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J. Biol. Chem., Vol. 277, Issue 51, 49143-49157, December 20, 2002

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The Fine Structure of Caenorhabditis elegans N-Glycans^*,

John F. Cipollo, Catherine E. Costello^§, and Carlos B. Hirschberg^¶

From the  Department of Molecular and Cell Biology, Boston University, Goldman School of Dental Medicine and ^§ Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118-2526

Received for publication, August 6, 2002, and in revised form, October 1, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We report the fine structure of a nearly contiguous series of N-glycans from the soil nematode Caenorhabditis elegans. Five major classes are revealed including high mannose, mammalian-type complex, hybrid, fuco-pausimannosidic (five mannose residues or fewer substituted with fucose), and phosphocholine oligosaccharides. The high mannose, complex, and hybrid N-glycan series show a high degree of conservation with the mammalian biosynthetic pathways. The fuco-pausimannosidic glycans contain a novel terminal fucose substitution of mannose. The phosphocholine oligosaccharides are high mannose type and are multiply substituted with phosphocholine. Although phosphocholine oligosaccharides are known immunomodulators in human nematode and trematode infections, C. elegans is unique as a non-parasitic nematode containing phosphocholine N-glycans. Therefore, studies in C. elegans should aid in the elucidation of the biosynthetic pathway(s) of this class of biomedically relevant compounds. Results presented here show that C. elegans has a functional orthologue for nearly every known enzyme found to be deficient in congenital disorders of glycosylation types I and II. This nematode is well characterized genetically and developmentally. Therefore, elucidation of its N-glycome, as shown in this report, may place it among the useful systems used to investigate human disorders of glycoconjugate synthesis such as the congenital disorders of glycosylation syndromes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Much of the current understanding of glycosylation processes in eukaryotes has been derived from yeast, mammalian cell lines, and organ tissues. Although these systems have been useful, they are not suited for the analysis of these processes in multicellular organisms as they relate to genetic, developmental, or environmentally interactive processes.

Caenorhabditis elegans is a developmentally well characterized model organism. Its genome has been sequenced, and the organism can be genetically manipulated. Several gene ablation services are currently available, and these should increase the rate of genetic and biochemical progress. The wealth of genetic information has allowed the cloning of some glycosyltransferases through reverse genetics (1, 2). However, the lack of C. elegans N-glycan structural information has made substrate specificity and enzyme activity difficult to identify. Recently, the major O-linked glycan structures were reported (3). The observation of a novel 1,6Glc substitution facilitated the identification of the elusive activity of the previously cloned enzyme (4). Therefore, the elucidation of the C. elegans N-glycan structures is warranted. Once this system is in place, the effect that aberrant glycan biosynthesis has at the multicellular level can be more clearly understood. Such an effect has been shown by the study of the sqv mutants where metabolic error in chondroitin biosynthesis leads to malformation of the nematode vulva (5, 6).

There is a growing number of human genetic disorders wherein an error in biosynthesis or metabolism leads to dysregulation of glycoconjugate synthesis and developmental impairment. This group of diseases has been classified under the general heading congenital disorders of glycosylation (CDG)¹ (7, 8). Yeast has facilitated the discovery of several causative gene lesions associated with these human disorders. However, because yeasts are simple eukaryotes, information provided by yeast systems is only inferential when extrapolated to the multicellular organism. C. elegans may aid in such investigations because the effects of errors in glycosylation manifest themselves throughout cell lineages and resultant tissue formation.

Here we present an overview of the N-glycans produced by C. elegans. From mixed stage worms we have characterized a nearly contiguous series of N-glycans that suggest the major N-glycosylation pathways in this nematode. Together with the sequenced genome and mapped cell fates of the organism, this information will facilitate understanding the role of glycosylation in development.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

All chemicals were ACS grade or better. Phosphocholine calcium salt was from Fluka (Fluka Chemie GmbH, Industriestrasse 25, CH-9471 Buchs SG, Switzerland). TEPC-15 antibody and anti-mouse horseradish peroxidase conjugate were from Sigma, and the chemiluminescence detection kit was from PerkinElmer Life Sciences. The PNGase F used in this study was the kind gift of Dr. Thomas Plummer.

Western and Carbohydrate Blotting

SDS-PAGE and blotting to polyvinylidene difluoride membranes was performed as described previously (9). Wheat germ agglutinin was purchased from EY (EY Laboratories, Inc., San Mateo, CA) and TEPC-15 from Sigma. Protein loading was normalized to protein concentration.

