Lun BallouS, Eugenio AlvaradoS, Pei-kuo TsaiSS, Anne Dellll, and Clinton E. BallouS)) ...... Barber, M., Bordoli, R. S., Sedgwick, R. D., and Tyler, A. N.. 12. Dell ...
Vol. 264, No. 20, Issue of July 15, pp. 11857-11864,1989 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, InC.
Protein Glycosylation Defects in the Saccharomyces cereuisiae mnn7 Mutant Class SUPPORT FOR THE STOP
SIGNAL PROPOSED FOR REGULATION OF OUTER CHAIN ELONGATION* (Received for publication, June 14, 1988)
Lun BallouS, Eugenio AlvaradoS, Pei-kuo TsaiSS, AnneDellll, and Clinton E. BallouS)) From the $Departmentof Biochemistry, Universityof California, Berkeley, California 94720 and the TDepartment of Biochemistry, ImperialCollege, London S W7, Great Britain
Total cell mannoprotein was isolated from Saccharomyces cerevisiae X2180 mutants that have defects in elongation of the outer chain attached to the Nlinked core oligosaccharides (mnn7, mnn8, mnn9, and mnnl0) (Ballou, L., Cohen, R. E., and Ballou, C. E. (1980) J. Biol. Chem. 255, 5986-5991). Comparison of the oligosaccharides released by endoglucosaminidase H digestion confirmed that the mnn9 mutation eliminates all but two mannoses of the outer chain, whereas the mnn8 and mnnlO strains produce outer chains of variable but similar lengths. The isolate designated mnn7 was found to be allelic with mnn8.Haploid mutants of the type mnn8 mnn9 or mnn9 mnnlO had the mnn9 phenotype, which established that the mnn9 defect is dominant and presumably acts ata processing step prior to the steps affected by mnn8 and mnnl0. Analysis of the mnnl mnn2 mnnlO oligosaccharides revealed that the heterogeneous outer chain contained 6-16 al+G-linked mannose units and each was terminated by asingle al42-linked mannose unit, whereas the core lacked one such unit that was present in the mnn9 oligosaccharide. The results are consistent with andsupport the hypothesis (Gopal, P. K., and Ballou, C. E. (1988) Proc. Natl.Acad. Sci. U.S. A. 84, 8824-8828) that addition of such a side-chain mannose unit is associated with termination of outer chain elongation in these mutants and may serve as a stop signal that regulates outer chain synthesis in the parent wildtype strain.
strated that all but two mannoses of the outer chain were lacking (3). Although the position of attachment of the outer chain to the ManeGlcNAcz core oligosaccharide has been revised to accommodate recent structural studies (4),the interpretation of the effects of these mutations is unchanged because they only affect the synthesis of the outer chain. Further studies on the mutants, reported herein, have revealed that mnn7 and mnn8 are allelic, whereas mnn8, mnn9, and mnnlO complement each other and must represent different alleles. Investigation of the N-linked oligosaccharides from the mnn8 and mnnlO mutants has confirmed that they have shortened outer chains that vary from 6 to 16 mannose units, compared to 50 or more in the wild type. Further analysis of the mnnlO oligosaccharide established that the outer chain is terminated by a single al+2-linked mannose, which may serve as a stop signal for regulating the elongation process as postulated by Gopal and Ballou (5). The structural relationships suggest that themnn9 mutant must act at an earlier step than mnn8 or mnnlO and should, therefore, be dominant to them. Analysis of the mannoproteins made by the various haploid double mutants confirmed this expectation. We believe that the explanation for the phenotypes displayed by this mutant class will be found in defects that affect the logistics of the processing pathway. EXPERIMENTALPROCEDURES
