THE JOURNAL 0 1984 by The American Society of Biological Chemists, Inc.

OF BIOLOGICAL CHEMISTRY Vol. 259, No. 6, Issue of March 25, pp. 3805-3811,1984 Printed in U. S. A.

Carbohydrate Structure of Saccharomyces cerevisiae mnn9 Mannoprotein"

(Received for publication, October 4, 1983)

Pei-Kuo Tsai, Jiirgen FrevertS, and Clinton E. Ballout From the Department of Biochemistry, University of California, Berkeley, California 94720

The neutral oligosaccharides from Saccharomyces cerevisiae mnnl mnn9, mnn2 mnn9, and mnn9 mutant mannoproteins, and from mnnl and wild type carbox- ypeptidase Y, have been characterized. The major oli- gosaccharide from the mnnl mnn9 mutant, ManloGlcNAc, has the structure

aMan+BaMan+6aMan+eflMan+4a,9GlcNAc

aMan aMan aMan aMan

aMan

&Man

t" t" T" t" t" ta

whereas the largest oligosaccharide from the mnn9 mutant, Man13GlcNAc, has the structure

aMan~saMan+BaM~+BflMan+'aflGlcNAc

aMan aMan aMan aMan

aMan aMan aMan aMan

aMan

t" T" t" t" t" t" t" t"

t'

the differences being due to the mnnl mutation. The smaller mnn9 homologs had lesser amounts of terminal nl+3-linked mannose and may be precursors of the mature oligosaccharide. The mnn2 mutation had no effect on the mnn9 oligosaccharide structures. Carbox- ypeptidase Y and mnn9 oligosaccharides were identi- cal, which suggests that the mnn9 mutation eliminates the differences in carbohydrate structure that distin- guish intra- from extracellular mannoproteins. One mnnl mnn9 oligosaccharide, ManllGlcNAc, retained the terminal al-2-linked mannose of the lipid-linked core precursor, which suggests that processing to give the larger oligosaccharides can occur without removal of this unit. A smaller mnnl mnn9 oligosaccharide, MansGlcNAc, was a mixture of isomers that must, in part, have arisen by action of an al-2-mannosidase.

Extracellular (secreted) mannoproteins in Saccharomyces cerevisiae are characterized by asparagine-linked polyman- nose chains that consist of a core oligosaccharide, with 8-13

PCM80-23388 and United States Public Health Service Grant AI- * This work was supported by National Science Foundation Grant

12522. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$Supported by a grant from the Deutsche Forschungsgemein- schaft.

3 To whom inquiries should be addressed.

mannose units, to which is attached an outer chain of 50 or more mannose units (1, 2). Through genetic manipulations, several mutants that affect side chain structure in the outer chain have been obtained (3-6), and none interferes with mannoprotein secretion or normal cellular functions.

A new class of mutant was isolated recently that is defective in polymerization of the outer chain backbone and that leads to a clumpy morphology and reduced cell viability (7). The most extensively affected of these mutants, designated mnn9, has now been studied in detail, and we show that its carbo- hydrate chains are altered in a way that eliminates all of the distinguishing difference between intracellular and extracel- lular mannoproteins. This result suggests that the outer chain part of the asparagine-linked carbohydrate of yeast manno- proteins has no obligatory role in determining their location in the cell; but the morphology of the mutants shows that the mannoproteins have an important role in cell-wall organiza- tion, particularly in the region of the septum where complex developmental changes occur during cell division (8).

In this paper, we describe the structures of the neutral oligosaccharides of the mnn9 mannoprotein based mainly on the anomeric proton NMR and fast atom bombardment mass spectra, and in a later paper structures of the phosphorylated oligosaccharides will be presented.' The analysis reveals sub- tle differences in carbohydrate structure between the classes of glycoprotein that may be related to the processing pathway in yeast (9).

EXPERIMENTAL PROCEDURES

Materials-All mutant yeast strains were derived from S. cereuisiae X2180 (Yeast Genetics Stock Center, University of California, Berke- ley CA) by treatment with ethylmethane sulfonate (3, 7). The mnnl strain lacks an c~1+3-mannosyltransferase activity that is involved in addition of terminal a1-3-linked mannose units in the core, outer chain and the oligosaccharides attached to serine and threonine (10, 11). The mnn2 strain has an unbranched outer chain of d 4 - l i n k e d mannose owing to a defective al+2-mannosyltransferase activity that is required for adding the first side chain unit to the backbone. The mnn7, mnn8, mnn9, and mnnlO mutants all have defects in elongation of the backbone, related in some way to an altered ability to form a 1 4 linkages.

