A New Saccharomyces cerevisiae mnn Mutant N-Linked ...

Vol. 264, No. 20, Issue of July 15,PP. 11649-11856,1989

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U.S.A.

0 1989by The American Society for Biochemistry and Molecular Biology, Inc.

A New Saccharomyces cerevisiae mnnMutant N-Linked Oligosaccharide Structure* (Received for publication, November 21, 1988)

Luis M. HernandezSQ, LunBallouS, Eugenio AlvaradoS, Beth L. Gillece-Castroq,A. L. Burlingamell, and ClintonE. BallouS1) From the $Department of Biochemistry, University of California, Berkeley, California 94720and the TDepartment of Pharmaceutical Chemistry, University of California, San Francisco, California 94143

We find that theN-linked MansGlcNAcz- coreoligo- have Structure I1 (where M is mannose and GNAc is GlcNAc) saccharide of Saccharomyces cerevisiae mnn mutant and that theouter chain is formed by mannoproteinsis enlarged by the addition of the outer chain to the al-3-linked mannose in the side chain aM+6aM+6PM+4apGNA~ aM+6aM+6pM+4a/3GNA~ that is attached to the B1-4-linked mannose rather 7' t3 t3 t' T3 t3 than by addition to the terminal arl-6-linked mannose.aM aM aM6+aM aM aM6+aM* This conclusionis derived from structural studies on a T' tz t' aM phosphorylatedoligosaccharidefractionandfrom aM mass spectral fragmentanalysis of neutral coreoligo7' t' aM aM saccharides. I11

I1

It was reported previously (1,2) that the N-linked oligosaccharide, released from Saccharomyces cerevisiae mnnl mnn2 mannoprotein by digestion with endo-pl4-N-acetylglucosaminidase H, when treated with endo-d-6-mannanase yielded a core oligosaccharide MangGlcNAc with Structure I.

an extension of the unsubstituted (starred) a l a - l i n k e d mannose unit. The results also support Structure I11 for the mnnl mnn9 oligosaccharide. The conclusions presented here are at variance with the results by Trimble and Atkinson (3) for yeast invertase oligosaccharides, which could mean that there is more than one site for addition of the outer chain to the core oligosaccharide.

aM+6aM+6aM+6pM+4acupGNA~

f'

aM aM aM

t3

t3

EXPERIMENTALPROCEDURES

t'

aM

t'

aM

I

From this, itwas inferred that theouter chainpolymannose unit was formed by elongation of the terminal al-6-linked mannose (M) of the Ma&GlcNAc2-Rcore (GNAc is GlcNAc). In recent studies, however, we have found that phosphorylated core fragments obtained from N-linked oligosaccharides with attached outer chains have phosphate linked to position 6 of this core mannose, which result precludes it from being a site for attachment of the d-6-linked outer chain. This finding has led us to consider alternative sites for attaching theouter chain to the core. We conclude that only one site is consistent with the methylation, acetolysis, and 'H NMR data previously published (1, 2) and with mass spectral fragmentationdata reported herein. We propose that the mnnl mnn2 and mnnl mnn2 mnnlO core oligosaccharides * This work was supported by National Science Foundation Grant PCM87-03141 and National Institutes of Health Grant AI-12522 (to C. E. B.) and by National Institutes of Health Grants RR-01614 and AM-27643 (to A. L. B.). 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 solelyto indicate this fact. 5 Visiting scholar from the Department of Microbiology,University of Extremadura, Badajoz, Spain. 11 To whom correspondence should be addressed.