Yeast Strains-The mnn7 class of mutant (1) was derived from mutagenesis of the mnn2-2 MAT a strain with ethylmethane sulfonate, and the isolates were identified by a reduced ability to react with rabbit antiserum directed against the unbranched a l 4 - l i n k e d We have described a new class of nonconditional Sacchu- mannose backbone of the latterstrain (6,7). Themnn7 mutant came from strain LB328, the mnn8 mutant from strain LB303, the mnn9 romycescerevisiae mutants, with defects in mannoprotein mutant from strain LB302, and themnnlO mutant from strain LB301 glycosylation, that share the phenotype of growing with a (1) (Table I). The four mutants all showed an increased ability to clumpy morphology owing to incomplete cell separation (1). react with a1+3-mannosyl-specific antiserum (7) owing to exposure These strains, designated mnn7, mnn8, mnn9, and mnnl0, of the core after removal of the outer chain, and they gained the were obtained by mutagenesis of the mnn2 strain, a mutant ability to bind alcian blue dye (8) owing to an increased content or that makes mannoproteins with an unbranched al-&-linked accessibility of mannosylphosphate groups in the mannoprotein. All the strains retained the mnn2 defect, although the effect of this outer chain (2), and these mutants all make mannoproteins of mutation was expressed only in the mnn7, mnn8, and mnnlO strains, that possess a truncated backbone as though they carry de- which still contained short sections of the outer chain that became fects in N-linked oligosaccharide outer chainelongation. Sub- branched in the absence of the mnn2 lesion. Strains containing the mnnl mutation (defective in al-&rnansequent characterization of the endoglucosaminidase H-released oligosaccharide fraction from the mnn9 strain demon- nosyltransferase) (9) were constructed by crossing a strain of the genotype mnnl mnn2 with, for example, mnn2 mnn9 and selecting for recombinant haploids from the sporulated diploid that were * This work was supported by National Science Foundation Grant clumpy (mnn9 phenotype) and failed to agglutinate with antiserum against al-3-linked mannose (mnnl phenotype). Such a recombiPCM87-03141 and United States Public Health Service Grant AI12522. The costs of publication of this article were defrayed in part nant should have the genotype mnnl mnn2 mnn9, and this was by the payment of page charges. This article must therefore be hereby confirmed by backcrossing it to the mnn2 strain and recovering the marked “aduertisement”in accordance with 18 U.S.C. Section 1734 expected double mutants (mnnl mnn2 and mnn2 mnn9) from the solely to indicate this fact. sporulated diploid. In a similar fashion, a cross between the mnnl § Present address: Syntro Corporation, San Diego, CA 92121. mnn2 mnn7 and mnnl mnn2 mnn9 strains gave a diploid that, (1 To whom correspondence should be addressed. following sporulation and dissection, yielded some nonparental di-
11857
Protein Yeast
11858
Glycosylation Mutants
TABLEI Saccharomyces cerevisiaestrains used in this study Strain
Genotype
S. cerevisiae X2180" LB1-3Bb LB1-33B' LB328-lBd LB303-lBd LB302-4A LB301-2D LB394-3A LB393-2A LB1437-2B LB1409-1C LB1414-12A LB1415-16D LB1418-1A
