Schizosaccharomyces pombe produces novel Gal0-2Man1-3 O-linked oligosaccharides

Trent R. Gemmill2,4 and Robert B. Trimble1,2,3

1Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509, USA and 2Department of Biomedical Sciences, School of Public Health, Albany, NY 12201, USA

Received on July 22, 1998; revised on October 2, 1998; accepted on October 2, 1998

Schizosaccharomyces pombe whole-cell glycoproteins, previously depleted of N-linked glycans by sequential treatment with endo-ß-N-acetylglucosaminidase H and peptide-N4-asparagine amidohydrolase F, were ß-eliminated with 0.1 M NaOH/1 M NaBH4 to release the O-linked oligosaccharides. The saccharide-alditols were separated by gel-exclusion chromatography into pools from Hexitol to Hex4Hexitol in size. Analysis of the Hexitol pool indicated Man to be the only sugar linked to Ser or Thr residues. The Hex1Hexitol pool contained two components, Gal[alpha]1,2Man-ol (2A) and Man[alpha]1,2Man-ol (2B). The Hex2Hexitol pool contained two components, Gal[alpha]1,2Man[alpha]1,2Man-ol (3A) and Man[alpha]1,2Man[alpha]1,2Man-ol (3B). The two Hex3Hexitol components were Gal[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man-ol (4A) and Man[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man-ol (4B). The Hex4Hexitol component was found to be a single isomer with the composition of Gal[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man[alpha]1,2Man-ol (5AB). Surprisingly, galactobiose was not detected in any of these oligosaccharides. The gma12 (T. G. Chappell and G. Warren (1989) J. Cell Biol., 109, 2693-2707) and gth1 (T. G. Chappell personal communication) [alpha]1,2-galactosyltransferase-deficient mutants and the gma12/gth1 double mutant S.pombe strains were similarly examined. The results indicated that gma12p is solely responsible for the addition of terminal [alpha]1,2-linked Gal in compound 2A, while one or both of gma12p and gth1p are required for the [alpha]1,2-linked Gal in 4A. Both transferases are largely responsible for terminal Gal in isomer 5AB. Neither gma12 nor gth1 had any discernible effect on the structure of the large N-linked galactomannans as determined by 1H NMR spectroscopy. Thus, while gth1p and gma12p appear responsible for adding [alpha]1,2-linked Gal to terminal Man, neither adds galactose side chains to the N-linked poly [alpha]1,6-Man outerchain, nor the O-linked branch-forming [alpha]1,3-linked Gal. Furthermore, the presence of Hex[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2- structures in the O-linked glycans implies the presence of a novel branch-forming [alpha]1,3-galactosyltransferase in S.pombe.

Key words: glycan 1H/13C NMR/O-linked oligosaccharides/S.pombe/yeast glycoproteins

Introduction

The fission yeast, Schizosaccharomyces pombe, has proven to be a useful model system for studying glycoprotein trafficking and processing (Ziegler et al., 1994). With the exception of the absence of an ER Man9-[alpha]1,2-mannosidase, and therefore the inability to trim Man9GlcNAc2 to Man8GlcNAc2, S.pombe synthesizes and processes lipid-linked and newly-transferred N-glycans in the ER as found in most eukaryotes studied to date. Like Saccharomyces cerevisiae, S.pombe elongates its N-linked oligosaccharides into a series of extended glycans in the Golgi apparatus, but, unlike S.cerevisiae and more like animal cells, S.pombe's Golgi apparatus is morphologically well-defined and contains several galactosyltransferases (Chappell and Warren, 1989), which add Gal to most of its N-linked oligosaccharides (Ziegler et al., 1994; Ballou and Ballou, 1995).

O-linked oligosaccharide synthesis and processing is not well characterized in this organism (Ballou et al., 1994; Ballou and Ballou, 1995). Like all fungi studied to date, S.pombe initiates O-glycan synthesis by the addition of Man to Ser or Thr residues, then elongates this monosaccharide with either Gal or Man to form a series of short oligosaccharide chains. Here we deduce the role of two galactosyltransferases, gma12p (Chappell and Warren, 1989; Chappell et al., 1994) and gth1p (Thomas G. Chappell, personal communication), in S.pombe O-glycan processing by defining the structures of ß-eliminated O-linked oligosaccharides from wild-type and galactosyltransferase-deficient S.pombe glycoproteins. Comparison of O-glycans from gma12, gth1, gma12/gth1 double mutant, and the wild-type strain revealed gma12p and gth1p in concert to be almost entirely responsible for the addition of [alpha]1,2-linked Gal to S.pombe O-linked oligosaccharides and do not appear involved in Gal addition to the outer chains of N-linked oligosaccharides. However, gma12p does appear responsible for addition of terminal [alpha]1,2-linked Gal to N-linked oligosaccharide cores in S.pombe (Ziegler et al., 1998). In addition, we identified a previously unreported Gal[alpha]1,3-linked epitope on disubstituted Man on these O-glycans, implying the presence of a novel 2-O-substituted-Man [alpha]1,3-galactosyltransferase in S.pombe. This [alpha]1,3-linked Gal is also found on a portion of the N-linked Hex10GlcNAc core oligosaccharides formed in S.pombe, suggesting the same enzyme is responsible for both N- and O-linked additions of this novel sugar epitope (Ziegler et al., 1998).