Materials and Methods-Core oligosaccharides from S. cerevisiae mnnl mnn2 (2), mnnl mnn2 mnnlO (4), and mnnl mnn9 (5) mannoproteins were from other studies. All three were obtained by digestion of the mannoproteins with endo-N-acetylglucosaminidase H, while the first two were also treated with a bacterial e n d o - a 1 4 mannanase (6) to remove the outer chain portion. The mnnl mnn2 core oligosaccharide,prepared in this manner, is from an earlier study (2). Neutral and acidic (phosphorylated) oligosaccharides were separated by gel filtration in water, and the phosphorylated oligosaccharides were fractionated by gradient elution from QAE-Sephadex (7). Methyl 4,6-benzylidene-a-~-mannopyranoside was prepared according to Buchanan and Schwarz (8), and it was phosphorylated with diphenylphosphorochloridate (Aldrich) according to Kilgour and Ballou (9). Exo-al+2-mannosidase (10) and a nonspecific a-mannosidase (11) were isolated from bacterial sources. The first enzyme removed only the a1-2-linked mannoses and left the mannosylphosphate groups intact, whereas the other bacterial mannosidase also removed some a1-3- and a 1 4 - l i n k e d mannose. Jack bean a-mannosidase, which removed all terminal a-linked mannose units, was obtained from V-LABS (Covington, LA). Separations of neutral and phosphorylated sugars and oligosaccharides were done on a Dionex BioLC carbohydrate system by gradient elution with sodium acetate in alkaline solution according to the manufacturer's manual. Oligosaccharides were acetolyzed and the acetylated fragments were analyzed by fast atom bombardment mass spectrometry (12). Oligosaccharide ABEE' and ABOE derivatives were prepared by reductive amination, purified by HPLC and analyzed by LSIMS (13, 14). Octyl p-aminobenzoate was synthesized as reported (15). 'H NMR (16-19) was done a t 500 MHz in DzO on a 'The abbreviations used are: ABEE, aminobenzoyl ethyl ester; ABOE, aminobenzoyl octyl ester; LSIMS, liquid secondary ion mass spectrometry; NOE, two-dimensional nuclear Overhauser effect; HPLC, high performance liquid chromatography.

11849

Yeast Oligosaccharide Structure

11850

Bruker spectrometer in the Department of Chemistry (University of California, Berkeley), and chemical shifts are referenced to internal acetone at 62.217 relative to sodium 3-(trimethylsily1)propanesulfonate (16). Others have used a value of 62.225 for acetone determined 1-sulfonate (18). relative to sodium 4,4-dimethyl-4-silapentane RESULTS

Mannosidase Digestion of the mnnl mnn2 mnnlO Oligosaccharide-Mannoprotein, prepared from themnnl mnn2 rnnnlO mutant (20) by extraction with hotcitrate buffer followedby chromatography on DEAE-Sephadex, was digested with endoglucosaminidase H to release the N-linked oligosaccharides. These were separated from residual protein by gel filtration on a Bio-Gel P-10 column in salt and were fractionated into neutral and acidic oligosaccharides by gel filtration on a Bio-Gel P-4 column by elution with water. The neutraland acidic phosphorylated fractions were digested separately with endo-a14-mannanase to remove the outer chain portion, and theglucosamine-containing core fragments were recoveredby gel filtration on a Bio-Gel P-4 column. The H-1 NMR spectra of the neutral and acidic oligosaccharides are identical (Fig. l ) , except for the mannosylphosphate signals in the latter at 65.43-5.45. All of the anomeric proton signals have previously been assigned to Man9GlcNAc isomer A as shown in Table I (2), but they would also agree with isomers B andC . The only difference between these structures is in the location of the unsubstituted a l 4 - l i n k e d mannose unit remaining from the outer chain, and itis known that an a 1 4 - l i n k e d mannose has relatively little effect on the H-1 chemical shift of a mannose to which it is linked (16,18). All three isomers are also consistent with the previously reported methylation data for the Man9GlcNAc oligosaccharide obtained from the mnnl mnn2 mannoprotein (1, 2) (data cited in the footnote of Table I).

The neutral and phosphorylated core fragments were each digested with a bacterial a-mannosidase preparationthat has an active exo-al-2-mannosidase but that also shows exoal-3- and exo-al4-mannosidase activities (11).The limit product from the action of this mannosidase on the neutral mnnl mnn2 mnnlO oligosaccharide had StructureIV, whereas the phosphorylated oligosaccharide gave a fragment that had Structure V (where M is mannose and GNAc is N-acetylglucosamine). LYM-~~LYM+~~M+'LY/~GNAC P+6~M-t6aM+6pM+4,401pGNA~