Oligosaccharide Purification

Glycan Extraction-- The procedure was monitored by Bearden's protein assay (10) and phenol-sulfuric assay for neutral hexose (11). C. elegans N2 worms (100 g wet weight) were suspended in 10 mM phosphate buffer, pH 7.0, which included 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, and 0.3% sodium azide. Worms were disrupted by bead beating with 425-600-µm glass beads in a BioSpec Bead Beater® 6 times for 1 min and were submerged in ice for 5 min between repetitions. Worm homogenate was lyophilized, followed by delipidation by 4000-fold extraction from 10:10:1 chloroform/methanol/water (CMW) and removal of free glycan by extensive solvent extraction from 50% methanol, which was monitored for neutral hexose by phenol-sulfuric assay. No protein was detectable in the methanol extract by Bearden's protein assay, indicating that the detected carbohydrate was not covalently linked to protein. Protein was solubilized in 125 mM Tris chloride, pH 6.8, 1% SDS, 5% -mercaptoethanol buffer (TSB), by boiling for 10 min followed by agitation on a rotary shaker for 3 h and then centrifugation at 10,000 × g for 10 min. The procedure was performed three times, and the soluble supernatants were combined, dialyzed, and digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma) for 4 h at 37 °C in 50 mM ammonium bicarbonate, pH 8.5. The reaction was stopped by boiling two times for 10 min. An adaptation of the solvent precipitation method for the isolation of oligosaccharides of Verostek et al. (12) was employed. The majority of peptides and glycopeptides (GP) were precipitated in 80% acetone. To recover the remaining peptides in the 80% acetone, the solvent was rotary evaporated to dryness, the residue resuspended in dH₂O, and peptides further purified over C-18 Sep-Pak® by step elution using 2 column volumes each of 20, 40, 60, and 100% isopropyl alcohol subsequent to an aqueous wash of dH₂O to first remove residual contaminants from the C-18-bound PG. Solvent and C-18-purified GP were combined and glycans released with PNGase F activity. The reaction was adjusted to pH 5.5 with acetic acid, and oligosaccharide was recovered by precipitation in 50% methanol at 20 °C for 4 h followed by centrifugation. The 50% methanol extract was rotary evaporated to dryness and reconstituted in dH₂O, and residual peptide/glycopeptides were removed using the same Sep-Pak® C-18 procedure described above. At this step the dH₂O eluate contained glycan, and residual GP was recovered in the isopropyl alcohol elution. The recovered GP was rotary evaporated, resuspended in 50 mM pH 5.5 ammonium acetate buffer, and digested with PNGase A at 2.5 milliunits/ml (Roche Diagnostics). The released oligosaccharides and GP were collected using the same procedures as employed for those released by the PNGase F. PNGase A released N-glycans with 1,3Fuc linked to the reducing end GlcNAc residue, whose linkages are not accessible to PNGase F.

Chromatography-- Size exclusion chromatography was performed on a 2.6 × 100-cm Bio-Gel P-4 column calibrated with cytochrome c as the void volume marker, Glc_0-3[³H]Man_5-9GlcNAc₂ oligosaccharides, and raffinose. HPAEC was performed on a Dionex DX-600 chromatography system equipped with a PA-100 analytical column and PAD detector using methods reported previously (13). Fractions were collected for off-line MS and NMR analysis (see below).

Chemical Procedures

Hydrofluoric Acid Hydrolysis-- Oligosaccharides were evaporated to dryness and resuspended in 48% hydrofluoric acid (HF) for 48 h. The reaction was stopped by neutralization with saturated LiOH solution. LiF salt precipitate was removed by centrifugation, and the hydrolyzed product was evaporated in a Savant® Speed evacuation apparatus.

Permethylation of Polysaccharides-- Approximately 0.10-3.0 nmol of each polysaccharide sample was dried in a Savant® speed evacuation apparatus and permethylated using a modification of the Me₂SO/NaOH method of Ciucanu and Kerek (14). Briefly, ~200 mg of NaOH was added per ml of Me₂SO. Dried polysaccharides were resuspended in the NaOH/Me₂SO solution (100 µl) and allowed to stand at room temperature for 1 h. One-half volume of iodomethane (Sigma) was added, and the reaction was allowed to proceed for 1 h, at which time an additional volume of NaOH/Me₂SO and one-half volume of iodomethane were added. The reactions were allowed to proceed for an additional hour and then were terminated by addition of 2 reaction volumes of dH₂O. An equal volume of chloroform was added, and the permethylated sugars, which partition into the hydrophobic phase, were extracted repeatedly against dH₂O. The chloroform was evaporated and sample reconstituted in 70% methanol.

For the permethylation of HF-treated glycans, the reaction was ended after the 1st h and the chloroform/dH₂O wash procedure was performed. The sample was evaporated by speed evacuation and again permethylated for an additional hour followed by reaction termination and processing as above.