A preliminary report of some of the work in this article has appeared previously (Gemmill and Trimble, 1997).

Results

Approximately 80 mg of glycoprotein was prepared from the wild-type, gma12, gth1, and the gma12/gth1 strains of S.pombe, ß-eliminated and desalted as described in Materials and methods. Negatively charged species were recovered from the QAE-Sephadex column as described previously (Gemmill and Trimble, 1996) and examined by HPAEC analysis. A single negatively charged component was found by HPAEC (not shown), which gave no phenol-sulfuric color, was unhydrolyzable by 2N HCl, and did not coelute with pyruvic acid or known pyruvylated saccharides, and therefore was inferred to be a PAD-active protein-degradation product. Aliquots of neutral oligosaccharide-alditols from all four S.pombe strains were then analyzed by HPAEC-PAD (Figure 1), and the remainders were sized by Bio-Gel P-4 chromatography (Figure 2).


Figure 1. HPAEC-PAD profiles of S.pombe O-linked oligosaccharides. O-Glycans released from the wild-type, gma12, gth1, and gma12/gth1 double mutant strains of S.pombe by ß-elimination were chromatographed by HPAEC on the analytical Dionex PA1 column with PAD as described in the text. The individual isomers are labeled as well as the elution position of 4U, a galactosidase product of 5AB, which was not found as a natural product in any of the strains examined.


Figure 2. Bio-Gel P-4 profiles of S.pombe O-linked oligosaccharides. O-glycans from the four S.pombe strains released by ß-elimination were sized by Bio-Gel P-4 chromatography as described in the text. Open circles indicate the number of nanomoles of hexose by phenol-sulfuric acid assay, and (solid circles) indicate the number of nanomoles of mannitol by HPAEC per aliquot.

The wild-type S.pombe O-glycans were pooled into the five (Hex1-5-ol) size classes, and each size class was separated into individual isomers by preparative HPEAC-PAD. The separation pattern on the PA-100 column (not shown) was similar to that shown on the PA1 column (Figure 1), with one exception; the Hex5-ol pool was separated into two peaks on the preparative column and only one peak on the analytical column. The Hex2-ol and Hex4-ol pools each separated into two isomers (2A, 2B, and 4A, 4B), respectively. However, the Hex3-ol pool could only be resolved into one peak (Ballou and Ballou, 1995), which was later determined to consist of two isomers (3A and 3B). Each isomer was examined by mass spectrometry (Table I), and all molecular weights were within 1 Da of those expected for the size assigned. Compositional analysis of hydrolysates of each isomer showed Gal, Man, and mannitol to be the only constituent monosaccharides.

Table I. Preliminary characterization of S.pombe O-glycan isomers including their proposed structural formulas
Pool Mr (Da) Amount obtained (µMol) Isomera Proposed structure
2A 344.1 18 2A Gal[alpha]1,2Man-ol
2B 343.7 12 2B Man[alpha]1,2Man-ol
3 505.8 10 3A
3B
Gal[alpha]1,2Man[alpha]1,2Man-ol
Man[alpha]1,2Man[alpha]1,2Man-ol
4A 668.2 65 4A Gal[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man-ol
4B 668.6 26 4B Man[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man-ol
5A
5B
831.6
829.6
4.8
2.4
5AB Gal[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man[alpha]1,2Man-ol
aPool 3 was composed of two inseparable isomers (3A and 3B), and 5AB separated into two distinct peaks (5A and 5B) when chromatographed by preparative HPAEC as described in Materials and methods but was confirmed to be a single isomer.

With the exception of 5AB, which required HMQC/HMBC NMR experiments, the remaining Hex2-4-ol pools were examined by 1D proton and 2D DQF COSY NMR spectroscopy with the results shown in Figure 3, and the linkage assignments summarized in Table I. Figure 4 shows the results of permethylation analyses performed on each of the S.pombe wild-type O-linked isomers. Note that the permethylation product of 2-substituted mannitol is not visualized in this TLC system, and that the availability of standards for identifying various di-O-methylsaccharides is limited, thus hampering their absolute identification. Finally, the isomers were examined by enzymatic digestion with [alpha]-mannosidase and [alpha]-galactosidase followed by HPAEC chromatography (Figure 5). Assignment of the oligosaccharide structures in each size pool is described individually in the following paragraphs.