f3

f

LYM

otM

IV

f3

LYM V

Thus, this enzyme yields the same incompletely digested fragment irrespective of the presence of the phosphate group. Tentative structures of these two fragments were inferred from the one-dimensional H-1 NMR spectra (Fig. 2, A and B ) that show signals for two mannose units in a 1 4 linkage, one being substituted by a mannose in a1-3 linkage, and one mannose in 81-4 linkage, while the phosphorylated fragment gives an additional signal for the a-mannosylphosphate unit. Both samples also give signals for the two anomeric forms of N-acetylglucosamine, while the signal for the al-3-linked mannose shows the characteristic splittingdue to a long range influence from the hexosamine anomers (18),which is diagnostic for this particular mannose unit. By two-dimensional homonuclear Hartmann-Hahn correlation spectroscopy (19), we were able to demonstrate conclusively that the mannosylphosphate unit was attached to the unsubstituted a l a - l i n k e d mannose rather than to theal3-linked mannose. Phosphorylation at position 6 of a hexose is known to shift the C-6 protons downfield to 64.05-4.15 (21), and new signals in the spectrum for compound V at 64.09-4.14 in brackets (Fig. 3, right panel) are clearly correlated with H-1 of the unsubstituted a l a - l i n k e d mannose unit. At the same time, signals observed at 63.80-3.90 (presumably due to H-6,6') in the spectrum of IV (leftpanel) are absent in V. Finally, the above conclusions were confirmed by digestion of the phosphorylated oligosaccharide with jack bean cu-mannosidase, which removed all terminalmannose units including that attached to the phosphate. The H-1 NMR spectrum of the fragment (Fig. 2C) shows signals at 64.89 and 4.90 for two B a 1 4 - l i n k e d mannoses and a signal at 64.77 for a 8-linked mannose, along with the signals for the anomers of N-acetylglucosamine, but no signal for an al-3-linked mannose or a mannosylphosphate group. This spectrum supportsthe structure P+6aMan+6aMan+6~Man+4a@GlcNAc. Confirmation of the Attachment of Phosphate to Position 6 of Mannose in Mannoproteins-Because it was possible that the phosphate was linked to a ring position of the unsubstituted a l 4 - l i n k e d mannose, rather than C-6, we sought a rigorous characterization of the mannose phosphate released by acid hydrolysis. In most published work, the evidence that phosphate islinked to position 6 of mannose in glycoproteins PPM is based on the release of mannose 6-phosphate during acid FIG. 1. H-1NMR spectra of the neutral and acidic oligosac- hydrolysis (22, 23). Since phosphate migrates between hycharides. A, the neutral Man9GlcNAc;B , the neutral ManloGlcNAc; and C, the phosphorylated MangGlcNAc oligosaccharide. The H-1 droxyl groups under acidic conditions, however, we felt it signals of A and C are identical, whereas the latter also shows signals important to determine how readily mannose 6-phosphate for a mannosylphosphate unit at 65.42-5.45. Table I shows the was formed from mannose that had a phosphate group on a assignments, which are consistent with three possible isomers. In B , ring position. In fact, one method for synthesizing glucose 6the signal at 64.92 is shifted to 65.12 and a new split signal is observed phosphate consists of the random phosphorylation of glucose at 65.045 and 5.055, overlapping other signals in this region. The with polyphosphoric acid followed by refluxing in strong acid slight heterogeneity in the N-acetylglucosamine H-1 signals and the small signal at 65.39 reflect the presence of a ManllGlcNAc contam- to convert all isomers quantitatively to the6-phosphate (24). Methyl-4,6-benzylidene a-D-mannopyranoside (8) was inant (5).

u

11851

Yeast OligosaccharideStructure TABLEI H-1 NMR data and methylation analysis of MahGlcNAc core oligosaccharide All three isomers give the same methylation pattern of 2,3,4,6-tetramethyl- (4 mol), 3,4,6-trimethyl- (2 mol), 2,4-dimethyl- (2 mol), and 3,4-di-methylmannose (1mol). The reported values (1, 2) are 2,3,4,64etramethyl- (4.0 mol), 3,4,6-trimethyl- (2.1 mol), 2,4-dimethyl- (2.0 mol), and 3,4-dimethylmannose (1.0 mol). A trace of 2,4,6trimethylmannose (0.07 mol) was also reported, and a small amount of 2,3,4-trimethylmannose is found if the sample contains some of the homolog with an additional a l 4 - l i n k e d mannose unit from the outer chain. M, mannose: GNAc. N-acetvlducosamine. Alternative assignments(8) Alternative structures and residue E D C B Aa A@ E D C B A PPm

5.125 4.7104.925 5.235 A. aM+6aM+6aM-6/3M~4a/3GNA~

t' t3 t3

aM aM

4.770

4.868

5.035 5.335

5.085 5.110

t' (YM t'

5.289 5.045

CYM

B.

