y was expected from earlier work (ll), but the presence of over 95% of the phosphate in this oligosaccharide pool was a novel observation. The Man/P ratio through the profie (Fig. 2 A ) increased from about 9 to over 50 indicating a mixture of species, some of which contained one or two phosphates while those smaller than about ManlaGlcNAc were probably phos- phate free.

The residual 20% of the carbohydrate on carboxypeptidase Y, which represented the inaccessible fourth chain, was re- leased with Endo H in SDS and its size was also determined on the Bio-Gel P-4 column. The profile (Fig. 2B) revealed MangGlcNAc and ManllGlcNAc to predominate with lesser amounts of ManloGlcNAc and ManlpGlcNAc. These results c o n f i i the prediction that the species in the fourth chain oligosaccharide pool (Fig. 2B) were several residues smaller than those accessible to Endo H under native conditions (Fig. 2A). A carbohydrate species not previously reported on yeast glycoproteins and present reproducibly in the Endo H-resist- ant oligosaccharides from carboxypeptidase Y was Mane- GlcNAc (Fig. 2B). Recent studies indicate that Mans- GlcNAc is the ultimate product of trimming of the GlcaMangGlcNAc species initially transferred to yeast proteins and is the minimum precursor for mannose chain elongation in this organism (10).

80 I00 120 FRACTION N U M B E R

FIG. 2. Chromatography of carboxypeptidase Y oligosac- charides on Bio-Gel P-4. A, the oligosaccharide pool representing the three chains released from native carboxypeptidase Y by Endo H. Mannose (Am) was determined on 50-pl aliquots. The Man/P ratio was determined by pooling 60 pl from each of 3 successive fractions, indicated by the horizontal bars, and assaying 12 p1 for mannose and the remainder for phosphate. B, the fourth oligosaccha- ride chain released by Endo H after denaturation of carboxypeptidase Y with SDS. Mannose was determined in 20O-~l aliquots along the profile. The Man/P ratio in the material loaded onto the column was over 50 and, therefore, phosphate was not assayed along the profile. The elution of the marker compounds is indicated at the top of A. M, Man; N , GlcNAc.

Phosphate Distribution on the Carboxypeptidase Y Oli- gosaccharides-To examine the phosphate distribution in the accessible oligosaccharides, fractions 80-125 in Fig. 2A were pooled and chromatographed on QAE-Sephadex. As shown in Fig. 3 A , approximately 23% of the mannose passed through the column (Pool I), 48% eluted with 12 m~ NaCl (Pool 11), and the remaining 29% eluted with 80 m~ NaCl (Pool 111). Based on the work of Varki and Kornfeld (5) and Hashimoto et al. (6), the oligosaccharides in Pool I should be phosphate free, while those in Pools 11 and I11 should contain one and two diesterified phosphates per chain, respectively. The three pools (Fig. 3 A ) were desalted and chromatographed separately on the calibrated Bio-Gel P-4 column. As shown in Fig. 3B, the phosphate-free neutral fraction (Pool I) was Mang-,s- GIcNAc species with ManllGlcNAc the predominant oligosac- charide. It should be noted that less than 15% of the mannose in Pool I (Fig. 3B) was represented by species smaller than ManllGlcNAc. Since the MansGlcNAc processing intermedi- ate (10) was not present in these oligosaccharides, it can be concluded that nearly all of the accessible chains on native carboxypeptidase Y had been elongated with at least three mannoses during glycoprotein synthesis.

The oligosaccharides in Pool I1 ranged in size from Man12_leGlcNAc, and, as shown in Fig. 3B, their Man/P ratio declined from about 15 to 10 through the profie, indicative of 1 phosphate residue per oligomannosyl chain. The oligosac- charides in Pool I11 had an apparent size of Manls-lsGlcNAc, and the Man/P ratio for these (Fig. 3B) declined from about 7.5 to 6 through the profde, confii ing 2 phosphate residues per chain. Comparison of the apparent size and Man/P ratio of the oligosaccharides from carboxypeptidase Y with the elution of phosphate-free markers of known size (Fig. 3B)

by guest on February 1, 2020http://w

ww

.jbc.org/D

ownloaded from

Oligosaccharide Accessibility and Processing 2565

FIG. 3. Analysis of the charge and size of the carboxypeptidase Y oli- gosaccharides accessible to Endo H. A, chromatography of the species pooled from Fig. 2.4 (tubes 80 to 125) on QAE- Sephadex. The oligosaccharide sample was loaded in 2 ml, and 2-ml fractions were collected. Changes in the concen- - tration of sodium chloride in the 2 mM x 0.3 Tris base elution buffer are indicated at 2 the top of A. Mannose was determined in 50-pl aliquots. B, resolution on Bio- Q,