The original isolation of the mnn9 mutant was done in the mnn2 background (7), and the mutant designated B15-50 was back-crossed to mnn2 strain LB1-3B to eliminate other possible mutations. The resultant mnn2 mnn9 strain was designated LB-302-4A(a) and LB- 302-1B(a) for the two mating types. The mnn2 mnn9 strain used in this study was LB-302-4A. To obtain the mnn9 single mutant, the latter strain was crossed to strain X2180 and the diploid was dissected to give LB-344-1B(a) and LB-347-1C(or). The a strain was used in this work. For construction of a mnnl mnn9 strain, LB-344-1B(a) was crossed to mnnl strain XW-451-3A(ol) and the recombinant double mutant was designated LB-361-1C(a). In the first report on this mutant class, preliminary analysis indicated that the mnn2 mnn8

P.-K. Tsai, J. Frevert, L. Ballou, and C. E. Ballou, manuscript in preparation.

3805

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3806 Yeast Mannoprotein Structure

(LB-303) and mnn2 mnnlO (LB-301) strains had shortened outer chains, but no analysis was available for the mnn2 mnn9 (B15-50) strain. In the present work, we show that the latter strain completely lacks the outer chain. None of the mutations has been shown to be in a structural transferase gene and, therefore, one or all may be of a regulatory nature. The carboxypeptidase Y oligosaccharides were from an earlier study (12) or were isolated by Dr. W. D. Sikkema in this laboratory.

Bio-Rad, while 100% DzO for NMR was from Aldrich. DEAE-Sepha- Materials for gel filtration and ion exchange were obtained from

cel was obtained from Pharmacia. A specific al-+2-mannosidase from Aspergillus saitoi (13) was provided by Dr. C. A. Dekker of this department and was a gift from Dr. A. Kobata, Department of Biochemistry, Kobe University, Kobe, Japan. Endo-N-acetyl-0-D- glucosaminidase H was purified from Streptomyces plicatus (14).

Methods-Carbohydrate was determined by the phenol/sulfuric acid reaction (15) and protein by absorbance at 280 nm. Thin layer chromatography was done on Whatman cellulose MN 300 sheets using ethyl acetate/pyridine/water (5:3:2, v/v) as solvent for mono- saccharides. Sugars were detected with the silver nitrate/sodium hydroxide dip reagent (16). The oligosaccharide core products (10 pg) were chromatographed on a Silica Gel G plate from Fisher. The plate was developed once in propranol/nitromethane/water (52.32.7, v/v) and once in propanol/nitromethane/water (5:2:3, v/v) (17). After chromatography, the plate was dried, sprayed with sulfuric acid, and heated at 105 "C for 20 min to detect the carbohydrate.

'H NMR was done according to Cohen and Ballou (18) on 200 and 250 MHz spectrometers in the Department of Chemistry, University of California, Berkeley, CA or on a 500 MHz spectrometer in the NMR Facility, Department of Chemistry, University of California, Davis, CA. Spectra were recorded at 40 "C, and the HOD signal was suppressed by a software program called "Losat" in the 500 MHz spectra. A pulse width of 90" was used with a sweep width of 2500

standard (62.217 at 40.0 "C). Hz. Chemical shifts are expressed relative to an internal acetone

Mass spectrometry was performed with a VG Analytical ZAB 1F High-Field Magnet mass spectrometer according to Dell and Ballou (19) in the Department of Biochemistry, Imperial College of Science and Technology, London, U. K. Samples of 1-10 pg were dissolved in 5% acetic acid and loaded into a drop of glycerol on the probe target. Thioglycerol was usually added to suppress the glycerol signals and enhance ion intensities. Ion masses were determined by counting signals on an oscillograph print-out of the spectrum.

Isolation of Mannoproteins-Mannoproteins were obtained from early stationary phase cells by hot citrate buffer extraction followed by precipitation of the mannoprotein first with methanol and then with cetyltrimethylammonium bromide (20). The cetyltrimethylam- monium bromide-precipitated fraction was further purified by chro- matography on DEAE-Sephacel, which was equilibrated with 50 mM Tris-HC1, pH 7.5. The column was eluted with a linear gradient of 0- 0.5 M NaCI, and the main carbohydrate peak was collected, dialyzed, and lyophilized.