5.125 aM+6aM-6/3M+4a/3GNAc

t'

aM

t3

aM

4.710

t3

aM'caM

5.235

4.770

4.868

4.925 5.035 5.335

5.085 5.110

t' aM t'

5.289 5.045

aM

C. aM

L U M + ~ ~ M - ~ / ~ M + ~ C U ~ G N4.710 AC

t' t3 t3

5.235 5.125 4.770 5.335 5.035

aM

t' aM6+aM t' aM

4.868 5.085 5.110 4.925

5.289 5.045

phosphorylated with 1molar equivalent of diphenylphosphorochloridate in pyridine, a reaction that is expected to yield the 2,3-cyclic phenylphosphate (9). The reaction mixture was evaporated to remove pyridine, the residue was refluxed with concentrated ammonium hydroxide for 2 h to eliminate the phenyl group, and, after evaporation of the ammonia, the residue was heated for 2 h on a steam bath in50% acetic acid to remove the benzylidene group and open the cyclic phosphate. The product, which was expected to be a mixture of methylmannoside 2- and 3-phosphates, was desalted on a BioGel P-2 column to give a main fraction with the characteristics of a mixture of phosphate monoesters of methyl-a-D-mannoB pyranoside. Two H-1 NMR signals were seen at 64.73 and 4.86 in the ratio of 1:2 that we assign to the 2-phosphate and 3-phosphate derivatives of methyl-a-D-mannoside, assuming that the cyclic phosphate would open preferentially to give the equatorial 3-phosphate and that H-1 of the 2-phosphate wouldbe most deshielded. The 31P spectrum showedtwo C signals at 64.79 and 4.90 in a 1:2 ratio, and two signals of unequal intensities for the methyl aglycon appeared at about 63.40 in the proton spectrum, both results being consistent with the presence of the two isomeric phosphate esters. PPM The methylmannoside phosphate was hydrolyzed in 1 M FIG. 2. H-1NMR spectra of fragments from a-mannosidase trifluoroacetic acid at 100 "C for 2 h, and the product was digestion of neutral and phosphorylated mnnl mnn2 mnnlO compared by HPLC with authentic mannose 6-phosphate, oligosaccharides. A, neutral Man4GlcNAc produced by Oerskouia mannosidase digestion that is consistent with structure IV; B , man- using a 15-min gradient from 100 to 200 mM sodium acetate nosylphosphate derivative V produced by Oerskouia mannosidase in 100 mM NaOH followed by a 15-min gradient from 200 to digestion; C, phosphorylated MansGlcNAc produced by jack bean 500 mM sodium acetate in 100 mM NaOH on a Dionex BioLC mannosidase digestion. Assignments of signals are indicated on the carbohydrate system. Mannose 6-phosphate was eluted at figure, but the origin of the slight contaminant in B at 65.10 is 20.76 min (Fig. 4A), while the synthetic mixture gave peaks unknown. at 20.62,23.24,23.52, and 24.22 min (Fig. 4B). Whenmannose 6-phosphate was added to the mixture, it was eluted coinci-

11852


4.70

4 80

4 90

5.00

5 10

5 20

5 30

5.40

5 50 DPN

4.10

400

390

3.80

3 1 0

360

PPI4

4 . 10

4.00

3 90

3 DO

3 IO

3 60

PPM

FIG. 3. Two-dimensional homonuclear Hartmann-Hahn correlation spectra of Man4GlcNAcand Man-P-Man4GlcNAc. Left panel, the neutral oligosaccharide; right p a n e l , the phosphorylated oligosaccharide. H-1 signals are shown across the left, while signals for other sugar protons are along the top. Crosspeaks appearing at about 64.0-4.2 are H-2 signals of the various sugars and can be correlated with the H-1signals at about 64.7-5.5. A group of signals (in brackets) in the left panel at 63.8-3.9, correlated with an H-1signal at 64.9, is shifted to 64.08-4.18 in the right p a d , an expected result of substitution at position 6 by phosphate.