Gel P-4 of the species separated on the $ o.2 basis of charge by chromatography in A Q:

into pool I (O), I1 (0). and 111 (0). Man- - nose was determined in 100pl aliquots 0, I along each profile. The Man/P ratio plotted above each peak was obtained by pooling 100 pl from each of 3 successive fractions and assaying 60 pI for mannose C and the remainder for phosphate. M , Man; N, GlcNAc.

5 IO 15

suggests that each phosphate contributes, probably by nega- tive charge exclusion, a size equivalent of 1 to 2 mannose residues on Bio-Gel P-4.

In contrast to the accessible oligosaccharides described above, more than 80% of the mannose in the pool which represented the fourth chain (Fig. 2B) passed through QAE- Sephadex (not shown) indicating the absence of charged phos- phate groups. The remaining mannose, which amounted to only 4% of that initially present on carboxypeptidase Y, eluted from QAE-Sephadex with 12 m NaCl, consistent with one phosphodiester unit per oligosaccharide. This latter fraction was insufficient in amount for further analysis beyond man- nose and phosphate determinations.

Accessibility a n d Location of the Fourth Carboxypeptidase Y Oligosaccharide-If protein folding restricts the processing of one of the four oligosaccharides on carboxypeptidase Y, not only should the periphery of this chain be hidden from mod- ifying enzymes, it also should be attached to one specific glycosylation site rather than being randomly located at all four sites. Evidence in support of both of these predictions has been obtained. Firstly, exhaustive digestion with a-man- nosidase failed to release appreciable mannose from the Endo H-inaccessible fourth chain, but once free of the protein, these oligosaccharides were hydrolyzed completely to mannose and Ma@( l+4)GlcNAc. In a parallel experiment, a-mannosidase removed 55% of the mannose from the accessible chains on native carboxypeptidase Y. However, on release of the Endo H-accessible chains from carboxypeptidase Y their suscepti- bility to a-mannosidase was not increased. This appears due to the fact that once mannose phosphate or phosphate alone becomes the nonreducing terminal residue, the action of the jack bean enzyme ceases (5,27).

Secondly, peptide mapping by limited trypsin digestion indicates that the fourth chain is associated with a single specific peptide (Fig. 4). From the recent sequencing studies of Svendsen et al. (29), carboxypeptidase Y has been shown to contain four N-glycosylation sequons located a t asparagine residues 13, 87, 177, and 350. A limited tryptic digest of citraconylated carboxypeptidase Y would predict from the proposed sequence (29) the production of four glycopeptides of about 8,000, 9,000, 10,000, and 18,000 daltons. Consistent with this prediction a limited tryptic digestion of native car- boxypeptidase Y (Fig. 4) yielded an 18,000-dalton peptide and a cluster of lower molecular weight poorly resolved species (lane I ) , most of which bound "'I-ConA (lane 2). By contrast, a comparable digest of carboxypeptidase Y containing the Endo H-resistant fourth oligosaccharide, revealed that only

