Novel Schizosaccharomyces pombe N-linked GalMan9GlcNAc isomers: role of the Golgi GMA12 galactosyltransferase in core glycan galactosylation

Frederick D. Ziegler1, John Cavanagh1,2, Catherine Lubowski1 and Robert B. Trimble1,2,3

1Wadsworth Center, New York State Department of Health, Albany, NY, USA and 2Department of Biomedical Sciences, State University of New York at Albany, 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 synthesizes very large N-linked galactomannans, which are elongated from the Man9GlcNAc2 core that remains after the trimming of three Glc residues from the Glc3Man9GlcNAc2 originally transferred from dolichyl pyrophosphate to nascent proteins in the endoplasmic reticulum. Prior to elongation of the galactomannan outer chain, the Man9GlcNAc2 core is modified into a family of Hex10-15GlcNAc2 structures by the addition of both Gal and Man residues (Ziegler et al. (1994) J. Biol. Chem., 269, 12527-12535). To understand the pathway of Man9GlcNAc2 modification, the Hex10GlcNAc-sized pool was isolated by Bio-Gel P-4 gel filtration from the endo H-released N-glycans of S.pombe glycoproteins. This pool yielded four major fractions, a, b, c, and g, on preparative high pH, anion exchange chromatography, that represented 10, 29, 46, and 13% of the total Hex10GlcNAc present, respectively. Structures of the glycan isomers present in each fraction were determined by one- and two-dimensional 1H NMR spectroscopy techniques. Fraction a is principally (~93%) a Man10GlcNAc with a new [alpha]1,2-linked Man cap on the upper-arm of Man9GlcNAc. Fraction b contained two isomers of GalMan9GlcNAc in which an [alpha]1,2-linked terminal Gal had been added either to the upper (b1, 30%) or middle-arm (b2, 70%) of Man9GlcNAc. The gma12- [alpha]1,2-galactosyltransferase-negative S.pombe strain (Chappell et al. (1994) Mol. Biol. Cell., 5, 519-528) did not make fraction b implying that the gma12p galactosyltransferase is responsible for synthesis of both isomers b1 and b2. Isomer c is Man10GlcNAc in which a new branching [alpha]1,6-linked Man had been added to the lower-arm [alpha]1,3-linked core residue as found earlier in Saccharomyces cerevisiae and Pichia pastoris. Fraction g had less than molar stoichiometry of both Gal and Glc. The major isomer (g1, 85%) is the Man9GlcNAc core with an [alpha]1,3-linked branching Gal on the penultimate 2-O-substituted Man of the lower arm. This residue is also found on a novel O-linked oligosaccharide recently described in S.pombe; Man[alpha]1,2(Gal[alpha]1,3)Man[alpha]1,2Mannitol (Gemmill and Trimble (1999) Glycobiology, 9, 507-515). The second isomer (g2, 15%) is the partially processed Glc2Man9GlcNAc intermediate. Defining these Hex10GlcNAc structures provides a starting point for understanding the enzymology of N-linked galactomannan core heterogeneity seen on S.pombe glycoproteins.

Keywords: glycan 1H NMR/oligosaccharide processing/S.pombe/yeast glycoproteins

Introduction

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).

Results

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 1 shows that the wild-type strain yielded a typical series of Hex9-15GlcNAc core glycans (Ziegler et al., 1994), while the oligosaccharide profile obtained from gma12- glycoproteins (not shown) appeared depleted in the core-filling species larger than Hex12GlcNAc. The final Bio-Gel P-4 profile of the isolated, pooled Hex10GlcNAc-sized glycans from each strain provided a symmetrical peak, which is shown for the wild-type in Figure 1B. MALDI-TOF MS of the pooled fractions from Figure 1B (tubes 129-133) gave a single signal with a m/z value of 1865, consistent with the calculated [Hex10GlcNAc+Na]+ mass of 1865.7 Da. (Figure 1C). Analytical HPAEC showed that Hex10GlcNAc from both the wild-type and galactosyltransferase-deficient strains contained up to seven resolvable peaks, labeled a-g in Figure 2. Although the gma12- strain was predominantly (85%) composed of peak c, it is noteworthy that peak b was not present in the N-linked Hex10GlcNAc pool from the transferase-deficient strain.