Monosaccharide Analysis

Monosaccharide composition of glycopeptide/peptide and released glycans was determined by GC/MS on a Finnigan TRACE GC 2000 series coupled to the GCQ plus electron ionization ion trap mass spectrometer. Monosaccharides generated by acid hydrolysis of glycans were trimethylsialylated and analyzed.

Mass Spectrometry

MALDI-TOF MS was performed on permethylated glycans using either a Finnogan Vision 2000 or Bruker Reflex IV instrument fitted with a nitrogen laser (337 nm, 3-ns pulse width) operated in positive ion linear and/or reflectron mode(s). To acquire branch information on select glycan species, jack bean -mannosidase digestions were performed using a method published previously (15) followed by permethylation and analysis by MALDI-TOF MS. Targets were spotted in a layered fashion allowing each layer to dry before adding the next. First, 1 µl of 6-aza-2-thiothymine (15 mg/ml in 70% methanol) was spotted. Next, an equal volume of 20 mM NaOAc was layered over the top, and finally 15-30 pmol of the permethylated oligosaccharide mixture in 70% methanol was layered. The TOF MS mass scales were calibrated externally with a mixture of malto-oligosaccharides in the size range of Glc_4-10 purchased from Sigma. Permethylated authentic Man₉GlcNAc₂ (Sigma) was also used as external standard. Average masses were reported for spectra collected in linear mode, and monoisotopic values for the ¹²C peak were reported for those collected in reflectron mode. Mass accuracy was ± 0.1%.

¹H NMR Spectroscopy

Polysaccharide samples were exchanged from 99.9% ²H₂O (Cambridge Isotope Laboratories, Andover, MA) three times followed by three additional exchanges from 99.96% ²H₂O (Cambridge Isotopes) three times. The samples were then reconstituted in 99.96% ²H₂O and lyophilized for 2 days and stored over P₂O₅ in vacuo for several days. Samples were reconstituted in 99.996% ²H₂O (Cambridge Isotopes) with 5 mM acetone added as internal standard. All spectra were collected on a Bruker Avance 500 MHz spectrometer. Spectral width for one-dimensional experiments was 3008 Hz collected with 8 K points digitization. Total scans were 1024. The two-dimensional DQF-COSY experiments were performed with spectral widths of 1608 Hz in both t₁ and t₂ dimensions. Digitization was 512 and 4096 points in t₁ and t₂, respectively. Line broadening of 3-5 Hz/Hz was used in both dimensions, and a sine bell squared adiposation function was applied in t₂. One-dimensional spectra were collected at 300 and 318 K and internally referenced to acetone at 2.225 and 2.217 ppm, respectively. DQF-COSY spectra were collected at 300 K and internally referenced to acetone at 2.225 ppm. For resonance intensity integrations, FIDs were collected for ~5 times t₁ relaxation time.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of C. elegans N-Glycans for Structural Analysis

By using wheat germ agglutinin for detection, we found that C. elegans N-glycans are releasable with PNGase F. Only a trace amount of N-glycan was detectable by ¹H NMR analysis of PNGase A released material performed subsequent to PNGase F treatment, suggesting that C. elegans mixed stage N-glycans contain little 1,3-linked core Fuc (data not shown).

TEPC-15 IgA detects phosphorylcholine (Pc). A TEPC-15 blot of the glycoprotein of the nematode showed a loss of antibody binding subsequent to PNGase F treatment suggesting that a portion of C. elegans N-glycans contain the Pc functional group.

The overall goal of this work was to provide an overview of N-glycosylation in C. elegans. PNGase F-released glycans were separated by size exclusion Bio-Gel P-4, and detection was by the phenol-sulfuric assay. The glycans were first pooled widely (Fig. 1, pools a-d) and analyzed by MALDI-TOF MS to determine major and minor component glycan compositions (Table I). Narrow pooling (Fig. 1, pools A-E) for fine structural determination of major pool isomers was accomplished (Table II, Figs. 3-8).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Bio-Gel P-4 chromatography of N-glycan released oligosaccharides. Glycans were pooled widely (pools a-d) and more narrowly (pools A-E). Pools a-d were analyzed by MALDI-TOF MS and results are shown in Table I. Pools A-E were investigated for fine structure determination. See text for details.

Little was known about N-linked oligosaccharide composition in C. elegans prior to this study. Seven µg of oligosaccharide from the included volume of the Bio-Gel P-4 eluate was analyzed by GC/MS. Fuc, Man, and GlcNAc were identified on the basis of their GC retention times and electron ionization mass spectra. It is noteworthy that no sialic acid was seen, a result consistent with the previous report for C. elegans (16).

