Schizosaccharomyces pombe shares with Saccharomyces cerevisiae the ER transfer of Glc3Man9GlcNAc2 to protein (Ziegler et al., 1994) and also has one or more Golgi-associated galactosyltransferases (Chappell et al., 1994) that modify core oligosaccharides in a manner more closely related to larger eukaryotes than S.cerevisiae. Our previous work (Ziegler et al., 1994), later corroborated by Ballou et al. (1994), demonstrated that S.pombe lacks the specific ER Man9-[alpha]-mannosidase found in S.cerevisiae and higher eukaryotic cells (Byrd et al., 1982; Jelinek-Kelly and Herscovics, 1988; Ziegler and Trimble, 1991). Thus, oligosaccharide chain elongation and core-filling proceed by the addition of Man and/or Gal to the oligosaccharide-lipid form of Man9GlcNAc2 (Ballou et al., 1994; Ziegler et al., 1994) (Scheme I) to produce mannans and galactomannans unique to this organism.
Scheme 1.
Figure 1. Isolation of the wild-type S.pombe Hex10GlcNAc oligosaccharide pool. (A) shows the initial Bio-Gel P-4 profile of the endo H-released S.pombe oligosaccharides present in the 60% methanol extract of acetone-precipitated digestions. (B) shows the Bio-Gel P-4 profile of pooled fractions 130-133 from (A). (C) demonstrates by MALDI-TOF MS the size homogeneity of the Hex10GlcNAc in pooled fractions 129-133 from (B).
Figure 2. Analytical HPAEC on a Dionex 4 × 250 mm PA-100 column of Hex10GlcNAc pools from wild-type (A) and gmal2- galactosyltransferase-negative (B) S.pombe. Approximately 3 nmol of total oligosaccharide was loaded onto the column in each case. (B) includes the typical sodium acetate gradient (dashed line) employed from 35 to 44 mM over the 40 min elution. PAD was used to monitor the profile.
In this report we show that there are four major Hex10GlcNAc isomers released by endo H from S.pombe glycoproteins, and these are formed by the addition of a Man or Gal residue in specific linkage to this Man9GlcNAc precursor. The isomers have been chromatographically purified and analyzed by 1H NMR spectroscopy. The results show that Man can be added [alpha]1,2- to the upper arm residue 9 in a core-filling reaction as seen in Pichia pastoris (Trimble et al., 1991) or [alpha]1,6- to the lower arm residue 5 to initiate outer chain-elongation as seen in both Pichia and S.cerevisiae (Trimble et al., 1991; Hernández et al., 1989; Trimble and Atkinson, 1992). Early in elongation, Gal is added in [alpha]1,2-linkage to mannose residues 9 and 10 of the upper and middle arms of the precursor, the latter of which is the target of ER Man9-[alpha] mannosidase in Pichia and S.cerevisiae, or in [alpha]1,3-linkage to residue 8 on the lower arm. There appears to be a specific galactosyltransferase responsible for each linkage addition, because the gma12- strain of S.pombe, in which a specific [alpha]1,2-galactosyltransferase gene has been deleted (Chappell et al., 1994), proved unable to galactosylate the upper- and middle-arm residues 9 and 10 but still attached [alpha]1,3-linked galactose to lower-arm residue 8. Thus, analysis of the Hex10GlcNAc isomers from wild-type and gma12- S.pombe strains identifies at least one specific core-filling activity for the gma12p galactosyltransferase, and implies the presence of a yet to be identified [alpha]1,3-galactosyltransferase. The latter activity may be the same enzyme that adds the branching Gal to the Hex(Gal[alpha]1,3)Man[alpha]1,2- structures also found in the O-linked oligosaccharides from S.pombe (Gemmill and Trimble, 1998). Resolution of Hex10 isomers
N-Linked oligosaccharides were released by endo H from wild-type and galactosyltransferase-deficient gma12- S.pombe glycoproteins and resolved by Bio-Gel P-4 chromatography. Figure
Table I summarizes the monosaccharide content and percent distribution of each Hex10GlcNAc peak isolated from wild-type and gma12- S.pombe by preparative HPAEC. Peaks a and c contained only Man and GlcNAc in a ratio of about 10:1 with no Gal present, and, as seen in Figure
Table I. NMR spectroscopy
Table I shows that the Hex10GlcNAc pool is composed mainly of Man10GlcNAc whether the source was the wild-type or the galactosyltransferase-deficient gma12- S.pombe strain. The absence of peak b in the gma12- strain suggested, however, that assigning structures to the individual Hex10GlcNAc isomers might serve to define the role that gma12p plays in core glycan synthesis in vivo. To accomplish this, sufficient a-c and g pools were prepared for examination by high-field 1D and 2D 1H NMR spectroscopic techniques.