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 2, both were present in the wild-type and the gma12- strains. Peak b was found only in the wild-type strain (Figure 2) and contained Man, Gal, and GlcNAc in a ratio of about 9:1:1. The last major peak, g, had Glc, Gal, Man, and GlcNAc in proportions consistent with Glc0.2Gal0.8Man9GlcNAc, suggesting the presence of at least two isomers in this fraction. Peak g's composition was essentially the same in the gma12- strain and wild-type, but the amount of this Hex10GlcNAc was about 3-fold lower in the former compared to the latter in the absence of peak b. Several minor peaks (d, e, and f) were observed at a level of a few percent each. Peak d may be the epimer of Man10GlcNAc formed in the presence of strong base used for HPAEC as described previously (Trimble et al., 1991), as this species had a sugar composition of Man10HexNAc. Peaks e and f were not present in sufficient quantity for further analysis but yielded monosaccharide compositions consistent with GalMan9GlcNAc and GlcMan9GlcNAc, respectively.

Table I. Monosaccharide composition and relative proportion of isomers in wild-type and gma12- S.pombe Hex10GlcNAc oligosaccharides
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
aDetermined on wild-type isomers preparatively-separated into the peaks shown in the analytical profile in Figure 2A. Details described are in Materials and methods.

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 3 and C1-H proton intensities obtained by integrating the 1D signals are summarized in Table II. Table II lists the approximate chemical shifts ([delta], p.p.m.) where anomeric protons for sugar residues in the specified linkages nominally are found. Fractional intensities from the 1D spectra (Table II) are used to apportion isomer amounts in each pool. The data for the precursor Man9GlcNAc (Scheme I) are included in Table II for reference. The actual C1-, C2-, and C3-H chemical shifts measured from the 2D DQF-COSY spectra for each of the ring systems in pools a-c and g are summarized in Table III. These shifts are needed to assign proton-proton interactions across glycosidic linkages using the ROESY experiments described in Figures 4 and 4 below. The comparable chemical shifts for sugar ring systems in the precursor Man9GlcNAc (Scheme I) also are included in Table III for reference. The structures deduced for the pool a-c and g components are shown in Scheme II.


Table II. C1-H intensity integrations for the S. Pombe core Man9GlcNAc and Hex10,11GlcNAc fractions a-c, and g separated by HPAEC
aAll linkages are [alpha] unless noted otherwise.
bS.Pombe Man9GlcNAc (Scheme I, ref. 1) is identical to that from soybean aglutinin, thyroglobulin, lactotransferrin and CHO cell membranes: Dorland,L., et al. (1981) J. Biol. Chem., 256, 7708-7711.
cThe combined resonance intensities for multiple residues at the same chemical shift are bracketed.
dNot determined.
eCore residue 3 could not be integrated accurately due to water suppression, but must be present in all N-linked glycan cores.

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. Assignment of the C1-, C2-, and C3-H chemical shifts to the sugar ring systems in S.pombe Man9GlcNAc core and Hex10,11 GlcNAc elongation products in HPAEC fractions a-c and g
aAll linkages are [alpha] unless noted otherwise.


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 3A is the normal signature for residue 6 when [alpha]1,2-Man residue 9 is terminal, as in Man9GlcNAc (Scheme I, Table II). This chemical shift correlates with the expected C2-H resonance at 4.006 p.p.m.. Thus, pool a appears to contain a minor Man10GlcNAc isomer that may have an additional terminal [alpha]1,2-linked Man on either residues 10 or 11 of the middle or lower arms, respectively (Scheme I). The characteristic upfield shift in residues 7 or 8 that would be expected on addition of an [alpha]1,2-Man to either residue 10 or 11, respectively, was too weak to allow rigorous assignment of this component.


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 2B). Thus, the role of gma12p in S.pombe core galactosylation should be revealed by defining the structure of this isomer.

The 1D 1H NMR spectrum (Figure 3B) integrated to 10 mol of anomeric hexose protons (Table II) and revealed three major differences compared to the Man9GlcNAc precursor (Ziegler et al., 1994, and Table II): (1) an additional mole of resonance intensity at about 5.40 p.p.m. indicative of a new 2-0-substituted [alpha]1,2 linked Man; (2) an additional mole of resonance intensity at 5.160 p.p.m. with a J1,2 coupling constant of 3.5 Hz expected for an [alpha]-anomeric proton and an axial C2-H as found in Gal; and (3) diminution of 1 mol of terminal [alpha]1,2-Man at 5.042 p.p.m. In conjunction with the compositional analysis (Table I), all of the NMR observations are consistent with the addition of a terminal [alpha]1,2-linked Gal to one of the terminal [alpha]1,2-linked Man residues, 9, 10, or 11, in Man9GlcNAc (Scheme I).