Structural Characterization

Characterization of C. elegans N-Glycans-- A major concern in this work was the detectability of both abundant and rare glycans. As our glycan source was from mixed stage worms, it was expected that glycans derived from the lower mass larval stages would be under-represented. To address this issue properly, an aliquot of Bio-Gel P-4 separated glycans was pooled widely to include all fractions of the included volume. The glycans were then derivatized by permethylation.

We have employed an HF hydrolysis and permethylation strategy in order to aid in providing an overview of glycoconjugates in this study. Permethylation tends to impart similar chemical properties over a wide range of oligosaccharides, which translates into similar detector response with added sensitivity in MALDI-TOF MS analysis. HF treatment has been used in combination with permethylation to aid in characterizing oligosaccharides in complex mixtures, where some components contain Pc (17, 18). As C. elegans protein extracts bind TEPC-15, we anticipated that some glycoconjugates would contain Pc and therefore used oligosaccharide permethylation, with and without prior hydrolysis with HF, to aid glycan detection.

Table I summarizes the MALDI-TOF MS spectra of permethylated and HF-treated and permethylated glycans and their compositions assigned on the basis of observed m/z values (Fig. 1, pools a-d). The compositions indicate the presence of five classes of N-glycan compounds, which include high mannose, mammalian-type hybrid and complex, fuco-pausimannosidic, and those containing Pc. Permethylation alone produced ions representative of all classes. Complex and hybrid glycans were more numerous than high mannose and fuco-pausimannosidic. HF treatment prior to permethylation produced a change in relative abundance of detected glycan types, where high mannose glycans were most abundant followed by fuco-pausimannosidic and hybrid glycans, and complex glycans were absent. The appearance of an ion at m/z 1768.4 was in agreement with Hex₅GlcNAc₂Pi₂, which is consistent with loss of two choline groups and substitution with methyl groups during the permethylation procedure, a modification that has been noted (19).

At this point in our investigation the following three lines of evidence pointed toward the presence of Pc oligosaccharides: a Western blot result that was positive for TEPC-15 reactive N-glycans; a change in glycan class detection upon HF treatment, presumably resulting from a change in surface activity due to release of phosphate esters; and appearance of the m/z 1768.4 ion described above. Further investigation of the Pc as well as the other classes of N-glycans is described below.

Characterization of High Abundance N-Glycans-- For a more detailed structural analysis of the major glycans present, the Bio-Gel P-4 eluate was pooled more narrowly as shown in Fig. 1. Permethylation ± HF treatment was performed, and the glycans were analyzed by MALDI-TOF MS to estimate pool complexity and relative abundance of glycan species. The narrowly pooled glycans (Fig. 1, pools A-E) contained the major N-glycans from mixed stage C. elegans (see Table I and Supplemental Material Fig. 2).

One-dimensional and two-dimensional ¹H DQF-COSY NMR spectroscopy of each pool revealed the glycosidic linkage and sequence of the pools A-E and confirmed the monosaccharide components determined previously by GC/MS. Anomeric proton chemical shifts and their relative proton intensities, as integrated from one-dimensional spectra, are shown in Table II. The two-dimensional ¹H DQF-COSY experiment allows C1-H/C2-H correlations to be well resolved which, in combination with the library of these chemical shifts and associated coupling constant data, allows the linkage arrangement of oligosaccharides in complex mixtures to be assigned accurately (20-31). The DQF-COSY C1-H/C2-H region of pools A-E is shown in Fig. 8.

The C1-H/C2-H correlation of glycan constituents are monosaccharide- and linkage-dependent. The results in Fig. 8 confirm the identities and linkage arrangement of the monosaccharide constituents of the pool. Apportionment of the anomeric resonance intensity of the one-dimensional NMR spectra gave each pool the constituent isomers and relative abundance (see Table II).

HPAEC was used to isolate each pool component, and the compositions of individual glycan species were verified by linear mode MALDI-TOF MS analysis (HPAEC + MS) (Figs. 2-5 and Fig. 7), which agreed well with the sequence and abundance of each isomer as determined by NMR analysis (see Tables II and III and Scheme II). In Scheme I four representative oligosaccharide structures, containing all residue types detected, are presented for the classes of N-glycans found in the high resolution study of narrowly pooled C. elegans glycans. Anomeric chemical shifts, linkage type, and the residue numbering scheme are provided in Scheme I. The NMR results will be discussed here along with HPAEC and MALDI-TOF MS results as these data are highly complementary. The isomers that were determined in each pool are shown in Scheme II.

View larger version (13K): [in this window] [in a new window]   Scheme I.   The representative structures of C. elegans N-glycan released oligosaccharides are shown. A, the conserved Man9GlcNAc2; B, high mannose-type; C, hybrid-type; and D, pausi-mannosidic. Residue identification number and anomeric chemical shift are shown next to each residue.