Manl-P

0

5

10

15 Time (rnin)

Man6-P

20

2

FIG. 4. Ion exchange separation of mannose phosphates. A, authentic mannose 1-phosphate and mannose 6-phosphate; B, acid hydrolysate (1 M trifluoroacetic acid, 100"C, 2 h) of methyl-a-Dmannopyranoside 2- and 3-phosphate; C, same as B except hydrolysis was for 4 h; D,acid hydrolysis of mnnl mnn2 mnnl0phosphorylated oligosaccharide as in B. The three peaks eluted at 23-25 min are assumed to be the 2-, 3-, and 4-phosphates of mannose.

dently with the first peak at 20.56 min. (Reproducibility of retention times was f0.2 min.) The four peaks were present in about equal amounts, but following a second acidic treatment under the same conditions, the peak at 20.56 min increased to 42% of thetotal (Fig. 4C), confirming that phosphate migration occurred slowly from the ring on to position 6. The mannose phosphate released by acid hydrolysis of oligosaccharide V under the above conditions gave a single hexose phosphate peak (Fig. 40) thatran coincidently with added mannose 6-phosphate. These results demonstrate that acid hydrolysis of a mannoside 6-phosphate under conditions used to hydrolyze glycoproteins will yield only mannose 6-phosphate, whereas hydrolysis of a mannoside with phosphate inposition 2 or 3 will yield a mixture of 4 isomers including some mannose 6phosphate. The presence of these other isomers proves that the phosphate initially occupied a position on the ring, since they cannotarise from mannose 6-phosphate. Conversely, the mere detection of mannose 6-phosphate by enzymatic analysis is equivocal proof for the presence of this structure in the glycoprotein because this isomer can be formed by phosphate migration from a ring position. Mass Spectral Fragmentation Patterns-Having demonstrated that the site of phosphorylation in the core of the mnnl mnn2 mnnlO oligosaccharide precludes addition of the outer chain to that position, we sought to locate the site of attachment of the outer chain from mass spectral fragment analysis of the neutral core fragment. Evidence has been presented that the ions of greatest abundance in liquid secondary ion mass spectrometry (LSIMS) of branched oligosaccharides are those that can result from single bond cleavages (13). This method, therefore, provides a way to distinguish between alternative isomeric oligosaccharides.Based on these observations, the fragment ions predicted for several alternatives for the mnnl mnn2 or mnnl mnn2 mnnlO oligosac-

Yeast OligosaccharideStructure

11853

charide are shown in Table I1 for negative mode LSIMS of allows discrimination between the 3 isomers in Table I1 by ABEE (25) and ABOE (14) derivatives. Of the isomers A, B, fast atom bombardment mass spectrometry of the acetylated and C, all of which fit the methylation and H-1 NMR data products (12). Thus, A should yield strong signals for gluco(Table I), only B fits the observed LSIMS pattern (Fig. 5), samine-containing fragmentsthat have lost 1,3, and5 hexose which shows most abundant ions for the loss of 1,2,4, and 9 units, but not 2 or 4, whereas B should give fragments that hexoses. Less abundant ions are seen for loss of 3, 5, and 6 have lost 1, 2, 3, 4, and 5 hexoses. The results in Table 111, hexoses, which ions may result from the presence of minor and particularly the abundance of the ions for loss of 2 and 4 isomeric components or from multiple bond cleavages. The hexoses, clearly support structure B. nonreducing-end fragments in greatest abundance contain 2 and 4 hexoses, a result consistentwith the predicted fragmenTABLEI1 tation pattern for isomer B. Major fragments expected in LSIMS Exo-al+2-mannosidase digestion of the neutral M, mannose; GNAc, N-acetylglucosamine. Man9GlcNAc oligosaccharide yields a ManeGlcNAc, which would have structure F if derived from isomer A, or structure Expected most abundant fragments' G, if derived from isomer B. The LSIMS pattern from this Oligosaccharide structure".b fragment shows major ions for loss of 1, 2, 3, and 6 hexoses M eD Type A (Fig. 6), which agrees with G, whereas F would show loss of Hex M-1Hex A. M-+6M-GM+6M+4GNA~ 1,2,4, and 6 hexoses. The major nonreducing-end (Type D) M - 2 Hex t' T3 t3 fragments formed have 2 and 3 hexoses, as predicted. Note Hex3 M - 3 Hex M M M Hex6 M-5Hex that isomer C is excluded because it would yield a t' 9 Hex M Man7GlcNAcwhen digested with this enzyme. M Support for isomer B as the structureof the core oligosact' M charide was also obtained by partial acetolysis. Treatment M - 1 Hex Hex B. with acetic anhydridelacetic acidlconcentrated sulfuric acid M+GM+6M+4GNAc Hexz M-2Hex t' t3 T3 (lOlO:l, v/v) at 40 "C for a limited time leads to theselective M-4Hex Hex, MM M'tM cleavage of a l a - l i n k a g e s in an oligosaccharide (26) and M - 9 Hex

t'

M

t' M I

M - 1 Hex

M+GM+6M+4GNAc C.