80 100 120 FRACTION NUMBER

FIG. 4. SDS-acrylamide step gel (12.5%-1596-17.58) electro- phoresis of the tryptic products of citraconylated carboxypep- tidase Y containing four (lanes 1 and 2) and one (lanes 3 and 4) oligosaccharide chains. Carboxypeptidase Y (0.1 mg) containing 37 nmol of lysine was reacted with a 50 M excess of citraconic anhydride (28) after denaturation by boiling for 3 min in 0.1% SDS and 0.1 M /3-mercaptoethanol. The citraconylated carboxypeptidase Y was hydrolyzed at the arginine residues in 0.4 M N-ethylmorpholine buffer, pH 8.5, with three I-pg additions of tosylphenylalanyl chloro- methyl ketone-treated trypsin at 2-h intervals over 6 h at 37 "C. The hydrolysate was lyophilized and dissolved in 20 n w Tris-HCl, pH 8.5, at a final peptide concentration of 2.5 mg/ml. Aliquots containing 10 to 15 pg of the peptides were analyzed by electrophoresis on a three- step gel slab consisting of 12.5, 15, and 17.5% polyacrylamide. After electrophoresis, the 15 and 17.5% gel sections containing the stained tryptic peptides were cut out and subjected to lectin-gel overlay using '"I-ConA (25). Lanes 1 and 3 represent stained bands (b), and lanes 2 and 4 show radioactive bands after '''I-ConA binding (D). The smaller tryptic bands were difficult to resolve even with the use of the step gel. The triangles approximate the location of three smallest glycopeptides based on their anticipated M, values (29); the arrow indicates the interface between the 15% and 17.5% gel sections. The positional markers are trypsin inhibitor (20,100) and a-lactalbumin (14,400).

the 18,000-dalton species bound l"I"conA (lanes 3 and 4). From the structure provided for carboxypeptidase Y (29) this peptide should encompass amino acid residues 39 through 174, which includes the Asn-containing sequon a t residue 87. Since the other smaller sequon-containing peptides from Endo H-treated carboxypeptidase Y do not contain carbohydrate (compare lanes 2 and 4 of Fig. 4), these results would appear t o con fm the unique location of the inaccessible oligosaccha- ride at Asn8-i.

Thus, the fourth oligosaccharide on carboxypeptidase Y is resistant to Endo H and a-mannosidase under native condi- tions but is susceptible to these enzymes once the tertiary structure of the molecule is perturbed. This chain which is smaller than the other three is essentially phosphate free and appears to be located consistently at the same glycosylation

by guest on February 1, 2020http://w

ww

.jbc.org/D

ownloaded from

2566 Oligosaccharide Access

site on the peptide backbone of each carboxypeptidase Y molecule.

Comparison of Accessible and Inaccessible Oligosaccha- rides from Invertase-To assess whether the Endo H-resist- ant chains associated with invertase were also smaller than those readily liberated under native conditions, oligosaccha- ride pools released before and after denaturation in SDS were compared on the Bio-Gel P-4 column. Fig. 5 shows that over 85% of the mannose in the accessible oligosaccharides was in species larger than ManzoGlcNAc, while, by contrast, essen- tially all of the residual oligosaccharides removed by Endo H under denaturing conditions were smaller than ManlzGlcNAc. ManloGlcNAc was the predominant oligosaccharide in the resistant pool from invertase, but ManllGlcNAc and Ma%- GlcNAc also seen in the inaccessible chain from carboxypep- tidase Y (Fig. 2B) were present in substantial amounts.

Mannose phosphate has been shown previously to be asso- ciated with invertase carbohydrate (30), and our assays con- f m e d the presence of three to four phosphates per 60,000- dalton subunit. All of the phosphate was associated with the large oligosaccharides released under native conditions by Endo H (Fig. 5 , 0 , tubes 58-80). The inaccessible oligosaccha- rides from invertase failed to bind to QAE-Sephadex and gave a negative phosphate analysis as found for the fourth chain on carboxypeptidase Y. Previously, the chains remaining on native invertase were shown to contain 9 a-mannosidase-re- sistant residues each (8). More recent experiments have con- f i e d that once removed by Endo H, these oligosaccharides are completely hydrolyzed by a-mannosidase to the expected products, mannose and ManPGlcNAc (not shown).

Endo H hydrolysis of native invertase in Fig. 5 (0.12 IU of Endo H X h/mg) should provide qualitatively the same prod- ucts seen in Fig. 1B (0.1 IU of Endo H X h/mg) where an average of two oligosaccharides of about 2,000 daltons each remained per subunit (lane 2). It should be noted that the presence of from one to three core oligosaccharides on native invertase subsequent to Endo H treatment (Fig. lB, lane 2) does not compromise the hypothesis that these chains were underprocessed as a result of steric restrictions during synthe- sis. Clearly, the conformation of native invertase is sufficiently “open” to permit at least 6 chains to be efficiently removed by Endo H in 1 h (yielding the 68,000-dalton species, Fig. lB, lane 2), and given enough time (20 h), Endo H will remove all of the short “resistant” oligosaccharides from at least some of the subunits (Fig. lB, lane 3). These results suggest that during the 5 to 10 min required for the synthesis of invertase