Figure 3. Montage of 500 MHz 1H NMR spectra of wild-type S.pombe Hex10GlcNAc pools a-c and g isolated by preparative HPAEC. Final sample concentrations in the NMR tubes were, in mM: a, 0.4; b, 0.8; c, 1.2; and g, 1.1. All samples included 1.1 mM acetone as an internal chemical shift marker (2.225 ppm). Labels beside signal peaks refer to the residues assigned in the structures deduced in Scheme II. Integrated values for C1-H proton intensities and C1-, C2-, and C3-H chemical shifts ([delta], ppm) are summarized in Tables II and III, respectively. A montage of the 1D 1H NMR spectra for a-c and g is shown in Figure
Table II. Pool a
Figure 4. Portions of the 2D DQF COSY and 1H-1H ROESY experiments used to assign the GalMan9GlcNAc structures in HPAEC pool b. (A) 2D DQF COSY crosspeaks. (B) 2D ROESY crosspeaks generated with a 200 ms mixing time. Labels in (A) are those used for residues in Schemes I and II and in Tables II and III. Boxed ROESY crosspeaks in (B) are identified by the residue's anomeric proton connectivity with its respective proton across the glycosidic linkage.
Figure 5. Portions of the 2D DQF COSY and 1H-1H ROESY experiments used to assign the Glc- and Gal-containing N-glycans in HPAEC pool g. (A) 2D DQF COSY crosspeaks. (B) 2D ROSEY crosspeaks generated with a 200 ms mixing time. Labels in (A) are those used in Schemes I and II and in Tables II and III. Boxed ROSEY crosspeaks in (B) are identified by the residue's anomeric proton connectivity with its respective proton across the glycosidic linkage.
Table III.
Scheme 2. This pool appears mostly to be the core Man9GlcNAc precursor (Ziegler et al., 1994) with an additional terminal [alpha]1,2-linked Man residue. Two significant differences in the 1H NMR spectrum of a compared to the precursor Man9GlcNAc are the shift in most of residue 6[prime]s C1-H from 5.144 p.p.m. in Man9GlcNAc to 5.123 p.p.m. and the downfield shift in residue 9[prime]s C1-H from 5.032 to 5.290 p.p.m. (Table III). These shifts are indicative of the addition of the new [alpha]1,2-linked Man residue 14 to upper arm residue 9 (see structure a, Scheme II). Addition of novel [alpha]1,2-Man termini to the Man8GlcNAc processing intermediate in Pichia pastoris was reported earlier (Trimble et al., 1991), and residue 6[prime]s upfield shift due to its anomeric center being shielded by residue 14[prime]s ring protons has been documented in detail elsewhere (Cohen and Ballou, 1980; Trimble et al., 1991). Residue 14[prime]s anomeric proton is found at 5.046 p.p.m., with that of residues 10 and 11, and integrates to the expected value of 3 mol (Table II). The small signal (0.07 mol) at 5.145 p.p.m. in Figure Pool b
Monosaccharide analysis of b provided a composition of GalMan9GlcNAc (Table I), and this peak was absent in the HPAEC analysis of the gma12- Hex10GlcNAc pool (Figure
The 1D 1H NMR spectrum (Figure
To determine which [alpha]1,2-linked Man residue(s) might be substituted by Gal, a 2D DQF-COSY experiment was performed to identify the chemical shifts of the C1-, C2-, and C3-Hs of all of the ring systems, followed by a ROESY experiment with a 200 ms mixing time to identify 1H-1H interresidue connectivities across the glycosidic linkages. The spectra for pool b are shown in Figure
Consistent with this assignment, note that 11[prime]s C1-H at 5.042 p.p.m. has the expected crosspeak with 8[prime]s C2-H at 4.105 p.p.m., and 9[prime]s C1-H at 5.035 has the expected crosspeak with 6[prime]s C2-H at 4.015 p.p.m. (b1 in Figure
Inspection of Figure
Introduction
Results
Peak identity
Monosaccharide compositiona
Yeast strain (% distribution)
972h-
gma12-
a.