Wild-type MATa mnn2 MATa mnnl mnn2 MATa mnn2 mnn7 MATa mnn2 mnn8 MATa mnn2 mnn9 MATa mnn2 mnnlO MATa mnnl mnn2 mnn7 MATa mnnl mnn2 mnn8 MATa mnnl mnn2 mnn9 MATa mnnl mnn2 mnnlO MATa mnnl mnn2 mnn7 mnn9 MATa mnnl mnn2 mnn8 mnn9 MATa mnnl mnn2 mnn9 mnnlO MATa "Mutagenized for original isolation of mnnl and mnn2 mutants (6). Mutagenized for isolation of mnn7 class of mutants (1). e Used for crossing mnnl mutation into mnn7class mutants. These two isolates proved to be allelic. types with two clumpy (mnnl mnn2 mnn7 mnn9) andtwo nonclumpy (mnnl mnn2) clones. The genotypes of such quadruple mnn mutants were checked by backcrosses to the mnnlmnn2 strain, with nonparental ditype recombinants showing four clumpy clones owing to the independent segregation of the mnn7 andmnn9 lesions. Subsequent studies, reported here, revealed that mnn7 and mnn8 were allelic in contradiction to the earlier report ( l ) , so further studies have been done with the mnn8 strain. The strainsused in this study are listed in Table I. Since the "wild-type" strain used in these studies had the mnnl mnn2 genotype, complementation between members of the mnn7 class in the diplophase led to the mnnl mnn2 phenotype. This phenotype was recognized by an enhanced reactivity of whole cells with an a14-mannosyl-specificantiserum (7),by a reduced binding of alcian blue dye (8), and by a reduced rate of migration on gel electrophoresis of the external invertase (lo), all consequences of the increased size of the N-linked polymannose chains. Independent segregation of the complementing mutations was demonstrated by the recoveryof mnnl mnn2 haploid clones after sporulation and dissection of the asci. Oligosaccharide Isolation-The yeast strains were grown at 30 "C on YEPD (1%yeast extract, 2% peptone, and 2% dextrose) in 2-liter Fernback flasks on a rotary shaker for 48 h. The cells, collected by centrifugation, were washed andthen extracted with hotcitrate buffer, the mannoprotein was precipitated from thesupernatant extract by adding methanol, the crude mannoprotein was purified by Cetavlon precipitation followed by chromatography on DEAE-Sephacel, and the carbohydrate peak eluted with salt was collected, dialyzed, and lyophilized (3). The pure mannoprotein was digested with endoglucosaminidase H, and the digest was fractionated on a Bio-Gel P10 column in saltfollowed by a Bio-Gel P-4 column eluted with water (3). Fractionation of the neutral oligosaccharides was carried out on the Dionex BioLC carbohydrate system by elution with a gradient of 50-200 mM acetate for 30 min in 100 mM NaOH. Methods-Procedures for determination of carbohydrate and protein and for paper and thinlayer chromatography of oligosaccharides have been described (3). Native gel electrophoresis of invertases was done on 7% gels, without a stacking gel, at 10 mA constant current. The running buffer was Tris borate pH 7.45, prepared by dissolving 5 gof boric acid and 1 g of Tris base in 1liter of water. The invertase bands were detected with an activity stain (9). To induce external invertase formation, cells were cultured in YEPD for 48 h by which time all of the glucose was consumed and invertase synthesis and secretion had occurred spontaneously. Fast atom bombardment mass spectrometry (11) was carried out on underivatized (12), peracetylated (13) or permethylated oligosaccharides (14) on a VG Analytical ZAB 1F high-field magnet mass spectrometer in the Department of Biochemistry, Imperial College, London, United Kingdom. 'H NMR was done at 40 "C in 100% DzO, referenced to acetone at 82.217 (15), on a 500 MHz spectrometer in the NMR facility, Department of Chemistry, University of California, Berkeley, CA (3, 15, 16). Methylation analysis was done according to Lee and Ballou (17).