60 70 80 90 100 110 120 130 FRACTION NUMBER

FIG. 5. Chromatography of yeast invertase oligosaccharides on Bio-Gel P-4. 0, oligosaccharides representing the seven chains released from native invertase by Endo H. 0, oligosaccharides rep- resenting the two chains released by Endo H from invertase after denaturation by boiling in SDS. Mannose was determined in 3O-pl aliquots of the fractions from each profile. Elution of marker oligo- saccharides is shown at the top of the figure. M, Man; N, GlcNAc.

,ibility and Processing

(31L three of its nine oligosaccharides are relatively inacces- sible to processing enzymes and, therefore, remain smaller than the others. On long term digestion with Endo H, however, subtle differences in the accessibility of the chitobiosyl core of the short chains become apparent. Thus under nondenaturing conditions, the slow release of some of the small oligosaccha- rides from the 68,000-dalton species produces the 66,000- and 64,000-dalton forms (Fig. lB, lane 2) and probably accounts for the Mans-lzGlcNAc oligosaccharides seen in the accessible carbohydrate pool depicted in Fig. 5 (0).

In conclusion, there appears to be a strong correlation between the N-linked oligosaccharides which are inaccessible to Endo H and a-mannosidase on native carboxypeptidase Y and invertase and those oligosaccharides which are uniformly smaller in size and lack peripheral phosphate residues. The simplest explanation for these differences, which is consistent with all observations made to date, is that shortly after the co-translational addition of carbohydrate to proteins (32) cer- tain oligosaccharides become inaccessible to the processing enzymes as a result of the polypeptide chain folding into its mature conformation. The susceptibility of the restricted oli- gosaccharides to a-mannosidase once they are removed from these enzymes indicates that before processing became lim- ited, the three glucoses and probably one mannose (10) were excised from the GlcsMan9GlcNAcz chains acquired in the initial protein glycosylation step. The presence of the MaQGlcNAc processing intermediate (10) only in the pool of oligosaccharides released by Endo H after protein denatura- tion is consistent with this hypothesis (Figs. 2 and 5 ) .

In higher eucaryotic cells, the presence of high mannose oligosaccharides appears to result from the lack of processing (MangGlcNAc) or only partial processing (Man5-sGlcNAc) of the Glc3Man9GlcNAcp precursor subsequent to glucose re- moval. While the precise factors that determine which sites are processed to complex chains and which remain high mannose have not been clarified, protein conformation or local amino acid sequence appears to play a key role (12, 13, 15,33). In support of this concept the recent studies of Green (34) demonstrate that the incorporation of amino acid analogs into immunoglobulins impairs the processing of high mannose to complex chains, possibly by altering polypeptide confor- mation. An additional example of impaired processing of high mannose chains due to steric factors arises during synthesis of the Sindbis virus glycoproteins and is described in the accom- panying papers (35, 36).

Acknowledgment-We express our appreciation to Dr. Ib Svendsen for providing us with the primary sequence of yeast carboxypeptidase Y.

REFERENCES

1. Struck, D. K., and Lennarz, W. J. (1980) in The Biochemistry of Glycoproteins and Proteolycans (Lennan, W. J., ed) pp. 35- 83, Plenum, New York

2. Hubbard, S. C., and Ivatt, R. J. (1981) Annu. Reu. Biochem. 50, 555-583

3. Schachter, H., and Roseman, S. (1980) in The Biochemistry of GhcoDroteins and Proteodycans (Lennarz, W. J., ed) pp. 85- IZ?, Plenum, New York

” ..