Isolation and Fractionation of Oligosaccharides-Mannoprotein (160 mg) was digested at 37 "C under a drop of toluene by endoglu- cosaminidase H in 1 ml of 50 mM sodium citrate buffer, pH 5.6. After 48 h, the reaction was stopped by heating the tube for 3 min at 100 "C, and the tube was centrifuged. The supernatant layer was applied to a Bio-Gel P-4 (-400 mesh) column (2 X 195 cm) and eluted with water in 1.2-ml fractions. The individual carbohydrate peaks representing neutral core fragments were collected, concentrated, and rechromatographed on the same column to establish their homoge- neity.

Glycosidase Digestion-Oligosaccharides were incubated with al-+ 2-mannosidase (13) in 0.1 M citrate buffer, pH 5.0, a t 37 'C for 34 h under toluene. The reaction was terminated by heating the sample in a boiling water bath for 1 min, the tube was centrifuged and the supernatant solution was chromatographed on a Bio-Gel P-4 column to recover the oligosaccharide product.

RESULTS

Zsolation of Mannoprotein Oligosaccharides-Mannoprotein extracted from whole cells by hot citrate buffer contains a mixture of molecules, and the precipitation by cetyltrimeth- ylammonium bromide enriches for those with high mannose content (20). Some difficulty is experienced in precipitating the mnn9 mannoproteins because the carbohydrate content

is much lower than that in wild type mannoproteins. Because most of the mannoprotein is located in the cell wall (I), material prepared in this way is assumed to represent extra- cellular mannoprotein. External invertase represents a more well defined extracellular mannoprotein (21), and a repre- sentative intracellular mannoprotein is available in the vac- uolar carboxypeptidase Y (22). In some instances we have fractionated the citrate-extracted mannoproteins by the chro- matographic procedures used for invertase and carboxypep- tidase Y isolation, but no change in the heterogeneity of the carbohydrate component is observed. As shown below, the oligosaccharide component from pure carboxypeptidase Y is as heterogeneous as that prepared from the bulk cell-wall mannoprotein.

The asparagine-linked oligosaccharides of the denatured mannoproteins were released by endoglucosaminidase H digestion (14) and were fractionated by gel filtration in water. For the mnnl mnn9 mutant, the pattern showed one major neutral oligosaccharide (core) along with two minor compo- nents that were eluted on each side of the major one (core+l and core-1) (Fig. 1A). The mnn9 or mnn2 mnn9 strains gave more complex patterns (Fig. lB), but they were similar to each other, which suggested that the mnn2 mutation had no effect on oligosaccharide structure in the mnn9 background. Clearly, the mnnl defect does reduce the size and heteroge- neity of the core oligosaccharides, an observation already noted (2). The similarity in size distribution between the mnn9 extracellular mannoprotein oligosaccharides and the wild type carboxypeptidase Y oligosaccharides (Fig. IC) pro- vided the first clue that these compounds might be identical.

In the void volume of the Bio-Gel P-4 column eluted with

I80 260 2io 240 2M)

Fraction

-

FIG. 1. Fractionation of oligosaccharide preparations by gel filtration. The products released by endoglucosaminidase digestion were fractionated on a Bio-Gel P-4 column (2 X 190 cm) in water. The patterns are for oligosaccharides mnnl mnn9 (A) , mnn2 mnn9 or mnn9 ( E ) , wild type or mnn2 carboxypeptidase Y (C), and mnnl carboxypeptidase Y (D). The fraction size was 1.2 ml.

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Yeast Mannoprotein Structure 3807

water is found the protein part of the mannoprotein and any phosphorylated oligosaccharides. The latter can be recovered by gel filtration of this excluded material in 0.1 M ammonium acetate, under which conditions the phosphorylated com- pounds are included. After desalting, the acidic oligosaccha- rides are fractionated on a QAE (quaternary aminoethy1)- Sephadex column (23) according to the number and degree of esterification of the phosphate groups. Both mnnl mnn9 and mnn9 mannoproteins yield mono- and diphosphorylated oli- gosaccharides, and the structures of these compounds will be described elsewhere.'

Heterogeneity and Size of Isolated Oligosaccharides-Al- though gel filtration can separate neutral oligosaccharides that differ by single hexose units and can give a good approx- imation of size (24), we have also used thin layer chromatog- raphy (17) and fast atom bombardment mass spectrometry (19) in the present study. Fig. 2 shows the chromatographic separation of reference oligosaccharides and the patterns given by isolated mnnl mnn9 oligosaccharide homologs. The latter compounds give distinct bands and appear to differ from each other by one hexose unit. Lane 2 shows the largest species (ManllGlcNAc) and lane 6 the smallest (Mans- GlcNAc) detected in this preparation.