-4 Hex 1341

-1 Hex

t'

I

M

t3 T3

M

M

t'

MG+M

M - 2 Hex M-3Hex M-4Hex M - 9 Hex

Hex Hex' He%

T'

M D. M+6M+6M+6M+4GNAc 855

1011

T'

T'

M

M

t3 t3 M

M

T'

M

FIG. 5. LSIMS of mnnlmnn2mnnlO oligosaccharide ManeGlcNAc.The oligosaccharide was coupled under reducing conditions to p-aminobenzoic acid ethyl ester (25);the ABEE derivative was purified by weak anion exchange HPLC and analyzed by mass spectrometry in the negative mode (13). More abundant ions are correlated with fragments that can result from single bond cleavages. Type A fragments are from the reducing end andare identified with a minus sign to indicate the loss of hexose(s); whereas Type D fragments are from the nonreducing end and have the general structure nHex-0-CH=CHO-.

T'

E.

M M+6M+6M+4GNAc

f'

MM

t3 f3 MGtM t' tz M

B

4s

51 400

FIG. 6. LSIMS of MansGlcNAc obtained by exo-crl+2-mannosidase digestion of 11. The ABOE derivative (14) was prepared and D fragments are identified. and analyzed as in Fig. 5. Both Type A A strongsignal at m/z 777 would beexpected for structure F of Table 11, whereas the observed ions support structure G .

M - 1 Hex M-2Hex M-4Hex M-5Hex M - 10 Hex

T'

M F.M+6M+6M+GM+4GNAc

t3t3

I

M

M - 1 Hex M - 2 Hex M-3Hex M-4Hex M - 6 Hex M - 10 Hex

M

M

M+6M+6M+4GNAc G.

t3 t3 M

M6+M

M-1Hex M - 2 Hex M - 4 Hex M - 6 Hex M-1Hex M-2Hex M-3Hex M - 6 Hex

Hex Hex' Hexr Hex Hexl Hex3

" A-C represent alternative structures for the MansGlcNAc that are consistent with the methylation and one-dimensional NMR data (see Table I). D and E are alternative structures for the Man&lcNAc. F is the oligosaccharide expected from exo-al+2-mannosidase digestion of A and D, whereas G is expected from digestion of B and E. C is excluded because it would yield a Man&lcNAc instead of the observed Man~GlcNAc. e Assuming single bond cleavage (13). In Type Aions the charge is retained ona reducing-end fragment that carries the derivatized (ABEE or ABOE) N-acetylglucosamine. In Type D ions the charge is retained on a nonreducing-end fragment with the structure Hex0-CH=CHO-.

11854


TABLE111 Acetylated fragments from partialacetolysis of mnnl mnn2 core oligosaccharide (MansGlcNAc) Ion intensity

Fragment mass”

Structureb Time

of acetolysis 30 min

J

’

90 min

w 2 14 [M - 59]+ [M - 59]+ - 1 Hex 9 0 [M - 59]+ - 2 Hex 9 27 [M - 59]+ - 3 Hex 14 10 [M - 59]+ - 4 Hex 16 12 [M - 59]+ - 5 Hex 20 67 The peracetylated ManeGlcNAc has M,= 2981, and each acetylated hexose unit is equivalent to a mass of 288. The positive ion is formed by the loss of acetate ion from C-1 of the peracetylated GlcNAc unit.