4. Kornfeld. R.. and Kornfeld, S. (1980) in The Biochemistry of Glycoproteins and Proteoglycans (Lennarz, W. J., ed) pp. 1- 34, Plenum, New York

5. Varki, A,, and Kornfeld, S. (1980) J. Biol. Chem. 255, 10847- 10858

6. Hashimoto, C., Cohen, R. E., Zhang, W.-J., and Ballou, C. E. (1981) Proc. Nutl. Acad. Sei. U. S. A. 78, 2244-2248

7. Tarentino, A. L., Plummer, T. H., Jr., and Maley, F. (1974) J. Biol. Chem. 249,818-824

8. Trimble, R. B., and Maley, F. (1977) J. Biol. Chem. 252, 4409- 4412

, , I

by guest on February 1, 2020http://w

ww

.jbc.org/D

ownloaded from

Oligosaccharide Accessibility and Processing 2567

9. Lehle, L., Cohen, R. E., and Ballou, C. E. (1979) J. Biol. Chem.

10. Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and

11. Trimble, R. B., and Maley, F. (1977) Biochem. Biophys. Res.

12. Nakamura, K., and Compans, R. W. (1979) Virology 95,8-23 13. Rosner, M. R., Grinna, L. S., and Robbins, P. W. (1980) Proc.

14. Aubert, J. P., Helbecque, N., and Loucheux-Lefebvre, N. H. (1981)

15. Wilson, I. A., Skehel, J. J., and Wiley, D. C. (1981) Nature 289,

16. Chu, F. K., and Maley, F. (1982) Arch. Biochem. Biophys. 214,

17. Ballou, C. E. (1976) Adu. Microb. Physiol. 14,93-I58 18. Tarentino, A. L., Trimble, R. B., and Maley, F. (1978) Methods

19. Trimble, R. B., Tarentino, A. L., Plummer, T. H., Jr., and Maley,

20. Kuhn, R. W., Walsh, K. A., and Neurath, H. (1974) Biochemistry

21. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and

254, 12209-12218

Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666

Commun. 78,935-944

Natl. Acad. Sci. U. S. A. 77,67-71

Arch. Biochem. Biophys. 208,20-29

366-373

134-139

Enzymol. 50,574-580

F. (1978) J. Biol. Chem. 253, 4508-4511

13, 3871-3877

Smith, F. (1956) Anal. Chem. 28, 350-356

22. Trimble, R. B., Maley, F:, and Tarentino, A. L. (1980) J. Biol.

23. Ames, B. N. (1966) Methods Enzymol. 8, 115-118 24. Laemmli, U. K. (1970) Nature 227,680-685 25. Chu, F. K., Maley, F., and Tarentino, A. L. (1981) Anal. Biochem.

26. Nakajima, T., and Ballou, C. E. (1975) Proc. Nutl. Acacl. Sei. ff.

27. Hashimoto, C., Cohen, R. E., and Ballou, C. E. (1980) Biochem-

28. Atassi, M. Z., and Habeeb, A. F. S. A. (1972) Methods Enzymol.

29. Svendsen, I., Martin, B. M., Viswanatha, T., and Johansen, J . T.

30. Smith, W. L., and Ballou, C. E. (1974) Biochemistry 13, 355-361 31. Novick, P., Ferro, S., and Schekman, R. (1981) Cell 25,461-469 32. Glabe, C. G., Hanover, J. A,, and Lennarz, W. J. (1980) J . Biol.

33. Kornfeld, R., and Wold, W. S. M. (1981) J. Virol. 40, 440-449 34. Green, M. (1982) J. Biol. Chem. 257,9039-9042 35. Hsieh, P., Rosner, M. R., and Robbins, P. W. (1982) J. Biol.

36. Hsieh, P., Rosner, M. R., and Robbins, P. W. (1982) J. Biol.

Chem. 255, 10232-10238

116, 152-160

S. A. 72, 3912-3916

istry 19, 5932-5938

25, 546-553

(1982) Carlsberg Res. Commun. 47, 15-27

Chem. 255,9236-9242

Chem. 258,2548-2554

Chem. 258,2555-2561

by guest on February 1, 2020http://w

ww

.jbc.org/D

ownloaded from

R B Trimble, F Maley and F K Chuhigh mannose oligosaccharides on carboxypeptidase Y and invertase.

GlycoProtein biosynthesis in yeast. protein conformation affects processing of

1983, 258:2562-2567.J. Biol. Chem. 

  http://www.jbc.org/content/258/4/2562.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/258/4/2562.citation.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on February 1, 2020http://w

ww

.jbc.org/D

ownloaded from