Man10GlcNAc
9.5
5.3
b.
GalMan9GlcNAc
28.5
0
c.
Man10GlcNAc
45.5
85.2
d.
Man10GlcNAc
1.2
3.1
e.
GalMan9GlcNAc
1.2
0
f.
GlcMan9GlcNAc
1.6
1.2
g.
Gal0.8Glc0.2Man9GlcNAc
12.5
5.2
10 7 4
Gal[alpha]1,2Man[alpha]1,2Man[alpha]1,3Man[alpha]1,6-
9 6
Gal[alpha]1,2Man[alpha]1,2Man[alpha]1,6-
This assignment is confirmed by the minor ROESY crosspeak between 9[prime]s C1-H at 5.396 p.p.m. and 6[prime]s C2-H at 4.003 p.p.m. (b2 in Figure
Note the small resonance intensity at 4.930 in the 1D spectrum of b in Figure
Pool c
The 1D 1H NMR spectrum for the Man10GlcNAc in pool c (Figure
Pool g
Compositional analysis of pool g (Table I) revealed both Glc and Gal in less than molar proportions compared to Man and GlcNAc suggesting the presence of more than one Hex10GlcNAc isomer. The 1D 1H NMR spectrum of pool g (Figure
The major chemical shift differences noted above could be due to the addition of Gal to either residue 8 or 11 (Scheme I), but the C1-H of a terminal [alpha]1,2-linked Gal would be expected at 5.160 to 5.180 p.p.m. (Table II, pool b and Ballou et al., 1994), not 5.288 p.p.m.. To ascertain the correct linkage assignment, the chemical shifts of the C1- to C3-Hs of the sugar ring systems and the glycosidic linkage 1H-1H couplings of pool g isomers were determined by 2D DQF-COSY and ROESY NMR experiments. The 2D DQF-COSY spectrum shows the 1.9 mol of C1-H resonance intensity at 5.282-5.288 p.p.m. (Table II) is correlated with two C2-Hs, one at 4.294 p.p.m. and the other at 3.850 p.p.m.. The 3.5 Hz coupling constant (J1,2) of the paired tetraplets 9-10 Hz apart at 5.288/3.850 p.p.m. (Figure
Interestingly, two additional C1-H/C2-H crosspeaks are apparent in g's 2D DQF-COSY (Figure
The data presented here show that the fission yeast, S.pombe, adds either mannose or galactose residues to the ER-form of Man9GlcNAc, whose structure was reported earlier by Ziegler et al. (1994). In a recent report, Ballou et al. (1994) observed the presence of Gal in core-filling glycans of the Hex13-14GlcNAc size, but detected only Man in the Hex10 GlcNAc size class. This is somewhat surprising as nearly 40% of the wild-type Hex10GlcNAc pool in this study contained isomers with Gal termini (Table I, Scheme II). The NMR results reported here show that a single Man is added [alpha]1,6- to residue 5 to form the most abundant isomer, c, which is the branch initiation for the outer chain first demonstrated by Ballou and co-workers (Hernández et al., 1989; Ballou et al., 1990) and is representative of the outer chain pattern found in both Pichia (Trimble et al., 1991) and Saccharomyces sp. (Byrd et al., 1982; Trimble and Atkinson, 1986, 1992). In S.cerevisiae this activity is provided by the Och1p (Nakanishi et al., 1993). This Hex10GlcNAc isomer also was observed in S.pombe by Ballou et al. (1994).
A second site of mannose addition to the Man9GlcNAc precursor is to upper-arm residue 9 (Scheme II, isomer a). This [alpha]1,2-linked addition to a Man already linked [alpha]1,2- to the core has not been found in S.cerevisiae Man8-13GlcNAc glycans (Trimble and Atkinson, 1986, 1992), but was found to be present as a minor component of Man10GlcNAc and Man11GlcNAc isomers from SUC2 invertase secreted by Pichia pastoris (Trimble et al., 1991).