Fast atom bombardment mass spectrometry gives molecular weights to one mass unit and, with present instrumentation, is capable of analyzing oligosaccharides with 20 or more hexose units (19). In Table I are listed the neutral oligosac- charides we analyzed, along with the most characteristic quasi-molecular ions. In the positive mode, the most common ions for a reducing oligosaccharide are the protonated [M + H+]+ or cationized [M + Na+]+ or [M + K+]+ forms or the carbonium ion [M - OH-]', whereas in the negative mode one usually observes [M - H+]- or [M + C1-1- ions. Because fragmentation usually occurs with sequential loss of hexose units, the presence of lower molecular weight ions is not an indication of size heterogeneity unless an appropriate deriv- ative is used that will reveal such fragmentation (25). As shown in Table I, the major oligosaccharide from mnnl mnn9 mannoprotein is ManloGlcNAc; the minor components are Man9GlcNAc and ManllGlcNAc; whereas the mnn9 manno-

FIG. 2. Thin layer chromatography of mnnl mnn9 oligo- saccharides. Lanes I and 9, partial acid hydrolysate of Dextran T- 10; lane 2, core+l (ManllGlcNAc); lune 3, core (ManloGlcNAc); lane 4, core-1 (MangGlcNAc); lane 5, mixture of the three oligosaccha- rides; lane 6, core-2 (Man&lcNAc); lane 7, a 1 4 - g l u c a n standards, Gs to GIs; and lane 8, mnnl mnn9 core after digestion with a1-2- mannosidase (Man&lcNAc).

TABLE I Fast atom bombardment maw spectral data for oligosaccharides

Comuound" Observed ionb Structural assignment

mnnl mnn9 homologs Core+l (f) Core (e) Core-1 (d) a1-2-Mannosidase-

digested core mnn2 mnn9 homologs

Fraction A (a)' Fraction B (b) Fraction C (c) Fraction D (d)

Fraction 91-103 (b) Fraction 104-107 (c) Fraction 108-113 (d)

Carboxypeptidase Yd

2002 (-) 1840 (-) 1678 (-) 1192 (-)

2512 (+) 2350 (+) 2188 (+) 2026 (+)

2350 (+) 2188 (+) 2026 (+)

[Manl,GlcNAc - H+]- [ManloGlcNAc - H+]- [MangGlcNAc - H+]- [MansGlcNAc - H+]-

[Man14GlcNAc + Na+]+ [Man&lcNAc + Na+]+ [Man12GlcNAc + Na+]+ [ManllGlcNAc + Na+]+

[Man&lcNAc + Na+]+ [ManlZGlcNAc + Na+]+ fManllGlcNAc + Na+]+

Fractions were obtained by gel filtration on Bio-Gel P-4. Letters in parentheses correspond to peaks in Fig. 1.

*The sign in parentheses indicates whether the mass spectrum was determined in positive or negative mode.

Although the molecular weight agrees with ManlrGlcNAc, the H- 1 NMR spectrum indicates that one of the hexose units is probably glucose.

dFrom a previous study (12) in which a small oligosaccharide, eluted from the Bio-Gel P-4 column in Fractions 114-122, was shown to be a mixture of MangGlcNAc and ManloClcNAc.

protein gives oligosaccharides with 11-14 hexose units and carboxypeptidase Y gives oligosaccharides with 9-13 hexose units.

Structural Assignments of the mnnl mnn9 Oligosaccha- rides-Mannose H-1 NMR chemical shifts can be used for linkage and sequence assignments of glycoprotein high man- nose oligosaccharides (18, 26, 27). Fig. 3 shows some repre- sentative spectra, and in Table I1 are listed assignments for the compounds we have studied. A characteristic feature of the spectra is the unit ratio between the signals a t 64.770, 4.868,5.286, and 5.325, which reflects the common occurrence of the tetrasaccharide unit +'~uMan+'@Man+~ that is linked

t3 t3 t' t'

aMan

aMan

to N-acetylglucosamine. Any excess over one proton in the signal a t 65.286 is attributable to additional units of the type +'aMan+', whereas units with the sequence +'aMan+'

t3 T'

aMan

aMan give a signal a t 65.376 instead of 65.325 (18, 26). The signal in the region of 65.04 is for units of the type aMan+' or +3aMan+2, whereas the signal a t 65.125 is for a l 4 - l i n k e d mannose substituted in position 2 (aMan+' or +'aMan+').

aMan+' gives a signal at 64.92 when linked to a mannose substituted a t position 2, or at 64.90 when linked to a mannose substituted a t position 3. As an illustration, the structure of the major mnnl mnn9 core oligosaccharide is shown with the chemical shifts for each mannose unit.