Figure 3. NMR spectra of S.pombe O-glycans. The individual O-glycan isomers purified by HPAEC (Figure 1) were analyzed by 1D proton (top of each panel) and 2D DQF COSY (bottom of each panel) NMR spectroscopy as described in the text. The amounts of each isomer pool in Table I were examined by 1H NMR at 500 MHz in 500 µl D2O.


Figure 4. Permethylation analysis of S.pombe O-glycans. The individual O-glycans and authentic S.cerevisiae Man[alpha]1,2Man-ol (YE) were permethylated, hydrolyzed, developed on silica gel TLC plates and visualized as described in the text. The region where di-O-methylhexoses elute is indicated (dMan), as are the positions of known standards: tMan, 2,3,4,6-tetra-O-methylmannose; tGal, 2,3,4,6-tetra-O-methylgalactose; 2Man, 3,4,6-tri-O-methylmannose.


Figure 5. Glycosidase treatment of S.pombe O-glycans. The purified O-glycans were treated with jack bean [alpha]-mannosidase (M[prime]ase) and coffee bean [alpha]-galactosidase (G[prime]ase), as described in Materials and methods, followed by HPAEC-PAD using the analytical PA1 column.

Monosaccharides

Mannitol was the only monosaccharide alditol found on ß-elimination of S.pombe glycoproteins by HPAEC/PAD analysis of the Hex1-ol pool using an MA1 column. This, combined with the finding that mannitol was the only monosaccharide alditol found on analysis of the Hex2-5-ol isomers, indicates that mannose is the only saccharide directly O-linked to proteins in S.pombe.

Disaccharides

The 2D DQF COSY spectra of 2A (Figure 3) indicated a terminal [alpha]Gal by the coupling of its 5.11 ppm anomeric proton with its axial C2-H (J1,2=4.0 Hz) at 3.83 ppm. Isomer 2A yielded Gal and mannitol upon [alpha]-galactosidase treatment (not shown), no reaction with [alpha]-mannosidase, and displayed only terminal Gal by permethylation (Figure 4). Gma12p is known to be an [alpha]1,2-galactosyl to Man transferase in vitro using defined acceptors (Chappell and Warren, 1989), and isomer 2A is absent from the gma12 knockout S.pombe (Figure 1), which is consistent with 2A being Gal[alpha]1,2Man-ol.

The NMR spectra for 2B (Figure 3) revealed a single anomeric proton at 5.02 ppm, which was coupled to an equatorial C2-H (J1,2 = 4.7 Hz) at 4.01 ppm. This signature identifies a single [alpha]Man terminus and is identical to the Man[alpha]1,2Man-ol samples prepared from S.cerevisiae (Materials and methods) and Hansenula polymorpha (Cohen and Ballou, 1980). Compound 2B yielded Man plus mannitol on [alpha]-mannosidase treatment (not shown), no reaction on [alpha]-galactosidase treatment, and was shown to have only terminal Man by permethylation (Figure 4). This confirms isomer 2B as Man[alpha]1,2Man-ol.

Trisaccharides

The 1D proton spectra of the 3AB pool (Figure 3) indicated the presence of two isomers, a Gal, Man, and mannitol containing species (3A, 82%) and a species containing two Man and mannitol (3B, 18%) on the basis of anomeric proton intensity integrations. The anomeric and C2-proton resonances of 3B's nonreducing terminal and penultimate residues were the same as those found for the H.polymorpha Man[alpha]1,2Man[alpha]1,2Man[alpha]ß (Cohen and Ballou, 1980). The COSY spectrum of 3A showed two anomeric protons, one at 5.19 ppm with axial C2-H coupling at 3.83 ppm (J1,2 = 3.8 Hz) and one at 5.36 ppm with equatorial coupling at 4.02 ppm (J1,2 4.9), which indicates the presence of [alpha]Gal and [alpha]Man, respectively. Permethylation of the Hex3-ol pool (Figure 4) showed 2-O-substituted Man, terminal Gal, and a small amount of terminal Man in agreement with the 4:1 ratio assigned by 1H NMR for the Gal- vs. Man-terminated isomers. The 3AB pool generated Man, mannitol and unchanged Hex3-ol upon [alpha]-mannosidase treatment, and Gal, 2B and unchanged Hex3-ol upon [alpha]-galactosidase treatment (Figure 5). These results also are consistent with the NMR findings and indicate the presence of two isomers; Gal[alpha]1,2Man[alpha]1,2Man-ol (3A, 82%) and Man[alpha]1,2Man[alpha]1,2Man-ol (3B, 18%).