Endo-al&+mannanase Digestion-This endomannanase was obtained from a soil bacterium isolated by enrichment culture on mnnl mnn2 mannoprotein, and the enzyme degrades the unsubstituted a l a - l i n k e d polymannose outer chain to mannose and mannobiose, with intermediate formation of fragments with 3-8 mannose units (18). The enzyme is unable to cleave next to a 2-substituted mannose, so it leaves one or more unsubstituted a 1 4 - l i n k e d mannose units at the reducing or nonreducing end of a branch-point, depending on the position of initial cleavage of the chain (19). For digestion of the mnnl mnn2 mnnIO oligosaccharide, 45 mg of M citrate/phosphate carbohydrate wasdissolved in 0.5mlof0.1 buffer, pH 7.0, containing 1mM CaC12. The endomannanase, 4.6 units in 0.8 mlof 1 mM potassium phosphate buffer, pH 7.0, was added and the reaction was incubated at 50 "C for 22 h. Additional enzyme was added (1.1 units) and the incubation was continued for 6.5 h, when it was terminated by heating the reaction in boiling water for 3 min. The cooled solution was centrifuged and the clear liquid fractionated on a Bio-Gel P-4 (-400 mesh) column (2 X 180 cm) by elution with water. Fractions of 1 ml were collected. Exo-al+2-mannosidase Digestion-This mannosidase was obtained from a strain of Aspergillus (20), and oligosaccharides were digested at 30 "C in 0.1 M sodium acetate buffer, pH 5. After release of reducing sugar ceased, the reaction wasboiled, centrifuged to remove any precipitate, and fractionated on a Bio-Gel P-4 column to separate mannose from larger fragments. RESULTS
Allelism of mnn7 and mnn8 Strains-At the time of their selection, the mnn7 (LB328-1B) and mnn8 isolates (LB3031B) appeared to be nonallelic by complementation of the mutant phenotypes in the heterozygous diploid as revealed by the reaction with mnn2 antiserum and with alcian blue dye (1).Subsequently, after additional backcrosses, a more detailed analysis of the presumed complementation has indicated that mnn7 and mnn8 are allelic (data not reported). Therefore, in the studies reported here we have confined the analyses tothe mnn8, mnn9and mnnlO strainsand,to simplify the structural comparisons, these mutations are superimposed on a background of the mnnlmnn2 mutationsso that theouter chain portion remains unbranched andthe core lacks terminal al+3-linked mannose. N-Linked Oligosaccharide Comparison-Mannoprotein, isolated by citrate buffer extraction of whole cells and purified by Cetavlon precipitation and DEAE-Sephacel column chromatography, was digested with endoglucosaminidase H. The released oligosaccharide fraction was isolated by gel filtration on a Bio-Gel P-10 column by elution with 0.1 M ammonium acetate, pH 7.0, and the included oligosaccharides were rechromatographed on Bio-Gel P-4 by elution with water. Allof the crude extractedmannoproteins gave a major carbohydrate-containing and ultraviolet-absorbing peak on the DEAE-Sephacel column that was eluted at low salt concentration (Fig. 1, peak B ) , whereas about one-third of the carbohydrate in the extract failed to bind to the column and was discarded (peak A). The majority of the protein in the extract was eluted at still higher salt concentration. Gel filtration of the endoglucosaminidase H digest of the mannoprotein fraction B gave the pattern shown in Fig. 2, the first peak corresponding to residual glycoprotein with 0linked carbohydrate and the second peak to the released oligosaccharides. Fractionation of the released oligosaccharides (peak B ) on a Bio-Gel P-4 column by elution with water separated an acidic phosphorylated fraction from the neutral oligosaccharides (Fig. 3). For comparison, the recovered neutral oligosaccharide fractions were rerun on a Bio-Gel P-4 column (Fig. 4),which patterns reveal distinct size differences between the mnn2, mnn9 and mnnlO mannoproteins. The mnnl mnn2 mnn9 strain yielded an oligosaccharide peak on the P-4 column that contained predominantly one homolog, ManloGlcNAc,as shown previously by gel filtration
Protein Yeast
Glycosylation Mutants
.!+
on Bio-Gel P-4 and by fast atom bombardment mass spectrometry (3). Its structure has been defined by NMR spectroscopy, mass spectrometry, and chemical and enzymatic analysis to be that shown here (4). aM
aM
'2
6 '3
a"+
11859
1.8
, ,
-
fj
pM-+ 4aPGNAc
6
a"t
6
t3
1.6
-
1.4
-
1.2
-
1.0
-
0.8
-
f)
aM+ a M
t2
t2 aM
0
aM
0
(D
t
0
T"
a
aM
f>
1.o
IiA
0.6
-
0.4
-
A
Fraction
FIG.3. Elution profile of the total mnnl mnn2 mnnlOoligosaccharide fraction B from Fig. 2. The combined material was
Fraction
FIG.1. DEAE-Sephacel fractionation of mannoprotein. The
chromatographed on a Bio-Gel P-4 column in water, under which conditions the phosphorylated oligosaccharides were eluted in apeak near the void volume( A )and theneutral oligosaccharideswere eluted later ( B ) . The H-1 NMR spectra of peaks A and B indicated that they were identical, with the exception of mannosyl phosphate groups in oligosaccharide fraction A. Peak B was collected for further study.