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 4, and the chemical shifts for ring protons are summarized in Table III. The ROESY interresidue interactions, mapped in Figure 4B, show a major crosspeak signal between Gal's C1-H (5.160 p.p.m.) and [alpha]1,2-linked Man residues 9, 10, and/or 11[prime]s C2-H (4.095 p.p.m.). That it is mostly 10[prime]s C1-H being shifted downfield from 5.058 p.p.m. to 5.396 p.p.m. on Gal addition is shown by the ROESY crosspeak between 10[prime]s C1-H at 5.396 p.p.m. and 7[prime]s C2-H at 4.074 p.p.m., confirming the glycosidic linkage between these two residues. Furthermore, 7[prime]s C1-H at 5.404 p.p.m. shows the expected crosspeak with 4[prime]s C3-H at 3.889 p.p.m.. These interactions indicate the following predominant middle arm structure,

                       10           7            4
          Gal[alpha]1,2Man[alpha]1,2Man[alpha]1,3Man[alpha]1,6-

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 4B).

Inspection of Figure 3B and Table II reveals about 0.3 mol of 6[prime]s C1-H intensity is shifted from its normal resonance at 5.145 p.p.m. in Man9GlcNAc to 5.123 p.p.m.. As discussed above (peak a) and earlier (Cohen and Ballou, 1980; Trimble et al., 1991), this results when residue 9 is 2-O-substituted and is seen in the 2D DQF-COSY spectrum (Figure 4A) as a minor C1-H/C2-H crosspeak at 5.128/4.003 p.p.m.. Thus, the ROESY crosspeak between Gal's C1-H at 5.160 p.p.m. and [alpha]1,2-linked Man C2-Hs at 4.095 p.p.m. (Figure 4B) also contains resonance intensity due to the 2-O-substitution of residue 9 by Gal resulting from the upper arm structure,

                           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 4B). Terminal Man 11 does not appear to be substituted by Gal in the peak b components on the basis of the intense crosspeak between 11[prime]s C1-H at 5.042 p.p.m. and 8[prime]s C2-H at 4.105 p.p.m., where each of these signals normally are found in the precursor Man9GlcNAc (Table III). Thus, peak b consists of the two Gal-containing isomers shown in Scheme II as b1 and b2 in a 7:3 ratio on the basis of integrations from the 1D spectrum (Table II).

Note the small resonance intensity at 4.930 in the 1D spectrum of b in Figure 3B. Due to pooling of fractions from separate preparative HPAEC runs, there appears to be a small amount of peak c in pool b (Figure 2A). This 4.930 p.p.m. signal is from residue 12, the new [alpha]1,6-linked Man branch added to lower arm residue 5 (see pool c results, below). In this isomer, residue 5 and 8[prime]s C2-Hs are shifted to 4.075 p.p.m. and 4.087, respectively, and provide the minor ROESY crosspeak between 8[prime]s C1-H at 5.308 p.p.m. and 5[prime]s C2-H at 4.087 p.p.m. (Figure 4B).

Pool c

The 1D 1H NMR spectrum for the Man10GlcNAc in pool c (Figure 3C) integrated to 10 anomeric protons (Table II) and had one additional signal compared to the precursor Man9GlcNAc; 1 mol of resonance at 4.932 p.p.m. indicative of a new terminal [alpha]1,6-linked Man (Cohen and Ballou, 1980). The C1- C2-, and C3-H assignments were derived from c's 2D DQF-COSY spectrum (not shown) and are listed in Table III. With the exception of retaining terminal [alpha]1,2-linked Man 10 (Scheme I), the spectrum of S.pombe Man10GlcNAc is identical to S.cerevisiae (Trimble and Atkinson, 1986; Hernández et al., 1989) and Pichia (Trimble et al., 1991) Man9GlcNAc processing intermediates, which have a new [alpha]1,6 linked Man residue 12 attached to core residue 5. Although early chemical, MS, and NMR data placed 12 on upper-arm 9 (Byrd et al., 1982; Cohen et al., 1982; Trimble and Atkinson, 1986), subsequent work has reassigned residue 12 to the lower arm (Hernández et al., 1989; Ballou et al., 1990; Reason et al., 1991), as shown in Scheme II, structure c. Additional historical aspects of the re-evaluation of this structure have been reviewed recently (Trimble and Verostek, 1995). Interestingly, this unique branching structure, to date found only in yeast, explains how alg3 S.cerevisiae, which lacks the upper arm residues 6, 7, 9, and 10 (Scheme I), can still generate complete outer chain mannan (Verostek et al., 1993a). The gene for the enzyme responsible for the addition of residue 12 in S.cerevisiae, OCH1, has recently been cloned (Nakanishi-Shindo et al., 1993).