Detailed Analysis of pool A Oligosaccharides-- The majority (96%) of the pool A components were high mannose glycans. 70% were Man₉GlcNAc₂ and were identical to the unprocessed form found in the ER prior to mannosidase trimming. Another 26% were Man₈GlcNAc₂ and differed from the previous glycoform only by the absence of the middle arm 1,2Man residue after, presumably, being trimmed by ER 1,2-mannosidase. Three trace components were detected by HPAEC + MS including two isomers of Fuc₂Man₅GlcNAc₂, eluting at separate positions by HPAEC, and one Man₃GlcNAc₃ isomer. The trace components were not in sufficient abundance to determine their fine structure. The pool isomers are shown in Scheme II. Key NMR proton resonances used in isomer assignments are underlined in Table II. Mass spectrometry, HPAEC + MS, exoglycosidase data, and NMR analyses that resulted isomer assignments are discussed in detail below.

View larger version (15K): [in this window] [in a new window]   Scheme II.   Derived N-glycan released oligosaccharide structures of mixed stage C. elegans Bio-Gel P-4 pools A-E. Pool abundances are given in %.

HPAEC separation of pool A glycans produced four well resolved peaks, which were centered at 4.6 (2%), 7.4 (2%), 15.1 (35%), and 19.7 (61%) min. MALDI-TOF MS analysis of the permethylated peak glycans produced ions at m/z 1931.2, 1930.3, 2192.4, and 2396.0, respectively (see Fig. 2). These are consistent with the sodium-adducted ions of two isomers of Fuc₂Man₅GlcNAc₂, Man₈GlcNAc₂, and Man₉GlcNAc₂, respectively. Reflectron mode MALDI-TOF MS analysis of the permethylated Bio-Gel P4 glycans did not detect the low abundance monoisotopic ions for the Fuc₂Man₅GlcNAc₂ isomers, but a previously undetected low abundance ion consistent with Man₃GlcNAc₄ was seen at m/z 1661.4 (see Table I and Supplemental Material Fig. 1).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   HPAEC + MS analysis of pool A glycans. The HPAEC trace is shown in A. MALDI-TOF MS analysis of permethylated glycans for each peak are shown as follows: B, 4.6 min; C, 7.4 min; D, 15.1 min; and E, 19.7 min.

The one-dimensional ¹H NMR spectra of pool A contained 10.47 mol of anomeric proton resonance intensity distributed among 5.41 (0.70 mol), 5.34 (0.96 mol), 5.31 (0.96 mol), 5.19 (0.43 mol), 5.14 (0.96 mol), 5.12 (0.02 mol), 5.09 (0.29 mol), 5.05 (2.64 mol), 4.87 (0.98 mol), 4.76 (1.00 mol, measured at 318 K), 4.70 (1.00 mol), and 4.60 ppm (0.55 mol). The above chemical shift values, along with the C1-H/C2-H correlations discussed below, are well documented identifiers for residues found within high mannose glycans (23, 27). Resonance intensity seen at 4.70 and 4.60 ppm are from core GlcNAc residues 1 and 2, and DQF-COSY cross-peaks were seen at 4.70 C1-H/3.63 C2-H and 4.60 C1H/3.78 C2-H ppm confirming this assignment. Notable is that 0.70 mol of resonance intensity was detected at 5.40 ppm in the one-dimensional spectra, and the DQF-COSY spectra of the pool A glycans revealed a cross-peak at 5.40 C1-H/4.09 C2-H. This resonance is produced by the central arm 2-O-substituted 1,3-linked Man residue. In nearly all eukaryotic systems studied to date, the 2-O-substituent Man residue is efficiently removed by ER -mannosidase (32) although 70% of pool A glycans escaped this activity here.

At 5.34 ppm, 0.96 mol of intensity was integrated. A DQF-COSY cross-peak was seen at 5.34 C1-H/4.10 C2-H, which is from the 2-O-substituted 1,3-linked Man residue 5. This assignment necessitates that 96% of pool isomers contain residue 5 in this form (see Scheme I). The DQF-COSY cross-peak seen at 5.31C1-H/4.10 C2-H ppm confirms that the 0.96 mol of resonance intensity is from the lower arm 2-O-substituted 1,2-linked Man residue 8 (see Scheme I and Fig. 8). A cross-peak was seen at 5.14 C1-H/4.02 C2-H ppm, which indicates that the integrated 0.96 mol of proton intensity in the one-dimensional spectrum at 5.14 ppm, was due to 2-O-substituted 1,6-linked Man residue 6 (see Scheme I and Fig. 8). The presence of 0.96 mol each of 2-O-substituted residues 6 and 8 necessitates the presence of their respective substituent residues 9 and 11 at 0.96 mol each, whose presence was seen in the one-dimensional spectrum at 5.05 ppm and confirmed by the DQF-COSY cross-peak seen at 5.055 C1-H/4.060 C2-H ppm. The 0.96 mol of anomeric resonance intensity at 4.87 ppm was coincident with the DQF-COSY cross-peak seen at 4.87 C1-H/4.15 C2-H ppm and was from 3,(±6)-O-substituted 1,6-linked core residue 4. The above proton allocations show the following Man₇GlcNAc₂ partial structure as being in 96% of pool isomers: Man1,2Man1,2Man1,3(Man1,2Man1,6Man1,6)Man1,4GlcNAc1,4GlcNAc/. The remaining 4% of pool isomers must contain the core triose Man1,4GlcNAc1,4GlcNAc/. Subtracting the resonance intensity in the two partial structures from pool resonance intensities leaves 0.70 mol at 5.40 ppm, 0.02 mol at 5.12 ppm, 0.29 mol at 5.09 ppm, 0.72 mol at 5.05 ppm, and 0.02 mol at 4.87 ppm.