mannoprotein (2.4 g) was applied to the column (3 X 18 cm) in 20 mM Tris-HC1 buffer, pH 7.5, and eluted with the same buffer in 11ml fractions to displace the first peak. A t fraction 70, a 1-liter linear gradient of 0-0.5 M NaCl in the same buffer was applied, which eluted a second carbohydrate-containing peak. Carbohydrate (0)was assayed by the phenol-sulfuric acid reaction, protein (0)as absorbance at 280 nm, and salt by conductivity ( X ) . The material in fractions 75-95 was combined for isolation of N-linked oligosaccharides.
0.8
-
0.8
-
0.4
-
0.2
-
0
$
4
'
O 120 0.1
140
100
100
200
220
240
200
280
300
320
340
I
1
I
I
I
1
I
I
1
900
Fractlon
Fraction
FIG.2. Elution profile on a Bio-Gel P-10 column of oligo-
FIG.4. Comparison of oligosaccharide elution profiles. The
saccharides released from mnnl mnn2 mnnlO mannoprotein
products from endoglucosaminidase digestion of the mannoproteins were fractionated on a Bio-Gel P-10 column (2 X 200 cm, -400 mesh), and the neutral oligosaccharide fraction was recovered and rerun on a Bio-Gel P-4 (-400 mesh) column (2 X 200 cm) by elution with water. The tubes were monitored for carbohydrate (A4w). Oligosaccharides from mnnl mnn2 ( A ) ,mnnl mnn2 mnnlO ( B ) ,and mnnl mnn2 mnn9 (C) mannoproteins.
by endoglucosaminidase digestion. Peak A is residual mannopro-
tein containing 0-linked carbohydrate, and peak B contains both acidic and neutral core oligosaccharides. The column was eluted with 0.1 M ammonium acetate and carbohydrate was assayed by the phenol-sulfuric acid method. All material absorbing at 280 nm was found under peak A.
Protein Yeast
11860
Glycosylation Mutants
The mnnl mnn2 mnnlO mannoprotein digest also gave a single broad oligosaccharide peak on the P-4 column (Fig. 4), but it was eluted earlier than the corresponding mnn9 oligosaccharide, indicative of a larger size. The H-1 NMR spectrum
A
B
suggested that theoligosaccharide had the same core structure as the mnnl mnn2 mnn9 oligosaccharide (Fig. 5 A ) but that it also had an unsubstituted heterogeneous cul4-linked outer chain consisting of an average of 10 mannoses that give the strong signal at 64.90 (Fig. 5 0 ) (1). The pattern obtained when this oligosaccharide fraction was rerun on Bio-Gel P-4 suggests that several homologs were present, but the column was unable to resolve oligosaccharides in this size range. The oligosaccharide fraction from the mnnl mnn2 mn& mannoprotein was similar tothat from the mnnl mnn2 mnnlO strain, a conclusion supported by the fast atom bombardment mass spectrum of the peak tube which agrees with a core oligosaccharide (ManloGlcNAc) with an outer chain of 12 mannose units (m/z = -4800 as thepermethyl ether). The assignment of such a structure was supported by the H-1 NMR spectrum (Fig. 5C), which showsall the expected signals for the core mannose units plus a strong signal at 64.90 for the outer chain. The mnnl mnn2 mn& and mnnl mnn2 mnnlO oligosaccharides were compared by the elution patterns they gave on the Dionex BioLC carbohydrate system (Fig. 6), which confirmed their heterogeneity and revealed that themnn8 oligosaccharides covered a slightly wider range of sizes. These patterns indicate that the oligosaccharides form an homologous series that differ by 1 mannose unit one from another. MannoproteinComplementation-The invertase synthesized in the mnnl mnn2 mnn9 mutant migrates on a native gel in a characteristic position that differs from that for the invertases from the mnnl mnn2 mutant or the corresponding mnn8 and mnnl0 mutants (10).Invertases from the mnnl mnn2mnn8mnn9 or mnnl mnn2mnn9mnnlO haploid
n
D
0
5.3
5.2
5.1
5:O
4:9
4.8
PPM
FIG. 5. Comparison of the 500 MHz H-1 NMR spectra for purified neutral oligosaccharide fractions from mutant mannoproteins. A, mnnl mnn2 mnn9; E , mnnl mnn2 mnn7; C, mnnl mnn2 mnn8; and D,mnnl mnn2 mnnl0. All signals in spectrum A are presentin B, C and D, whereas the latterthree have an additional signal at 64.897 that is characteristic of the unsubstituted a l 4 linked polymannose chain.