Table II. Chemical shifts and coupling constants for monosaccharide components of S.pombe O-linked glycans
Pool Ringa Sugar (ppm) (Hz) (ppm)
C1-H (J1,2)b C2-H C3-H C4-H C5-H
2A Ga Gal[alpha]1,2-> 5.11 (4.0) 3.83 3.89 4.02 4.13
2B Mt Man[alpha]1,2-> 5.02 (4.7) 4.01 3.88 3.81  
3A Mx ->2Man[alpha]1,2-> 5.36 (4.9) 4.02 3.92 3.83  
Ga Gal[alpha]1,2-> 5.19 (3.8) 3.83 3.93 4.03 4.13
3B Mx ->2Man[alpha]1,2-> 5.27 (4.9) 4.03 3.71    
Mt Man[alpha]1,2-> 5.07 (4.8) 4.09 3.86    
4A Md ->2,3Man[alpha]1,2-> 5.33 (4.8) 4.16 4.11 3.98 3.87
Ga Gal[alpha]1,2-> 5.25 (4.1) 3.81 3.93 3.99 4.11
Gb Gal[alpha]1,3-> 5.28 (2.9) 3.87      
4B Md ->2,3Man[alpha]1,2-> 5.24 (4.9) 4.21 4.11 3.95 3.88
Mt Man[alpha]1,2-> 5.21 (4.7) 4.04 3.87 3.85 3.87
Gb Gal[alpha]1,3-> 5.28 (3.5) 3.87      
5AB Md ->2,3Man[alpha]1,2-> 5.43 (4.9) 4.23 4.09 3.97 3.87
Mx ->2Man[alpha]1,2-> 5.26 (4.9) 3.99 3.98 3.75 3.83
Ga Gal[alpha]1,2-> 5.25 (4.0) 3.82 3.93 4.02 4.10
Gb Gal[alpha]1,3-> 5.31 (3.1) 3.85 3.89 4.02 4.14
aThe naming convention for each ring system is the same as in Figures 6 and 8: Mt, terminal Man; Mx, 2-substituted Man; Md, 2,3-disubstituted Man; Ga, 2-linked Gal; Gb, 3-linked Gal.
bChemical shifts ([delta], ppm) and J1-2 coupling constants (Hz) are from the 2D DQF-COSY experiments part of which are shown in Figures 3 and 6.

Tetrasaccharides

The NMR spectra of 4A (Figure 3) indicated the presence of three anomeric protons. The 5.33 ppm signal had a 4.16 ppm C2-H COSY crosspeak tetraplet, indicative of a substituted Man. The C1-H through C4-H signals (Table II) are within the chemical shift range expected for a 2,3-disubstituted Man. For example, the 2-O-,3-O-disubstituted Man in Man[alpha]1,2(Galf[alpha]1,3)Man[alpha]1,2Man found in Trichophyton mentagrophytes oligosaccharides (Ikuta et al., 1997) has C1-H through C4-H resonances at 5.19, 4.31, 4.01, and 3.78 ppm, respectively, and the 2-O-,3-O-disubstituted Man in Gal[alpha]1,3(poly-GlcA[alpha]1,2)Man[alpha]1,3Galß1-, found in certain Klebsiella K10 oligosaccharides (Dutton et al., 1989), provides C1-H through C4-H resonances at 5.12, 4.25, 4.11, and 3.94 ppm, respectively. The 5.25 ppm signal and its axial 3.81 ppm C2-H resonance indicate an [alpha]Gal, which is either on the nonreducing terminus or is minimally 2,3-unsubstituted. The third signal, at 5.28 ppm, with a 3.87 ppm C2-H DQF COSY crosspeak resonance, is characteristic of [alpha]Gal. However, its overlap with the other Gal signal makes the typical axial C1-H to C2-H DQF COSY paired tetraplets, split due to passive coupling to Gal's C3-Hs, less pronounced. Isomer 4A reacted only with [alpha]-galactosidase giving Gal and 3AB on HPAEC analysis (Figure 5), and permethylation showed the presence of terminal Gal and a disubstituted species (Figure 4), indicating its composition to be Gal[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man-ol.


Figure 6. NMR analysis of the Hex5-ol isomer. The Hex5-ol isomer was purified as described in the text and examined by NMR; some of the data is summarized in Table II. Labels are as described in Table II and Figure 8: Mx, 2-substituted Man; Md, 2,3-disubstituted Man; Ga, 2-linked Gal; Gb, 3-linked Gal; Mol, mannitol. Numbers after a residue, e.g., Md2, indicate the ring carbon proton location, in this case a di-substituted Man's C2-H. The atom numbers of the individual mannitol signals were not labeled. Correlations between atoms in each spectra are indicated by hyphens. (A) details the 1D proton NMR spectrum. (B) shows a slice of the 2D DQF COSY experiment. (C) shows a slice of the 2D TOCSY experiment. (D) shows a 2D ROESY experiment, with fortuitous TOCSY signals indicated by T. (E) shows a composite of an HMBC experiment (inside gray boxes) overlaid onto an HMQC experiment (outside of gray boxes).