2922 2634 2346 2058 1770 1482

Two-dimensional NOE Spectrum-To confirm other features of the new structure we propose for the mnnlmnn2 and mnnl mnn2 mnnlO eo;? oligosaccharide,we have established that it is also consistent with a two-dimensional NOE spectrum (17) that shows through-space interresidue connectivities between the H-1of one mannose and H-2 or H-3 protons of an adjacent hexose. The spectrum in Fig. 7 shows interresidue H-1-H-2’ connectivities between the mannoses with H1 signals at 65.045 and 65.289 (A-B*-C), and between the latter and amannose with an H-1signal at 65.335 (C-D*-E). Moreover, a weak interaction is seen between the mannose H-1 signal at 65.335 and thatfor H-2 of the /?-linkedmannose with an H-1 signal at 64.770 (E-F*-G).Finally, the a l 4 linked mannose with an H-1signal at 65.125 shows interresidue connectivity only to anunsubstituted al-2-linked mannose with an H-1 signal at 65.035 (H-I*-J).Thus, the structural features of the MamGlcNAc- oligosaccharide precursor are retained in these MangGlcNAc fragments. Structure of the ManloGlcNAcOligosaccharide from the mnnl mnn9 Mutant-The ‘H NMR spectrum of this oligosaccharide (Fig. 1)differs from structure I1 in that itlacks an H-1 signal at 64.925 for an unsubstituted a l a - l i n k e d mannose, and it shows two additional signals at 65.045/5.055 and 5.125 (5). Thesechanges result from substitution of the al+ 6-linked mannose in I1 at position 2 to give structure 111. In support of this conclusion, the LSIMS spectrum shows most abundant ions for the loss of 1, 2, 4, and 5 mannoses (Fig. 8 and Table 11). DISCUSSION

In the early studies on yeast “mannan,” methylation analysis demonstrated that the molecule was highlybranched and contained a142, a1-3, and a 1 4 linkages (27), and glycopeptides were isolated that suggested the polymer was a glycoprotein (28). Jones and Ballou (29) isolated a bacterial mannosidase that stripped all side chains from the mannan and yielded an a l 4 - l i n k e d polymannose chain, which established the backbone structure of the polysaccharide. When it was found (1) that yeast mannoprotein contained a “core” oligosaccharide by which the polymannose chain was linked to asparagine as in other eucaryotic glycoproteins, it was proposed t h a t t h e a l a - l i n k echain d extended directly to the /?lA-linked mannose attached to the chitobiose unit. This proposal was consistent with all available data, including subsequent analyses byhigh resolution ‘H NMR (2). Our present studies on the neutral and phosphorylated core oligosaccharides from the mnn mutantmannoproteins, however, have made the original structure untenable because one site

L 4.80

L 4.90

” 5.00

5.10

5.20

5.30

J

L”5.40

v PPM

rrn

FIG. 7. Partial two-dimensional NOE (NOESY)spectrum of the neutral core oligosaccharide ManoGlcNAc. The H-1 NMR spectrum is shown along the left side of the figure and a partial spectrum for ring protons along the top. EachH-1 signal is identified with a mannose unit of the oligosaccharide (2). Urntarredletters indicate intraresidue H-1-H-2 NOE cross-peaks andthe starred letters indicate interresidue H-1-H-2’ NOE cross-peaks. Interresidue cross-peaks due to dipolar coupling are identified by their absence in a two-dimensional correlation spectrum (not shown), which shows only the intraresidue cross-peaks due to J-coupling (17). The interresidue peak B* results from an NOE between H-1 of an aMan+’ unit and H-2 of a +‘aMan+’ unit, the intraresidue H-1-H-2 peaks being A and C, respectively. The interresidue peak D* results from an NOE between H-1 of a +‘aMan+’ unit andH-2 of a unit, whichgive intraresidue H-1-H-2 peaks C and E. The weak interresidue peak F*, due to an NOE between H-1 of a unit and H-2 of the +6PMan+4 unit, reflect the fact that these two

t3

mannoses are al+B-linked. These connectivities are consistent only with the structural unit c~Man+‘aMan~’aMan+~PMan+~.Consistent with this conclusion, the spectrum shows an interresidue cross-peak I* due to an NOE between an aMan+’ unit an aMan+‘ unit, as expected for a cYMan+6 side chain. Intraresiduepeaks for the

t’

aMan two aMan+* units (A and H) appear as anunresolved contour, and other unlabeled cross-peaks are for intraresidue H-1-H-2 and H-lH-3 NOE interactions.

of phosphorylation is the same as that to which the polymannose outer chain was thought to be linked. In considering alternative locations for attachment of the outer chain structureto thecore oligosaccharide, we find that only one position is consistent with the methylation data, ‘H NMR analysis, the results of exo-~~+2-mannosidasedigestion, and fragmentation analysis by LSIMS and by partial acetolysis. The results reported here and elsewhere (1, 2, 5) support the following representation of the N - and 0-linked carbohydrate components of these S. cereoisioe mannoproteins (Fig. 9). We have adopted an orientation of the chains

11855


I

'p

la63

-5

3 1101 -6 Hex

HEX

1425 -4 Hex

1581

-3 Her

FIG. 8. LSIMS of mnnl mnn9 oligosaccharide Man,oGlcNAc. The ABOE derivative (14) was prepared and analyzed in the negative mode (13). The parent ion is at m/z 2073, and the most abundant fragment ions correspond to the loss of 1, 2, 4, and 5 hexoses. Only Type A fragments are shown.