5
10
15
20
25
Time (min)
FIG. 6. Fractionation of oligosaccharides by HPLC. The separations were done on the Dionex BioLC carbohydrate systems as described under “Experimental Procedures.” A, mnnl mnn2 m n 8 oligosaccharides; B, mnnl mnn2 mnnlO oligosaccharides. The first peak eluted in both patterns is estimated to contain about 14 mannose units, corresponding to an outer chain of 6 mannoses, and each successive peak contains 1 additional mannose. The exact number of peaks depended on how much material was loaded on the column.
Yeast Protein Glycosylation Mutants mutants all have the mnnl mnn2 mnn9 phenotype, as expected if mnn9 were dominant. The mannoprotein oligosaccharide structures support this conclusion since the H-1NMR spectra reveal the absence of a signal at 64.90 for the a 1 4 linked outer chain units (Fig. 7). The mnnl mnn2 mnn7 and mnnl mnn2 mnn8 haploids and the mnnl mnn2 mnn7/mnnl mnn2 mnn8 diploid all give the same invertase and oligosaccharide patterns, which confirms the absence of complementation. On the other hand, the mnnl mnn2 mnn8/mnnI mnn2mnnlO diploid gives a pattern very similar to thatshown by the mnnl mnn2 mutant (notshown), which demonstrates complementation even though the two mutants make mannoprotein with similar oligosaccharide structures. The mnnlO defect appears to be dominant to mnn8 because the Dionex BioLC pattern of the oligosaccharides from the mnnl mnn2 mnn8 mnnlO haploid strain was similar to that of the mnnl mnn2 mnnlO strain. Detailed Structure of the mnnl mnn2 mnnlO Oliosaccha-
A
B
11861
ride-Although the H-1 NMR spectrum of this oligosaccharide (Fig. 5 0 ) was consistent with it having a core that was identical to thatof the mnnl mnn2 mnn9oligosaccharide and that the molecule differed only by the addition of an unbranched a l 4 - l i n k e d outer chain, the alternative existed that one of the al+2-linked mannoses was attached to the outer chain rather than thecore. Such a difference in structure would not be revealed by the NMR spectrum. To test this alternative, the mnnl mnn2 mnnlO oligosaccharide was digested with an endo-al4-mannanase (18) and the products were separated on a Bio-Gel P-4 column (Fig. 8). The starting oligosaccharide would be eluted at about fraction 190 on this column whereas the digest gave three peaks that were eluted in theregion for Mans-llGlcNAc and four peaks in the region of mannose to mannotetraose. Paper chromatography of the latter four peaks (ethyl acetate/pyridine/watr, 5:3:2, v/v) supported these assignments, the R,,, values of peaks A , B, C, and D of Fig. 8 being 1.0, 0.77, 0.62, and 0.48, respectively. The H-1 NMR spectra revealed that peak B was a l a mannobiose and peak C was al4-mannotriose, whereas the spectrum of peak D was that expected for a n a l 4 - m a n n o triose substituted by an al+2-linked mannose unit (Fig. 9 andTable 11). The spectra for peaks G (Fig. 10) andH identified them as homologs of MansGlcNAc with one or two attached a 1 4 - l i n k e d mannose units (Table 11). These resiilts clearly demonstrate that thecore of this oligosaccharide had a single al-2-linked mannose attached to an a l 4 linked mannose of the backbone and that the