NMR analysis of 4B (Figure 3) also showed three anomeric signals. The signal at 5.24 ppm had C2-H through C4-H resonances similar to the disubstituted Man in isomer 4A, while the 5.28 ppm resonance belonged to a ring system like the [alpha]1,3-linked Gal in 4A (Table II). The 5.21 ppm signal had J1,2 (4.7 Hz) and J2,3 (4.9 Hz) coupling constants identifying this as a terminal [alpha]1,2-linked Man (Koerner et al., 1987), whose anomeric signal was shifted upfield due to the steric constraints of the neighboring [alpha]1,3-linked Gal. NOE-by-difference NMR spectroscopy (data not shown) showed that the C1-H of the Gal was close in space to the C3-H of the di-substituted Man species. Permethylation of 4B (Figure 4) indicated the presence of terminal Man, terminal Gal, and a disubstituted species. Isomer 4B was cleaved by [alpha]-galactosidase giving Gal and 3AB on HPAEC (Figure 5). The terminal [alpha]1,2-linked mannose was not cleaved from 4B by [alpha]-mannosidase, presumably due to steric hindrance by the [alpha]1,3-linked Gal. It is probable that the saccharide substitution on the equatorial C3 position of Man prevents access by the [alpha]-mannosidase just as steric hindrance retards the [alpha]-mannosidase digestion of Glc3Man5GlcNAc2 to Glc3Man4GlcNAc2 (Ziegler et al., 1994). These data indicated the structure of 4B to be Man[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man-ol.

Pentasaccharide

The 5A and 5B peaks from the preparative PA1 column coeluted when rechromatographed on the PA1 analytical column and had the same molecular weight by mass spectrometry (Table I). They had identical permethylation patterns (Figure 4) and 1D proton and 2D DQF COSY NMR spectra (Figure 6A,B). It was concluded that the two peaks represented conformational isomers of the same compound (5AB) stabilized by interaction with the anionic column packing resin at the pH conditions employed in HPAEC. Permethylation showed the presence of 2-O-substituted Man, terminal Gal and a disubstituted monosaccharide (Figure 4). NMR identified four anomeric protons. The ring protons of the Man in isomer 5AB with the anomeric proton signal at 5.25 ppm had analogous resonances and couplings to the Man residues in isomers 2A, 3A, and 4A (compare Figure 3 with Figure 6), whose anomeric protons resonated at 5.19, 5.19, and 5.25 ppm, respectively. Similarly, the 5.30 ppm Gal ring-system protons of isomer 5AB were similar to the 5.28 ppm ring-system protons in isomers 4A and 4B, and the 5.26 ppm resonance system in 5AB was similar to the 2-substituted Man ring systems in 3A and 3B. The ring protons of the 5AB Man whose C1-H resonated at 5.43 ppm was similar to the disubstituted Man rings described above. Pools 5A and 5B did not react with [alpha]-mannosidase, but did react with [alpha]-galactosidase giving Gal, 3AB, and an unknown Hex4-ol size species (4U in Figure 5). Since these data are consistent with one of two structures for the pentasaccharide: (1) Gal[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man[alpha]1,2Man-ol, or (2) Gal[alpha]1,2Man[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man-ol, several additional NMR experiments were employed to determine its configuration.

Figure 6C-E show sections of TOCSY, ROESY, HMQC, and HMBC NMR experiments for isomer 5AB. No ROESY interactions were observed between the anomeric proton of either Man and their respective C3- or C5-Hs, confirming their [alpha]-configuration. However, a strong ROESY interaction was seen (Figure 6D) between the C1-H of the disubstituted Man at 5.43 ppm and the C2-H of 2-substituted Man at 3.98 ppm (Md1-Mx2), indicating that this is the penultimate non-reducing-end residue. The additional ROESY interactions between Ga1 (5.25 ppm) and Md2 (4.22 ppm) and between Gb1 (5.30 ppm) and Md3 (4.11 ppm) indicate that this residue is 2,3-di-O-substituted (Figure 6D). This supports 5AB isomer (1) as the correct structure.

The HMQC experiment displays the resonance of each proton along the x-axis and the resonance of the carbon it is attached to along the y-axis. The type of HMBC experiment performed here, in essence, shows connectivities between the protons on carbons involved in glycosidic linkages (x-axis) and the carbon on the other side of the glycosidic bond (y-axis). The experiments in Figure 6E indicated four glycosidic linkages: (1) C1 of the 2-linked Gal to C2 of the disubstituted Man (Ga1-Md2); (2) the anomeric center of the 3-linked Gal to C3 of the disubstituted Man (Gb1-Md3); (3) the anomeric center of the disubstituted Man to C2 of the 2-substituted Man (Md1-Mx2); and (4) the anomeric center of the 2-substituted Man to a carbon in the mannitol (Mx1-Mol). Although overlaps, which could not be resolved at any temperature from 277 K to 330 K, exist along the x- and y-axes in these spectra, careful examination of both axes in the HMBC spectrum indicate these are the only glycosidic linkages in the molecule. All other resonances in Figure 6E are accounted for as C1-C3 and C1-C5 three-bond intra-ring interactions. Thus, the multiple-bond correlation experiment (Figure 6E) confirms that the 5AB isomer has the structure Gal[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man[alpha]1,2Man-ol originally deduced and assigned from the COSY and ROESY experiments. Since [alpha]-galactosidase removes the [alpha]1,3-linked Gal from 4B and generates 3A and Gal from 4A, it seems unlikely that it would preferentially remove a 2-linked Gal from a 2,3-disubstituted Man. Thus, the product shown in Figure 5, 4U, is likely to be Gal[alpha]1,2Man[alpha]1,2Man[alpha]1,2Man-ol, but insufficient product was isolated to confirm this supposition by NMR techniques.