Two core Gal linkage additions were observed in the current study, [alpha]1,2- and [alpha]1,3-. The [alpha]1,2- linked Gal was present predominantly on the middle-arm mannose residue 10 (Scheme II, b1), with a lesser amount on residue 9 (Scheme II, b2). These residues also were shown by Ballou and co-workers to be present in their Hex14GlcNAc structures (Ballou et al., 1994) and appear from the current work to be specified by gma12p (Chappel et al., 1994). Interestingly, this [alpha]1,2- galactosyltransferase appears to be different from the [alpha]1,3-transferase that attaches Gal in the second linkage, [alpha]1,3-, to Man residue 8 of the lower arm in isomer g1 (Scheme II) leading to the structure,
11 8 5 Man[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man[alpha]1,3-
This is identical to a tetrasaccharide recently described among the S.pombe O-linked glycans released by [beta]-elimination (Gemmill and Trimble, 1998). The complete family of O-linked structures suggests, along with finding an [alpha]1,3-linked Gal on residue 8 of the preformed N-linked core Man9GlcNAc (Scheme II, isomer g), that the [alpha]1,3-galactosyltransferase responsible for this linkage is a 'branch forming" activity, which prefers to di-O-substitute the penultimate Man residue in an [alpha]1,2-linked polymer.
Current work is focused on determining how the Hex10GlcNAc isomers are further elongated by assigning structures to isomers present in the S.pombe Hex11GlcNAc pool from wild-type and galactosyltransferase-deficient strains and to isolate and characterize the novel branching [alpha]1,3-galactosyltransferase.
Materials
Schizosaccharomyces pombe wild-type strain 972h- was from the ATCC (strain 24843), and an [alpha]-galactosyltransferase-deficient derivative (gma12- D10::ura4, leu1-32-, ura 4-D18-, his3-; strain TCP-3001) was kindly provided by Dr. T. Chappell, MRC Laboratory for Molecular and Cell Biology, London. Yeast extract was obtained from Difco Laboratories, Detroit, MI; glucose from Mallinkrodt Baker Chemical Co., Paris, KY, and adenine hemisulfate and 99.9% D2O were from Sigma Chemical Co., St. Louis, MO. Dowex ion exchange resins were from Bio-Rad Laboratories, Hercules, CA, as was the Bio-Gel P-4 (extra fine resin, lot 49671A) gel filtration medium. The 99.96% and 99.996% D2O were supplied by Cambridge Isotopes Lab, Inc., Andover, MA. Labeled oligosaccharide markers were from a previous study (Trimble and Atkinson, 1992), and endo H was a product of this laboratory (Trumbly et al., 1985). HPAEC was performed using CarboPac PA1 or PA-100 columns with the Dionex I-450 system (Dionex Corp., Sunnyvale, CA). Monosaccharide standards were obtained from Dionex. All solvents and reagents were the purest commercially available, and dH2O was polished using a Milli-Q UF Plus system from Millipore Corp., Bedford, MA.
Cell cultures
S.pombe cells were grown in 2 l flasks containing 1 l of yeast extract medium as described previously (Ziegler et al., 1994). For this study ~350 g (wet weight) of wild-type or gma12- S.pombe cells served as starting material. For final 1H NMR analysis, oligosaccharide pools a and g were supplemented with additional material from another 350-500 g of cells.
Oligosaccharide preparation
Cell disruption with glass beads, soluble glycoprotein preparation, and endo H deglycosylation have been described previously in detail (Verostek et al., 1993; Ziegler et al., 1994). Released oligosaccharides were isolated as the fraction soluble in 60% methanol after acetone precipitation of the bulk endo H digestions. The methanol fraction was taken to dryness, dissolved in dH2O, and desalted and freed of small peptides by passage over a 1×3 cm column of Dowex mixed-bed ion exchange resin (Dowex 1-formate over Dowex 50-H+; Ziegler et al., 1994). Oligosaccharide size classes were resolved and purified using a 1.6 × 94 cm Bio-Gel P-4 column using 0.1 M acetic acid/1% butanol as the eluent at a flow rate of about 8 ml/h. Column profiles were scanned for neutral hexose by a scaled-down modification (Byrd et al., 1982) of the phenol sulfuric acid method (Dubois et al., 1956). Hex10GlcNAc, identified by its elution position relative to marker oligosaccharides, was pooled and rechromatographed on the Bio-Gel P-4 column. The central 85% of the final Hex10GlcNAc pool (about 900 nmol oligosaccharide) was evaporated to dryness, taken up in dH2O and chromatographed on a preparative Dionex CarboPak PA-100 column (9 × 250 mm). A 100 µl sample was injected using a 200 µl sample loop on the Rheodyne autoinjection valve. Nearly 500 nmol of oligosaccharide could easily be accommodated using this method. The effluent was desalted with a Dionex anion self-regenerating suppressor (ASRSI-4 mm), and fractions of 0.5 ml were collected. The profile was scanned for neutral hexose by phenol sulfuric acid assay as above. PAD cell response settings were E1 = 0.05, E2 = 0.6, E3 = -0.6; T1 = 480 ms, T2 = 120 ms, T3 = 360 ms. The attenuation was increased from 1000 nA to 3000 nA on preparative runs.