The 0.70 mol of resonance at 5.05 ppm exactly matches the 0.70 mol of resonance present at 5.40 ppm, indicating that 70% of pool isomers contain the 1,3-linked Man residue 7 and its 2-O-substituent Man 10. Thus, to the Man₆GlcNAc₂ partial structure defined above is assigned the resonance intensity from residues 7 and 10 to be added yielding archetypal Man₉GlcNAc₂ as isomer A1 and is 70% of pool isomers, which closely agrees with the HPAEC + MS analysis of the pool that gave a 19.7-min peak with a relative abundance of ~61% and m/z consistent with the Man₉GlcNAc₂ composition (see Fig. 2). This leaves 0.26 mol of intensity at 5.09 ppm and 0.02 mol of resonance each at 5.05, 5.12, and 4.87 ppm and 0.29 mol at 5.09 ppm. To the remaining 0.26 mol of the Man₇GlcNAc₂ partial structure is added 0.26 mol of 5.09 ppm yielding Man₈GlcNAc₂ isomer A2 as 26% of pool glycans, in good agreement with mass (m/z 2192.4) and relative abundance (~34%) detected for the HPAEC + MS peak eluting at 15.1 min. This oligosaccharide is identical to ER-processed Man₈GlcNAc₂. On balance 0.02 mol each of 5.05, 5.12, and 4.87 ppm and 0.03 mol of 5.09 ppm resonance remains to be assigned.

As stated above, the HPAEC + MS analysis of pool A showed the presence of trace amounts of Fuc₂Man₅GlcNAc₂ and Man₃GlcNAc₄ ranging from ~1 to 2% of total pool isomers. In addition to the above remaining resonance intensity, a trace amount of NAc resonance was seen at 2.09 ppm close to those of core GlcNAc residues 1 and 2, consistent with 1,2GlcNAc 13 (see Table II). A slight foot at the downfield edge of the resonance at 4.87 ppm was seen, which may be from terminal 1,6-linked residue 4 and 1,6-Fuc (33). As discussed below in pools B and C, anomeric resonance was seen centered at 5.33 for 1,2Fuc ppm, and thus a trace amount of this Fuc linkage may be present in this pool. A trace amount of Fuc methyl proton resonance was seen at 1.20 ppm. To the Man1,4GlcNAc1,4GlcNAc/ partial structure the remaining resonance is assigned. However, the remaining resonance does not allow linkage assignment with completeness and certainty. Trace resonance at 5.12 ppm hints that the Man₃GlcNAc₄ likely contains residue 13 and is assigned here as A3, which is supported by the trace amount of m/z 1661.4 ion seen in the reflectron MALDI-TOF MS analysis of the pool A isomers (see Table I and Schemes I and II). The low intensity DQF-COSY 4.63 C1-H/3.85 C2-H resonance shows the presence of trace 1,6Fuc, which causes a spatially dependent downfield shift of GlcNAc residue 2 (34). The Fuc residue was likely to be present in the Fuc₂Man₅GlcNAc₂ and is assigned here as A4, which is consistent with the compositions detected by MS (m/z 1931.2 and 1930.3) of pool HPAEC peaks seen at 4.6 and 7.4 min, respectively_. As seen in Table III, the glycan isomer compositions and relative abundance derived by ¹H NMR closely match those detected by HPAEC + MS.