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 3G) showed two major differences compared to the precursor Man9GlcNAc (Scheme I, Table II). The first was 1.9 mol of resonance intensity at 5.282-5.288 p.p.m., suggesting addition of a new residue, perhaps the Gal, associated in some way with the shift of most of residue 8[prime]s resonance intensity upfield from 5.304 p.p.m.. The second difference was the appearance of about 0.9 mol of intensity at 5.178 p.p.m., which may be most of residue 11[prime]s signature shifted downfield from its normal position at 5.042 p.p.m.. Note the presence of a small resonance signal at 5.255 p.p.m., as expected for the C1-H of a Glc residue (Tsai et al., 1984) predicted to be a minor constituent from pool g's compositional analysis (Table I).

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 5A) is characteristic of monosaccharides such as [alpha]-Gal with an axial C2-H. The ROESY spectrum (Figure 5B) reveals a crosspeak between Gal's C1-H at 5.288 p.p.m. and Man 8[prime]s C3-H at 4.071 p.p.m., assigned from the 2D DQF COSY experiment (Table III). This assignment confirms that it is residue 11 that provides the 2D DQF-COSY crosspeak at 5.178/4.029 p.p.m. (C1-H/C2-H in Figure 5A), because of the ROESY crosspeak seen between Man 11[prime]s C1-H at 5.178 p.p.m. and Man 8[prime]s C2-H at 4.294 p.p.m. (Figure 5B) to which it is attached in the core structure (Scheme I). All other 2D DQF-COSY and ROESY crosspeaks for remaining core residues 3-7, 9, and 10 are found where expected and labeled accordingly in Figure 5B (see also Table III). The integrated areas indicate that the major isomer, representing 85-90% of pool g, is the core Man9GlcNAc with an [alpha]1,3-linked Gal on Man residue 8 as configured in Scheme II, structure g1.

Interestingly, two additional C1-H/C2-H crosspeaks are apparent in g's 2D DQF-COSY (Figure 5A), one at 5.255/3.550 p.p.m., mentioned above, and the other at 5.402/3.555 p.p.m., which are the expected C1-H/C2-H crosspeaks for the two [alpha]1,3-linked Glc residues present in the Glc3Man9GlcNAc originally transferred in the ER to proteins that have survived glucosidase II trimming (Verostek et al., 1993b; Tsai et al., 1984). The weak crosspeak at 5.039/4.235 p.p.m. is the C1-H/C2-H correlation for residue 11 when 3-O-substituted by Glc, and accounts for the excess over 2 mol integrated at 5.03-5.06 p.p.m. for residues 9 and 10 in the 1D spectrum (Figure 3, Table II). This downfield shift in 11[prime]s C2-H also occurs on addition of [alpha]1,3-linked Man to S.cerevisiae core mannans (Trimble and Atkinson, 1986; Verostek et al., 1993b). As with 11, there is a weak signal at 5.304/4.110 p.p.m. for the C1-H/C2-H crosspeak of residue 8, which is not 2,3-disubstituted with Man and Gal (Figure 5A). The presence of Glc2Man9GlcNAc in pool g was surprising, given the absence of an obvious Hex11GlcNAc signal in the MALDI-TOF MS spectrum of the Hex10GlcNAc pool (Figure 1C). However, since this glucosylated species represents at most about 15% of pool g, which itself is less than 15% of the total Hex10GlcNAc pool analyzed by MS (Table I), it was likely below the limit of detection by this method. The structure of this component of pool g is shown in Scheme II as g2.

Discussion

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.

Materals and methods

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.

Acknowledgments

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.

Abbreviations

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.

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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


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