Monosaccharide analyses
Duplicate samples of each Hex10GlcNAc (1-3 nmol) were vacuum evaporated to dryness in conical screw-cap tubes, then taken up in 300 µl of 2N HCl. After degassing with a stream of prepurified N2 for about 1 min, the vials were sealed and hydrolysis performed at 100°C for 2 h in a heating block. HCl was removed by repeated vacuum evaporation from 300 µl of dH20, and the samples were taken up in a final volume of 100 µl dH20. Analysis of 25 µl aliquots was performed by HPAEC using the Dionex CarboPack PA1 column. Quantitation was accomplished by comparison with Dionex-supplied standards of known concentration.
NMR spectroscopy
The Hex10GlcNAc isomers isolated by HPAEC were rechromatographed on Bio-Gel P-4 to ensure size homogeneity and to remove traces of sodium acetate. They then were exchanged several times by rotary evaporation from 1 ml of 99.9% D2O and lyophilized twice from 0.6 ml 99.96% D2O. After storage in vacuo over P2O5 for several days, the samples were taken up in 490 µl of 99.996% D2O and flame-sealed in 5 mm 535-pp NMR tubes (Wilmad Glass Co., Buena, NJ). 1H NMR spectra were obtained at 500 MHz with a Bruker DRX Avance instrument at 296°K in the Wadsworth Center NMR Structural Biology Facility. One-dimensional 1H NMR spectra were acquired using a simple presaturation-90°-acquire sequence. Relevant acquisition parameters are: 1024 scans; 4096 data points collected over a spectral width of 1502 Hz; 1.5 s of very low power presaturation on the residual HDO signal at 4.79 p.p.m. giving an overall recycle time of 2.7 s. Homonuclear two-dimensional DQF- COSY (Rance et al., 1983) and ROESY (Bothner-by et al., 1984) spectra also were recorded. In all cases, the spectral width in both dimensions was 1502 Hz. For both experiments 4096 data points were taken during acquisition (t2) and 512 complex data points were taken in the indirect dimension (t1). Quadrature detection in the indirect dimension was achieved using the States-TPPI procedure (Marion et al., 1989). The ROESY spin-lock field strength was 1.5 kHz and was applied for 200 ms.
Mass spectrometry
The positive-ion time-of-flight mass spectra of the Hex10GlcNAc isomers were obtained on a Bruker Reflex Instrument using about 100 pmol of material cocrystallized with 2,5-dilydroxy benzoic acid as a matrix. Approximately 20 shots were averaged for each determination.
We are indebted to Dr. Gordon Rule, University of Virginia, Charlottesville, for providing preliminary one- and two-dimensional 1H NMR spectra of the S.pombe Hex10GlcNAc fractions, while our NMR facility was under development. We thank Ms. Lynn McNaughton of the Wadsworth Center Structural NMR Facility for collecting and processing the NMR data in this article and Dr. Charles Hauer and Mr. Robert Stack of the Wadsworth Center Biological Mass Spectrometry Facility for providing the MALDI-TOF MS spectra. We thank Dr. Trent Gemmill for providing Schemes I and II and for critically reading the manuscript. The efforts of Ms. Lynda Jury, Ms. Judy Valentino, and Ms. Tracy Godfrey in preparing the manuscript are gratefully acknowledged. This work was supported in part by USPHS NIH Grant GM23900 to R.B.T.
ER, endoplasmic reticulum; HPAEC, high-pH, anion-exchange chromatography; PAD, pulsed amperometric detection; MALDI-TOF, matrix-assisted, laser desorption ionization time-of-flight; MS, mass spectrometry; endo H, endo-[beta]-N-acetylglucosaminidase H (EC 3.2.1.96); dH2O, distilled deionized water; 1D, one dimensional; 2D, two dimensional; DQF, double quantum filtered; COSY, correlated spectroscopy; ROESY, rotating frame Overhauser enhancement spectroscopy.
3To whom correspondence should be addressed at: Wadsworth Center, C-547, N.Y.S. Department of Health, P.O. Box 509, Albany, NY 12201-0509