Detailed Analysis of Pool B Oligosaccharides-- Pool B contained high mannose, hybrid, and fuco-pausimannosidic glycans. The high mannose isomer, Man₇GlcNAc₂, was most abundant. This glycan was apparently trimmed by mannosidases with activities similar to mammalian ER and Golgi 1,2-mannosidases. The hybrid isomer was Fuc₁Man₃GlcNAc₃ and contained core-bisecting 1,4GlcNAc and core-linked 1,6Fuc. The fuco-pausimannosidic isomers retained the ER Man₉GlcNAc₂ central or lower arm, which was terminally substituted with Fuc. The pool isomers are shown in Scheme II. Key NMR proton resonances used in isomer assignments are underlined in Table II. Mass spectrometry data, HPAEC + MS, exoglycosidase data, and NMR analysis that resulted in isomer assignments are discussed in detail below.

Reflectron MALDI-TOF MS of the Bio-Gel P4 pool B glycans was performed (Table I). Abundant molecular ions were seen at m/z 1987.3, 1753.3, and 1590.4, which were consistent with sodium-adducted ions of Man₇GlcNAc₂, Fuc₁Man₅GlcNAc₂, and Fuc₁Man₃GlcNAc₃. Low abundance ions were seen at m/z 1549.4, 1497.3, and 1007.0, which are consistent with Fuc₁Man₄GlcNAc₂, Man₇, and Man₁GlcNAc₃, respectively. With HF treatment of pool glycans prior to permethylation, ions were seen at m/z 1989.4, 1754.8, 1580.4, 1550.4, 1376.0, and 1006.6, which are consistent with the sodium-adducted ions of Man₇GlcNAc₂, Fuc₁Man₅GlcNAc₂, Man₅GlcNAc₂, Fuc₁Man₄GlcNAc₂, Man₄GlcNAc₂, and Man₁GlcNAc₃, respectively.

HPAEC + MS analysis of pool B isomers produced four resolved peaks (see Fig. 3), which eluted at 3.5, 4.3, 11.4, and 12.3 min. The first peak eluted at 3.5 min in the void volume, and MS analysis of the permethylated sample produced no well resolved spectra. However, the 4.3-min peak was collected to contain the tail of the previous peak and contained a low abundance ion at m/z 1592.0, which is consistent with Fuc₁Man₃GlcNAc₃ as seen in the MALDI-TOF MS analysis of pool B described above. A second major peak detected in the 4.3-min pool was seen at m/z 1755.0, which is consistent with a Fuc₁Man₅GlcNAc₂ composition. A third peak was seen at m/z 1510.1, which is consistent with Fuc₁Man₅GlcNAc₁, which was likely produced by loss of GlcNAc₁ from Fuc₁Man₅GlcNAc₂ during the permethylation procedure and hints that the Fuc residue is not linked to the core-reducing end GlcNAc in the Fuc₁Man₅GlcNAc₂ isomer (m/z 1755.0). The 11.4-min pool produced an sodium-adducted ion at m/z 1988.3, which is consistent with Man₇GlcNAc₂. Upon MALDI-TOF MS analysis the peak at 12.3 min produced a sodiated ion at m/z 1755.8, also consistent with Fuc₁Man₅GlcNAc₂, indicating that two isomers with this composition exist in the pool B. Therefore, by HPAEC + MS pool B contains Fuc₁Man₃GlcNAc₃ (13%), Man₇GlcNAc₂ (56%), and two isomers of Fuc₁Man₅GlcNAc₂ (23 and 8%) as the four major components.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   HPAEC + MS analysis of pool B glycans. The HPAEC trace is shown in A. MALDI-TOF MS analysis of permethylated glycans for each peak are shown as follows: B, 4.3 min; C, 11.4 min; and D, 12.3 min.

The one-dimensional ¹H NMR spectra of pool B contained resonance intensity at 5.41 (0.15 mol, residue 10), 5.34 (0.65 mol, residue 5), 5.33 (0.27 mol, residue 15, see below), 5.31 (0.12 mol, residue 8), 5.19 (0.44 mol, residue 1a), 5.14 (0.53 mol, residue 6), 5.09 (0.88 mol, residue 7, and 5), 5.05 (1.33 mol, residue 8, 9, and 11), 4.92 (0.32 mol, terminal residue 4), 4.89 (0.20 mol, residue 12), 4.87 (0.68 mol, residue 4,), 4.70 (0.56 mol, residue 1b), 4.60 (1.00 mol, residue 2), and 4.46 ppm (0.20 mol, residue 14). At 318 K residue 3 was present at 1.00 mol of integrated resonance intensity at 4.76 ppm but was obscured at 300 K by the HDO peak (see Table II). This gives a total integrated anomeric intensity of 8.33 mol. Residues 1-3 are present in all structures and will not be considered in the allocations below. In the DQF-COSY spectra of the pool, a strong cross-peak was seen at 5.34 C1-H/4.10C2-H and was from 2-O-substituted residue 5. A unique cross-peak was seen at 5.33 C1H/3.86 C2-H ppm, and the C2-H/C3-H cross-peak of the scalar coupled system was seen at 3.86/4.00 ppm. The measured coupling constants for the system were J_1,2, 3.5 Hz and J_2,3, 9.9 Hz, which are consistent with -L-Fuc (35). The presence of 2-O-substituted 1,6-linked upper arm residue 6 assignment is confirmed by the presence of a strong DQF-COSY cross-peak at 5.14 C1H/4.02C2-H ppm. The cross-peak at 5.05 C1-H/4.07 C2-H ppm confirms the presence of terminal 1,2Man residues 8-10. A low cut close to base line in the DQF-COSY spectrum of the pool revealed a cross-peak at 4.89 C1-H/3.78 C2-H ppm, coinciding with the 0.20 mol resonance at 4.89 ppm in the one-dimensional spectrum (boxed in Fig. 8). This is diagnostic of core-linked 1,6-Fuc (33). The cross-peak at 4.87 C1-H/4.13 C2-H ppm confirms the presence of 3,(±6)-di-O-substituted Man residue 4 (25). The presence of core bisecting GlcNAc residue 14 is confirmed by DQF-COSY cross-peak 4.45 C1-H/3.65 C2-H ppm (36).