5.125 5.125 4.868 4.770 aMan-6aMan-6aMan-6/3Man-4a/3GlcNAc 5.23, 4.67

aMan aMan &Man aMan 5.325 5.04 5.04 5.112 T'

5.087 aMan 5.286

t' t'

t2 T' t3 T3

T' aMan 5.04

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3808 Yeast Mannoprotein Structure

F II

, , , , ; 5.4 , , , , , 5.3 ;,,U!L 5.2 5.1 5.0 4.9 48

PPM FIG. 3. H-1 NMR spectra of purified oligosaccharides. A,

mnnl mnn9 core; B, mnnl mnn9 core+l; C, mnn2 mnn9 core fraction b; D, mnnl mnn9 core after exhaustive digestion with al-2-man- nosidase; E, reduced mnnl mnn9 core; and F, reduced al+2-man- nosidase-digested mnnl mnn9 core.

An interesting feature of the spectrum of the unreduced oligosaccharide is the split in the signal of the a1+3-linked mannose at 65.112/5.087, which is attributed to a long range influence exerted by the two anomeric forms of the N-acetyl- glucosamine (18, 26). After reduction, this mannose gives a

single signal at 65.112 (Fig. 3E). In addition, the signal at 65.325 moves upfield to 65.313, and the signal at 64.770 moves to 64.787, which indicates that these two neighboring ano- meric protons are also affected.

Confirmation of this structure for mnnl mnn9 oligosaccha- ride was obtained by digestion of the compound with an a1+ 2-mannosidase (13), which removed four mannose units and yielded a MansGlcNAc ( l a n e 8 of Fig. 2 and Table I). The NMR spectrum shows two protons centered at 64.90, two centered at 65.10, and one each at 64.87 and 64.78 (Fig. 30) . These are in agreement with assignments based on model compounds (18) and support the following structure.

aMan-+6aMan+6aMan+6flMan+4a@GlcNAc t3 f3

aMan aMan

After reduction, the signal at 65.10 shifted to 65.13 and the signal at 64.78 shifted to 64.80, indicating that there is less shielding by the reduced GlcNAc residue (Fig. 327).

The core-1 fraction from mnnl mnn9 mannoprotein (Man9GlcNAc) is a mixture of three isomers. The H-1 NMR spectrum shows two small signals at 64.90 and 64.92. From studies on reference compounds (18), the signal at 64.90 is assigned to isomer A and that at 64.92 to isomer B. The presence of isomer C is suggested by the reduced intensity of the signal at 65.287 and the shift in part of the signal at 65.325 to 65.336 due to removal of the terminal al+Z-Man.

aMan+6aMan+6aMan+6flMan+'aflGlcNAc

aMan aMan aMan

aMan

aMan

t' t3 t3 t' t'

ISOMER A

aMan+6aMan+6aMan+6flMan+6aflGlcNAc t' T3 t3

T'

t'

aMan aMan aMan

aMan

aMan ISOMER B

aMan-+6aMan+6aMan+6flMan+4aflGlcNAc

aMan aMan aMan aMan

aMan

tz t' f3 T3

t' ISOMER C

All of these isomers could have arisen from the major core oligosaccharide by mannosidase attack during isolation, but we determined by a long term digestion of the mnnl mnn9 Man,,GlcNAc with a large excess of endoglucosaminidase H that such degradation did not occur. Thus, these isomers appear to be normal processing intermediates.

Our studies suggest that the core+l component (Manll- GlcNAc) of the mnnl mnn9 mannoprotein has the following structure. The oligosaccharide is unusual in that it

aMan+6aMan+6aMan+6PMan+4aflGlcNA~

aMan aMan aMan aMan

aMan aMan

aMan

t' t' T3 f3

t' t'

rz

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Yeast Mannoprotein Structure 3809

TABLE I1 Oligosaccharide H-I NMR assignments

Linkage assignments and integrations'

oligosaccharide Mannoprotein

sourceb

mnnl mnn2 homologsb Small Large

rnnnZg mnnl mnn9 homologs

Core-1 (f) Core (e) Core+l (d)

Fraction D (d) Fraction C (c) Fraction B (b) Fraction A (a)h

mnn2 mnn9 homologs

mnn2 mnngg mnn2 mnn9 invertas@ mnnY carboxypeptidase homologs

ManloGlcNAc (e) ManllGlcNAc (d) ManlzGlcNAc (c) Manl,GlcNAc (b)

MangGlcNAc (f) Man,,GlcNAc (e)