other al-2linked mannose must be in the outer chain where it can be released in the form of a tetrasaccharide by action of the endomannanase. The structure of the tetrasaccharide from peak D is based on the H-1NMR spectrum (Fig. 9, bottom) that shows signals for aterminal a1-2-linked mannose (65.035), an a l 4 linked mannose substituted at position 2 (65.125), an a l a linked nonreducing mannose (64.883), anda 6-substituted reducing end mannose (65.160 and 4.890) (Table 11). The integrations show that each type of mannose is present in unit ratio. After digestion with exo-al-2-mannosidase, the a1+2-linked mannose was removed and a trisaccharide was recovered that had an ‘H spectrum identical to that of au-
C
100
200
240 220
200
200
300
320
340
300
300
400
420
Fraction
5.3
5.2
5.1
5.0
4.9
4.8
PPM
FIG. 7. Comparison of the 500 MHz H-1NMR spectra of purified oligosaccharide fractions from mannoprotein multiple mutants. A , mnnl mnn2 mnn9; B , mnnl mnn2 mnn8 mnn9; and C, mnnl mnn2 mnn9 mnnl0.
FIG. 8. Fractionation of the endomannanase digest of the mnnl mnn2 mnnlO oligosaccharide. The digest (“Experimental Procedures”) was separated on a Bio-Gel P-4 column, and the fractions were analyzed for carbohydrate by the phenol-sulfuric acid method. Peaks A-F, oligomannosides obtained from the outer chain, and peaks G-I, N-acetylglucosamine-containingcore fragments, are identified in Table 11.
ucture
11862
Protein Yeast
Glycosylation Mutants thentic ala-mannotriose (Fig. 9, top). The attachment of the al+2-linked mannose to themannotriose unit was established by methylation analysis (17), which gave a ratio of tri0-methyl to tetra-0-methylmannose of 3.5 (expected 3.0 for an unbranched tetrasaccharide), andno di-0-methylmannose was formed. An al+Z-linked mannose on either of the other two hexoses would givedi-0-methyl-, tri-0-methyl- and tetra0-methylmannose in the ratio 1:1:2, Taken together, the results prove that themnnl mnn2 mnnlOoligosaccharidehas the general structure OM
aM
"
6 134 6 aM+ aM+ DM+ a@GNAc
t3
6
6
cuM+ aM+ aM"t
. . . .aM+ 6a M T2
6
T2 CYM I
5.0 55 .1 50 .1 55 .2 50 .25
J
~
'
~
1
'
~
'
'
1
'
~
'
PPI4
~
1
~
'
5.4 0 .0840. 045. 940. 9 5
~
~
1
'
~
~
~
1
~
FIG. 9. H-1NMR spectra of the mannotriose and mannotetraose fragments produced by endomannanase digestion of the mnnZ mnn2 mnnZO oligosaccharide. The trisaccharide (top) shows signals expected for a14-mannotriose at64.883 (1.1protons), 64.905 (1.1protons), and 65.160 (0.7 protons) and 64.877 (0.3 protons), the latter two signals representing the a/@ anomers of the reducing end. The tetrasaccharide (bottom) shows signals a t 65.125 (0.99 protons), 65.035 (1.01 protons), 64.883(1.0 protons), 65.160 (0.67 protons), and64.890 (0.33 protons) as expected for an al&-mannotriose
~
~
'
1
'
"
~
aM 1
~
"
J
I
'
~
~
'
I
'
'
t2 olM
in which the two parts are connected by a chain of unsubstituted a l A - l i n k e d mannoses to give the intact molecule. substituted a t position 2 of one of the mannose units (15). All signals in the spectra are assigned in Table 11. Following exo-al+2-mannosidase digestion, the tetrasaccharide yielded a trisaccharide with an H-1 spectrum identical to thatin the top panel.