Role of gma12p and gth1p in N-glycan modeling

To determine the effects of the gma12 and gth1 mutations on the production of large N-linked galactomannans, the Bio-Gel P-4 V0 glycan fraction from all four S.pombe strains was subjected to 1D proton NMR analysis (Figure 7). The presence of 6-monosubstituted Man, found at 4.9 ppm, is a measure of incomplete [alpha]1,2-galactosylation of the poly-[alpha]1,6-mannan backbone. This signal would show an increase, and the terminal Gal and 2,6-di-O-substituted Man a proportional decrease, if the backbone was undergalactosylated (Tabuchi et al., 1997). Analyzing the NMR spectra (Figure 7) reveals the oligosaccharides from all four strains to have nearly identical signatures, indicating that gma12p and gth1p play little or no role in the galactosylation of the mannan outerchain.


Figure 7. NMR analysis of S.pombe N-linked galactomannans from all four S.pombe strains. The large galactomannans purified from the S.pombe strains as described in the text were analyzed by 600 MHz 1D proton NMR spectroscopy. Selected signals are assigned as described elsewhere (Ziegler et al., 1998). The resonances of Gal's C1 proton (TG1), the 2,6-disubstituted Man backbone C1-H (DM1), The 3-substituted Gal C4 proton (3G4), the pyruvate CH3 protons (Pv), and the pyruvylated ß-Gal C1 (ßG1) and C4 (ßG4) protons are labeled.

Discussion

In agreement with earlier work (Ballou and Ballou, 1995), mannose was the only sugar detectable in O-glycan linkage to protein, Gal[alpha]1,2Man-ol (2A) and Man[alpha]1,2Man-ol (2B) were the only Hex2-ol components, while a Gal[alpha]1,2Man[alpha]1,2Man-ol species (3A) was present in S.pombe. However, our study found no evidence for substituted Gal, by either NMR or permethylation analyses, in any of the O-glycans isolated. This stands in contrast to the tetrasaccharide species reported elsewhere as Gal[alpha]1,2Gal[alpha]1,2Man[alpha]1,2Man-ol (Ballou and Ballou, 1995), which had the same anomeric proton resonances as seen here for isomer 4A (Figure 3), and we assigned as Gal[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man-ol. In addition to these structures, Man[alpha]1,2Man[alpha]1,2Man-ol (3B), and two other novel species, Man[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man-ol (4B) and Gal[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man[alpha]1,2Man-ol (5AB), were released from S.pombe glycoproteins by ß-elimination. The interrelationship of all of these isomers leads to the O-glycan biosynthetic pathway proposed in Figure 8.


Figure 8. Proposed pathway for O-glycosylation in S.pombe. Residues are labeled as in Table II and Figure 6: Mt, nonreducing terminal Man; Mx, 2-substituted Man; Md, 2,3-disubstituted Man; Ga, 2-linked Gal; Gb, 3-linked Gal; Mol, Mannitol. Arrows up, [alpha]1,2-linkage. Diagonal arrows, [alpha]1,3-linkage. Forward reaction arrows indicate the deduced processing pathway, with the enzymes involved indicated: Gma, gma12p; Gth/Gma, either gma12p or gth1p; Gtm, the unknown enzyme(s) which adds Gal to the 3-position of 2-O-substituted Man. Left reaction arrows indicate products obtained after digestion with either jack bean [alpha]-mannosidase (M[prime]ase) or coffee bean [alpha]-galactosidase (G[prime]ase) as described in Materials and methods. Structure 3U represents a theoretical glycosidase digestion product, and structure 4U represents a presumed biosynthetic O-linked intermediate not found on S.pombe glycoproteins in this current work.

In comparing the O-linked glycans from all the S.pombe strains (Figure 1), several functions for gma12p and gth1p could be assigned (Figure 8). Isomer 2A was absent in the gma12 strain, and isomers 4A and 5AB were greatly reduced, but not absent. Gma12p is known to be a UDP-Gal::Gal[alpha]1,2Man galactosyltransferase in vitro (Chappell and Warren, 1989), and appears to be solely responsible for the galactosylation of isomer 2A, Gal[alpha]1,2Man-ol, in vivo. The gth1 strain had reduced amounts of 3AB, 4A, and 5AB, while the double mutant lacked 2A and 4A entirely, and the amount of 5AB was greatly reduced, if not entirely, absent (Figure 1). This indicates that gth1 encodes a functional [alpha]1,2-galactosyltransferase, and that its specificity must overlap that of gma12p. This finding is similar to the [alpha]1,2-mannosyltransferase family with overlapping specificities recently detailed in S.cerevisiae (Lussier et al., 1997). The negligible amount of Gal[alpha]1,2Man-containing species in the double mutant indicates that these two enzymes account for most, if not all, of the [alpha]1,2-linked Gal in S.pombe O-glycans.