Carboxypeptidase mnnl homologs

mnn2 mnnl0' cul+2-Mannosidase-digested

mnnl mnn9 core

aMan+' aMan aMan+'

64.77

1 1 1 1 1 1 1

64.87 64.9014.92'

1 2 1

0.210.2 0 0

0 0 0 0 0 0 0

0.3 0.3 0.3 0

0.610 0

13

2

65.04d 65.14' 65.29 65.3315.38'

2 2 1 110 2 2 1 110 2 3.5 1 1 10

2.8 3 4

3 2.75

3.3 3.4 3 3 3

2.5 3 2.7 3

2.3 3 2

4.24 5 5.6 5.6 4.7 4.3 4.3

2.8 3.8 4.6 5.7

0.44 1 1

1 1 1.2 1.1 1 1 1

2.5 2.5 0.6 110 3 3 1.1 110 3 4 1 110

0 2 0 010

Values were rounded off to whole integers when within 0.1 of the number shown. Letters in parentheses refer to oligosaccharide peaks in Fig. 1. The mnnl mnn2 homologs were obtained by

gel filtration on Bio-Gel P-4 (Ref. 2, Fig. 5). ' The signal is at 64.92 when terminal and attached to a mannose substituted at position 2, but it is at 64.90

when attached to a mannose substituted at position 3 or when in a chain of a1A-linked mannose otherwise unsubstituted.

The values are for integration over the region from 65.01 to 5.07. 'The values are for integration over the region 65.07 to 5.17. In some spectra this region is resolved into a

signal at 65.124, which we assign to aMan+', +'aMan+'j, and +3~Man+3, and one at 65.15 which we assign to t' T'

aMan aMan aMar~-~. In the mnnl mnn9 mutant, which lacks most of the terminal al-3-linked mannose, the signal at 65.14 is small, whereas the mnn2 mnn2 mutant oligosaccharide preparation can be resolved into homologs in which the forms of increasing size show an increasingly intense signal at 65.14. If the unit aMan+'aMan+'aMan+' is present, a signal at 65.12 is shifted to 65.10. ' H-1 signal of this mannose resonates at 65.325 if attached to the 8-linked mannose and at 65.376 when attached to an a1A-linked mannose.

Unfractionated mixture of oligosaccharides obtained by endoglucosaminidase H digestion of the mannoprotein. For isolation of mnn2 core, endo-al4-mannanase digestion is also required.

Although fractions A and B have similar H-1 NMR spectra, they differ in size by one hexose unit (Table I ) . Thus, A contains another sugar in unidentified linkage that is probably accounted for by small signals a t 65.520 (0.4), 65.265 (0.23), and 65.168 (0.15). The splitting of these signals suggests that the sugar could be a-glucose.

still possesses the unit +'aMan+', identified by the signal

aMan

aMan

at 65.376 (Fig. 3B), that is characteristic of the lipid-linked core precursor (28). This finding contradicts a recent proposal (29) that removal of this terminal al-&-linked mannose is essential before enlargement of the core can occur.

t3 t2

Structural Assignments of the mnn9, mnn2 mnn9 Manrw- protein and Wild Type Carboxypeptidase Y Oligosacchrides- The neutral oligosaccharides from the extracellular mnn2 mnn9 or mnn9 mutant mannoproteins lack a typical outer chain but are still several mannoses larger than the core fragment from the mnn2 mannoprotein (Table 11) (2). Thus, the mnn9 mutation interferes with the synthesis of the outer chain but it does allow enlargement of the core by a process that is not prevented by the mnn2 mutation. Although the

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3810 Yeast Mannoprotein Structure

presence or absence of the mnn2 lesion has no effect on the structure of these oligosaccharides, it is apparent that the absence of the mnnl defect allows the synthesis of larger carbohydrate chains. The increased intensity of the signal at 65.14, without an increased intensity at the 65.04 region, demonstrates that this is a consequence of additional mannose in a1+3 linkage. Although we are unable to assign these mannose units to specific locations in the ManllGlcNAc and ManlzGlcNAc, we assume the latter represent biosynthetic intermediates in the formation of the Man13GlcNAc of mnn9 (fraction B) to which we assign the following structure.

aMan+6aMan+6aMan+6pMan+4a@GlcNAc

aMan aMan aMan aMan

aMan aMan aMan aMan

aMan

t' t' t3 t3 t3 t3 t3 t'

t'

A mnn9 oligosaccharide (Table I, fraction A) with one hexose more than Man13GlcNAc, but with mannose H-1 sig- nals identical to fraction B (Table 11), showed additional small signals at 65.52, 5.26, and 5.17 that together total one proton. This compound appears to contain a derivative of Man13GlcNAc with glucose units attached in three different linkages.