I M2M-[T "

M

~

M

~

M

-

~2~2~2"

FIG. 9. Schematic representation of the N - and 0-linked carbohydrate of S. cerevisiae mannoproteins inferred from studies on mutant and wild-type mannoproteins. Small oligomannosides linked to serine and threonine are characteristically labile to alkaline @-elimination.The N-linked polymannose chains consist of the ManaGlcNAcz-core, in block letters, to which the outer chain is attached. In the mnnl mnn9 mutant, theouter chain consists only of the two italicized mannose ( M ) units, whereas in the absence of the mnn2 defect the outer chain is extended and the single block italicized mannose is not added. Instead, an equivalent mannose unit is present at the nonreducing end of the outer chain (4). Branching of the outer chain is prevented by the mnn2 lesion, while the mnnl mutation eliminates all terminal al+3-linked mannoses in the core, outer chain, and on 0-linked oligosaccharides. GNAc, N-acetylglucosamine.

that facilitates comparison with the previously published structure (30), and the starred mannose is a site of phosphorylation that precludes addition of the outer chain. Two other sites of phosphorylation, in addition to that shown in the outer chain, have been identified and will be reported elsewhere.' In this model, the MansGlcNAcz- core is highlighted in block letters, whereas the two italicized mannose units define the extentof elongation of the outer chain inthe mnn9 mutant (5). In the mnn2 mutant, the block italicized mannose isnot added and an unbranchedouterchain is formed in which z is about 10. Our revised structure is different from that proposed by Trimbleand Atkinson (3), who support isomer I for the MangGlcNActhey contained from yeast invertase. A simple explanation for this difference could be that one type of

* Hernandez, L. M., Ballou, L., Alvarado, E., Tsai, P.-K., and Ballou, C. E. (1989) J. Biol. Chem. 264, in press.

structure is made in the mnn mutants we have studied and another in the wild-type strain they analyzed. As far as is known, however, the mnnl and mnn2 mutations affect only the branching of the outer chain and not the initiation or elongation steps. Moreover, there are striking similarities in the 'H NMR spectra for the oligosaccharides obtained in both laboratories. Although one can rationalize the same spectra to fit the different isomers, we would expect significant differences in the actual spectra of isomers I and I1 owing to subtle long range effects. Trimbleand Atkinson (3) have commented on the anomalous upfield shift of H-2 of the 8linked mannose on addition of the a l 4 - l i n k e d mannose to the MansGlcNAc core to yield the MangGlcNAc, an effect noted in both their andour preparation. In thespectra of the ManloGlcNAc,both we and they note that, upon addition of an al-2-linked mannose to this a14-linkedmannose, the newly formed H-1 signal for one of the terminal al-2-linked mannoses is split into two doublets centered at 65.045 and 5.055. This effect is due, apparently, to an interaction with the N-acetylglucosamine anomers. Although our mass spectra for the intact oligosaccharide show ions expected for both I and I1 and could reflect the presence of a mixture of isomers, the relative intensities clearly favor I1 as the preponderant form. Even more supportive of thisstructure is the mass spectrum for the MansGlcNAc fragment obtained by exo-al-2-mannosidase digestion of the Man&lcNAc, which spectrum is consistent only with the single structure we propose. Our new structure is engaging in that itsuggests a plausible explanation for the mnn9 mutant phen~type.~ When the outer chain was thought to be an extension of the a l 4 - l i n k e d backbone, it was difficult to visualize a mechanism to explain why the elongation reaction would stop with the addition of a single mannose unit. If initiation of the outer chaininvolves a mannosyltransferasethat is specific for addition to anal+ 3-linked mannose, however, then this step should be distinct from the elongation reaction in which the mannose acceptor is a n a l 4 - l i n k e dmannose. Consequently, one would predict the existence of mutants that would affect the two processes, initiation and elongation, independently. REFERENCES

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We thank Dr. Finn Wold for bringing this point to our attention.

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