Although examination of large galactmannans from gma12, gth1, gma12/gth1, and wild-type S.pombe showed no clear-cut role for these encoded galactosyltransferases in their synthesis, gma12p has been shown to add [alpha]1,2-linked Gal to the Man9GlcNAc2 precursor to generate novel GalMan9GlcNAc2 core N-glycans (Ziegler et al., 1998). Additionally, the presence of branching Gal[alpha]1,3Man epitope on O-linked isomers 4A, 4B, and 5AB indicates the presence of an [alpha]1,3-galactosyltransferase activity previously unreported in S.pombe. The failure to find here any O-linked isomers with a lone [alpha]1,3-linked Gal terminus in the oligosaccharide structures, leads to the speculation that this galactosyltransferase introduces an [alpha]1,3-branch on an [alpha]1,2-linked mannose already 2-O-substituted by either Gal or Man. Interestingly, this enzyme also may be responsible for addition of an [alpha]1,3-linked Gal to the N-linked Man9GlcNAc2 core in S.pombe to form a GalMan9GlcNAc2 isomer in which the lower [alpha]1,3-linked arm has the structure Man[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Man[alpha]1- (Ziegler et al., 1998) as found in O-linked isomer 4B (Figure 8). Efforts are in progress to develop an assay for and to isolate and characterize this novel [alpha]1,3-galactosyltransferase.

Materials and methods

Materials

The gma12-deficient TCP3001 (h-,leu1-32, ura4-D12, his3-327, gma12-D10::ura4), gth1-deficient TCP3017 (h-,leu1-32, ura4-D12, his3-327, ade 1-D25, ade6-M210, gth1-D9::ura4), and gma12/gth1-deficient TCP3019 (h-,leu1-32, ura4-D12, his3-327, ade 1-D25, ade6-M210, gth1-D9::ura4, gma12-D10::ura4) strains of S.pombe were gifts of Dr. Thomas G.Chappell of The MRC Laboratory for Molecular and Cell Biology, London, England. The wild-type (gma12+/gth1+) S.pombe strain, 972 (h-), was from the American Type Culture Collection (ATCC #24843).

QAE-Sephadex and Sephadex G-50 were from Pharmacia Biotech, Piscataway, NJ. Bio-Gel P-4 and Bio-Gel P-2 gel filtration resins were from Bio-Rad Laboratories, Hercules, CA. All solvents were reagent grade or the purest grade available from commercial sources. Endo H was a cloned Escherichia coli product from this laboratory (Trumbly et al., 1985). PNGase F was a kind gift of Drs. Plummer and Tarentino of the Wadsworth Center. Coffee bean [alpha]-galactosidase was from Boehringer Mannheim Corp., Indianapolis, IN, and jack bean [alpha]-mannosidase was purified as described previously (Li and Li, 1972). Sep-Pak C18 disposable cartridges were obtained from Millipore Corp., Bedford, MA. Man[alpha]1,2Man-ol was isolated from S.cerevisiae by ß-elimination and characterized by 1H NMR and permethylation analysis (Cohen and Ballou, 1980). Galß1,3Galß1-OCH3, Man[alpha]1,(2,3 and 6)Man methyl glycosides and 99.96% 2H2O were from Sigma Chemical Corp., St. Louis, MO. Cambridge Isotopes Lab Inc., Woburn, MA, supplied the 99.996% 2H2O. Monosaccharide standards and the MA1, PA1, and PA-100 HPAEC columns were procured from Dionex Corp., Sunnyvale, CA. V-Labs inc. of Covington, LA provided the 2[prime]-fucosyllactose and the Gal[alpha]1,3Galß1,4Gal[alpha]1,3Gal.

Cell growth, glycoprotein preparation, and N-deglycosylation

Cells were grown in YEA medium at 32°C, and whole-cell glycoproteins were extracted and purified as described previously (Ziegler et al., 1994; Gemmill and Trimble, 1996). N-glycans were removed from the glycoproteins as described elsewhere (Gemmill and Trimble, 1996) by sequential treatment with endo H and PNGase F. The endo H-released oligosaccharides were recovered and purified by Bio-Gel P-4 chromatography. Little oligosaccharide was removed by the PNGase F treatment, and it was assumed that this residual glycan was resistant to endo H because of inaccessibility due to protein conformation.