As with the mnn9 structures, the intracellular carboxypep- tidase Y neutral oligosaccharides also form a homologous series. H-1 NMR spectra for these oligosaccharides are very similar to those for the mnn9 mutant, with the exception that some of the smaller homologs show signals for fractions of a proton centered at 64.91. This is attributed to a small amount of unsubstituted backbone mannose in a 1 4 linkage, and we conclude that these small oligosaccharides are precursors of the Man13GlcNAc, which itself appears to be identical with the mnn9 oligosaccharide of the same size. We presume that similar precursors of the mnn9 oligosaccharide must exist but are not always detectable. The mnnl mutation reduced the average size of the carboxypeptidase Y oligosaccharides (Fig. ID, Table 11), and the most abundant species had an NMR spectrum identical with that of the mnnl mnn9 core. The smallest core had a spectrum like the mnnl mnn9 core-1, but the single signal in the region 64.9 indicates that it is not a mixture of isomers.

All mature mannoprotein oligosaccharides occasionally contain a slight excess of mannose in +'aMan+* linkage (65.286), and we conclude that this represents the elongations of some of the aMan+2aMan-6 units to aMan+2aMan-2 aMan-t6. Such side chains can then accept c~Man+~ units to give the typical outer chain tetrasaccharide unit aMand3 aMan+2aMan+2aMan+6 (2).

DISCUSSION

All of the oligosaccharides we studied show single H-1 resonances at 64.77, 4.87, and 5.33, which are correlated in the presence of each of the trimannosyl units linked to N- acetylglucosamine. That these signals are found in a 1:l:l ratio rules out the presence elsewhere in the oligosaccharides of additional 2-substituted al-3-linked mannose. This is important in view of the published structure of the mammal- ian dolichol-linked oligosaccharide precursor (28), which con- tains 2 al-3-linked mannoses that are substituted at position 2 and gives two signals above 65.30 (18). One of these linkages (the one at lowest field) is removed at an early stage in the processing of the core unit (6,29), but our results suggest that this is not an obligatory modification (see below).

The H-1 signal at 64.9 is given by a l 4 - l i n k e d man- nose not substituted in position 2, 3, or 4 (aMan+' or +6aMan+6), and it is always found in oligosaccharides pro- duced from mnn2 extracellular mannoproteins by endoman- nanase/endoglucosaminidase digestion because the endom- annanase is unable to remove all of the outer chain (30). Fractions of a proton at this position are also seen in mnnl mnn9 core-1 and in some of the carboxypeptidase Y oligo- saccharides, however, and we assume that these are interme- diates in the biosynthesis of the largest homolog, which itself lacks a signal centered at 64.90.

The H-1 signal at 65.29 is due to an al-2-linked mannose substituted in position 2, and normally only one such mannose is observed in these oligosaccharides. This signal is charac- teristic of the mannotetraose linked to N-acetylglucosamine, and only in the larger homologs does it exceed one proton due to elongation of some side chains to form an outer chainlike structure.

The greatest ambiguity in assignments concerns the signals at 65.04 and 5.135. The signal at 65.04 is for a terminal a1+ 2-linked mannose or an al-2-linked mannose substituted at position 3. There should always be at least 2 mannose units in a1-2 linkage because 2-substitution is required to give the signals at 65.289 and 5.325. If the signal at 65.04 exceeds one and those at 65.289 and 5.325 equal only one each, the excess in intensity at 65.04 is due to mannose in a1+2 linkage to an a l 4 - l i n k e d mannose. Therefore, this signal is a marker for the formation of additional side chains on the a l 4 - l i n k e d backbone.

The signal centered at 65.135 in reduced oligosaccharides is a composite of terminal al-3-linked mannose, al-3-linked mannose substituted at position 3, and a l 4 - l i n k e d mannose substituted at position 2. The number of protons at this position due to units of the last type can be inferred after assignment of the signal at the 65.04 region is discussed above. Any remaining signals at 65.135 must represent the two types of al+S-linked mannose and +3aMan-3). In un- reduced oligosaccharides, a fraction of this signal is shifted to 65.112/5.087 owing to interaction with the N-acetylglucosa- mine unit (18, 26).