TABLE I1 H-1 NMR chemical shifts for endomannannse-producedfragments and
residue Peak from Fig. I"
Sugar D
6b
C
B
D
A
C
B
Aa
A@
5.165
4.879
ppm
A (3.3) B (1.1) C (1.5) D (0.82)'
4.877 4.890
5.160 5.160
4.901 5.157 4.883 4.905 4.883 5.125
4.893
5.035 5.085d G (0.55)
5.110
aMaM 42
13
4.710 aM+'aM+'@M+4a@GNA~ 5.235 4.770 4.868
5.035 5.125
f3
aM+'aM 5.335
4.925
t'
5.289
aM
t'
5.045
aM M Ha(0.35)'
aM
1'
5.085d
5.035
43
4.715 aM+'aM+'@"ilcy@GNA~ 5.235 4.770 4.870 a M+'aM+'a
f3
5.335 M
4.907
5.125
5.110
4.910
t'
5.285
aM
f'
5.045
(YM
Molar ratios, in parentheses, are normalized to the sum of D, E, and F as 1.0. *All signals were present in unit ratios, although those for the reducing hexose were divided between a and @ anomers in a 7:3 ratio. Referenced to acetone (62.217) at 40°C. e Peaks E and F gave signals expected for the next two higher homologs of this tetrasaccharide, but they also contained large amounts of the linear al&-linked isomers. Methylation of D gave a ratio of3.5 for tri- to tetramethylmannose. The sienal for this al-3-linked mannose is split by a long-range interaction with the anomers of N acetylglucosamine (16). Peak I is the next higher homolog with an additional a 1 4 - l i n k e d mannose that gives a signal at 64.889. The sum of G, H, and I was 11.1. "
I
11863
Yeast ProteinGlycosylation Mutants
FIG. 10. H-1 NMR spectra of the mnnl mnn2 mnnlOoligosaccharide and the core fragment producedby endomannanase digestion. The spectrum of the intact oligosaccharide (top) shows a major signal at 64.90 equivalent to about 12 protons that represents the unbranched outer chain and a signal at 65.125 equivalent to 2 protons that represents n l 4 - l i n k e d mannoses substituted atposition 2. The endomannanaseresistant core fragment G (bottom) shows signals for a single outer chain mannose (64.925) and a single %substituted n l 4 - l i n k e d mannose (65.125). Other signals are assigned in Table 11. The signal a t 64.70 ( t o p ) is distorted by a spinning sideband.
From integration of the areas under the peaks in Fig. 8, we find aboutequimolar amounts of peaks D and G H, whereas about 1.5 molar equivalents of mannotriose, 1of mannobiose, and 3of mannose were formed (Table 11).This would provide 9-10 mannoses to fill in the middle of the chain and would give a molecule containing 20 to 21 mannose units, in agreement with the average size of the mnnl mnn2 mnnlO oligosaccharide (Figs. 4 and 6). The mnnl mnn2 mnn8 oligosaccharide gave an H-1NMR spectrum that was identical to the mnnl mnn2 mnnlO strain and yielded similar fragments by endomannanase digestion, although the average length of the outer chain is slightly greater (Fig. 6). As a control to ensure that the endo-al4-mannanase is unable to remove an al-+2-linked mannose unit from the core oligosaccharide, a sample of the mnnl mnn2mnn9 oligosaccharide (ManloGlcNAc)was digested with the enzyme under the conditions used in this experiment. Mannose was not released and theoligosaccharide was recoveredunchanged after gel filtration on a Bio-Gel P-4 column (data notshown).
+
mM
12
nM
crM
13
aM-'nM-'~M-'~dCh.Aci-R
-
12