O-Glycan preparation

The residual glycoprotein was washed with 50% methanol three times to remove any remaining soluble carbohydrate, then dried and dissolved in 40 ml 0.1 M NaOH containing 1 M NaBH4 and allowed to stand 16 h at 45°C. The solutions were acidified by dropwise addition of acetic acid. Cations were removed by passage through two 20 ml columns of Dowex 50 H+. Borate was removed by three rotary evaporations with 1% acetic acid in methanol. The samples were then passaged through 5 ml QAE-Sephadex carbonate columns as described previously (Gemmill and Trimble, 1996).

Glycosidase reactions

Jack bean [alpha]-mannosidase and coffee bean [alpha]-galactosidase digestions were performed on 10 nmol of oligosaccharide in 100 µl PCR tubes for 3 days at 37°C in 50 µl of 20 mM sodium acetate containing 2 mM CaCl2 at pH 4.5 and 6.5, respectively. The reactions were stopped by the addition of 450 µl of methanol and centrifuged to remove precipitated protein. The supernatants were evaporated to dryness under vacuum and resuspended in 100 µl 90% methanol. The samples were again centrifuged to remove insoluble protein and the supernatants evaporated under vacuum, resuspended in 50 µl water, and analyzed directly by HPAEC-PAD as described below. The unusual glycosidase buffer described here was used to allow analysis of small amounts of analytes without risking possible loss due to the ion exchange columns normally used to remove protein and buffer salts. The enzymes were at least 80% as active with their respective PNP-substrates under these conditions when compared with standard buffering conditions, and they retained at least 50% of their original activity after 3 days at 37°C.

HPAEC-PAD oligosaccharide chromatography

HPAEC-PAD oligosaccharide chromatography was performed on a Dionex system using one of two methods. Analytical determinations were performed using a 4 × 250 mm PA1 column at 1 ml/min using 20 mM NaOH as the eluant. Preparative chromatography was performed using a 10 × 250 mm PA-100 column with the same conditions, except at a flow rate of 2 ml/min. In both cases PAD settings were E1 = 0.05, E2 = 0.7, E3 = -0.15; T1 = 4, T2 = 3, T3 = 6 with attenuation set at either 1000 or 3000 nA full scale, depending on sample load.

Permethylation analysis

Samples were permethylated by the NaOH method (Ciucanu and Karek, 1984) and hydrolyzed in 4M CF3CO2H for 4 h at 100°C. The resulting partially methylated monosaccharides were separated by TLC and visualized as described previously (Mukerjea et al., 1996).

NMR methods

Samples were prepared as described previously (Ziegler et al., 1994) and examined on a 500 MHz Bruker Avance spectrometer at 300 K using acetone as an internal reference ([delta], ppm = 2.225). The 1D proton and 2D DQF COSY experiments were performed as described previously (Ziegler et al., 1998). The HMQC experiment, which detects coupling between protons and the carbons they are bonded to, was performed using a JCH of 145 Hz. The HMBC experiment, which detects coupling between the C1-H protons and the carbons attached by a glycosidic linkage, was performed using a JCH of 145 Hz and a 3JCH of 14.8 Hz. The carbon dimensions in both the HMBC and HMQC experiments were referenced to the spectrometer frequency and not to an internal chemical shift marker.

Other methods

Bio-Gel P-4 chromatography methods have been described previously (Ziegler et al., 1994). Neutral hexose quantitation was by a modification (Byrd et al., 1982) of the phenol-sulfuric acid method (Dubois et al., 1956).

Acknowledgments

We thank Dr. John Cavanagh, Ms. Lynn McNaughton, and Dr. Susan Baxter of the Wadsworth Structural NMR Facility for help with the NMR data acquisition and Ms. Cathy Lubowski for preparing a portion of the S.cerevisiae Man[alpha]1,2Man-ol. We are indebted to Dr. Charles Hauer and Mr. Robert Stack of the Wadsworth Biological Mass Spectrometry Laboratory for providing the MALDI/TOF mass spectra. We gratefully acknowledge the Wadsworth Center photography unit for help with the illustrations and Ms. Tracy Godfrey for final manuscript preparation. This work was supported, in part, by USPHS Grant GM-23900 to R.B.T.

Abbreviations

ER, endoplasmic reticulum; Endo H, endo-ß-N-acetylglucosaminidase H (E.C. 3.2.1.96); PNGase F, peptide-N4-asparagine amidohydrolase F (E.C. 3.2.2.52); HPAEC, high pH anion exchange chromatography; PAD, Pulsed amperometric detection; TLC, thin-layer chromatography; MALDI/TOF, matrix assisted laser desorption ionization time of flight; MS, mass spectrometry; Man-ol, mannitol; HexN-ol, HexN-1Hexitol; Galf, galactofuranose; DQF, double quantum filtered; HMBC, heteronuclear multiple bond connectivity; HMQC, heteronuclear multiple quantum coherence.

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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
4In partial fulfillment of the Ph.D. degree requirements of T.R.G.


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