The mnn9 mutation in S. cereuisiae (7) leads to the secre- tion of mannoproteins that are indistinguishable in carbohy- drate structure from the intracellular carboxypeptidase Y. Thus, this mutation in some way interferes with the process- ing step by which the outer chain is added to the core oligo- saccharide. It is clear, however, that some enlargement of the core precursor does occur even on the mannoproteins that remain inside the cell.

Structural studies on the mnn2 mannoprotein (2) and la- beling studies (29) earlier suggested that the initial processing of the core oligosaccharide proceeds as shown in Scheme 1. Thus, removal of the 3 glucose units and 1 mannose yields a MansGlcNAc unit, and enlargement of this unit proceeds by addition of a single a l 4 - l i n k e d mannose, which is then substituted by mannose at position 2. Finally, each of the short side chains is substituted by mannose at position 3 to yield the mature oligosaccharide.

aMan+6aMan+6aMan+6aMan+4~Gl~NA~

aMan aMan aMan aMan

aMan aMan aMan aMan

aMan

t' t' t3 T3 t3 t3 t3 T'

t'

The order in which these latter steps occur is still undefined,

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Yeast Mannoprotein Structure

aMan+6aMan+6,9Man+4,9GlcNAc+ aMan+6aMan+6fiMan+4pGkNAc+

aMan aMan aMan + aMan aMan aMan

aMan aMan aMan

aGIc+* aGlc +3 aGlc +'aMan 1 a L n

c~Man+~aMan-+~aMan+~,9Man+~flGlcNAc+ c~Man-*'aMan+~c~Man+'flMan+~GlcNAc-,

aMan aMan aMan aMan aMan aMan aMan

t' t3 t3 t' t3 t3 t' t' t'

t'

t' t' t3 t3 t' t3 t3 P' + tZ

3811

aMan

aMan t'

SCHEME 1

but the fact that the oligosaccharides of intermediate size are heterogeneous in structure indicates that the process could be random. The H-1 NMR spectra also show that some of the mature molecules may have an additional al+2-linked man- nose added to one of the aMan+2aMan+6 units. It has been proposed (29) that the MamGlcNAc unit is an essential intermediate in all of these processing steps, but the fact that one of the larger oligosaccharides from the mnnl mnn9 mu- tant still retains the ~~Man+~aMan+~aMan+~ unit shows that this viewpoint is untenable.

The major oligosaccharide made in the mnnl mnn9 mutant has the structure

aMar~+~aMan+~aMan+~flMan+~flGlcNAc

aMan aMan aMan aMan

aMan

aMan

T' t' t3 t3 t' t'

which shows that the mnnl mutation prevents addition of al+3-linked mannose to mannose in both a1+2 and a1-3 linkages. This mutation also affects addition of a1-3-linked mannose to mannosylphosphate and mannotriose units in the outer chain and to mannotriose units attached to serine and threonine (2), and we now find that it also affects the carbox- ypeptidase Y oligosaccharides.

A minor component in the mnnl mnn9 oligosaccharide preparation had the structure MangGlcNAc and was a mixture of three isomers, two of which must have resulted from mannosidase action. In the synthesis of complex type glyco- proteins, a-mannosidases from animal sources have been described that remove the al-2-linked mannose residues from MangGlcNAc (31, 32), and there is some specificity to the order in which the mannoses are removed (33). Similar activities have not been reported in yeast.

Although the outer chain is characteristic of extracellular mannoproteins, only a few of the 9 carbohydrate chains on secreted invertase subunits are so modified (34, 35), perhaps only those on the surface of the folded glycoprotein (36). Whatever the function of the outer chain, the clumpy mor- phology of the mnn9 mutant suggests that this part of the mannoprotein has a role in wall organization. Since mnnl mnn9 mannoproteins possess chains that are 2 mannose units larger than the MamGlcNAc precursor, it appears that the defect is not in initiation of the outer chain but in its elon- gation. Because the outer chain is formed by the sequential addition of single mannose units (37), it is not obvious what the defect could be. Perhaps additional processing of the mnn9

&an t'

aMan

carbohydrate chain is required to act as a signal for completion of the outer chain. Alternatively, the mnn9 defect may divert the mannoprotein chains from the normal route so that the molecules bypass the compartment in which the polysaccha- ride chains of secreted mannoproteins are assembled.

2. 1.

3.

4.

5. 6.

7.

8.

9.

10.

11. 12.

13.

14.

15.

16. 17.

18. 19. 20. 21. 22.

23. 24. 25.

26.

27. 28. 29.

30.

31.

32. 33. 34.

35.

36.

37.

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P K Tsai, J Frevert and C E BallouCarbohydrate structure of Saccharomyces cerevisiae mnn9 mannoprotein.

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