Residues 7 and 8 are present at only 0.15 and 0.12 mol in 2-O-substitued forms, as indicated by their relative resonance intensities at 5.41 and 5.31 ppm, respectively. Therefore, the major pool isomer, Man₇GlcNAc₂, detected in the Bio-Gel P4 pool by MS and subsequently by pool HPAEC + MS analysis (~56% peak area at 11.4 min) must contain 2-O-substituted residues 6 (at 5.14 ppm substituted by residue 9 at 5.05 ppm) and 5 (at 5.34 ppm substituted by terminal residue 8 at 5.05 ppm) in order to reach the requisite size. The only Fuc₁Man₅GlcNAc₂ that can contain 6 or 9 must be devoid of residue 5 in order to contain the required number of Man residues. The absence of 5 causes the C2-H/C3-H resonance of 3 to shift to 4.09/3.66 ppm (37). The moderate J_2,3 (~3.5) and strong J_3,4 (~10 Hz) coupling constants of mannose allow detection of low abundance resonance in the C2-H/C3-H region of the two-dimensional NMR spectrum. Because no resonance was seen at 4.09 C2-H/3.66 C3-H ppm, all pool glycans contain residue 5. Thus, due to size constraints the Fuc₁Man₅GlcNAc₂ components in the pool cannot contain 2-O-substituted 6 or its substituent residue 9. Therefore, all of the 0.53 mol of resonance at 5.14 ppm from 2-O-substituted 6 exists in the Man₇GlcNAc₂ pool B isomer. The 56% pool abundance of the 11.4 min HPAEC + MS detected Man₇GlcNAc₂ peak supports the size and 53% relative abundance of this NMR assignment. Hence, the major pool isomer is Man1,2Man1,3(Man1,2Man1,6(Man1,3)Man1,6)Man1,4-GlcNAc1,4GlcNAc/ assigned here as isomer B1 (see Scheme II and Table II). This assignment consumes 0.53 mol of intensity at 5.34, 5.14, 5.09, and 4.87 ppm for residues 5-7 and 4 and 1.06 mol at 5.05 ppm for residues 8 and 9. On balance this leaves the following intensities to assign: 0.12 mol at 5.34 ppm; 0.27 mol at 5.33 ppm; 0.12 mol at 5.31 ppm; 0.35 mol at 5.09 ppm; 0.27 mol at 5.05 ppm; 0.32 mol at 4.92 ppm; 0.20 mol at 4.89 ppm; 0.15 mol at 4.87 ppm; and 0.20 mol at 4.46 ppm, respectively. The HPAEC + MS detected Fuc₁Man₃GlcNAc₃ peak represented ~13% of components. NMR analysis revealed 0.20 mol each of 1,6Fuc residue 12 and 1,4GlcNAc residue 14. Assignment of these residues to the archetypal core mannotriose yields Man1,3(Man1,6)(GlcNAc1,4)Man1,4-GlcNAc1,4(Fuc1,6)GlcNAc/ assigned here as isomer B2. This assignment consumes 0.20 mol of resonance intensity for terminal 1,3Man 5 (5.09 ppm), terminal 1,6Man 4(4.92 ppm), 1,4GlcNAc 14 (4.46 ppm), and core 1,6Fuc 12 (4.89 ppm). On balance this leaves 0.12 mol at 5.34, 5.31, and 4.92 ppm, 0.15 mol at 5.41, 5.09, and 4.87 ppm, and 0.27 mol at 5.33 ppm, and 5.05 ppm with 27% of components left to assign.

The two remaining components have the composition Fuc₁Man₅GlcNAc₂ as determined by HPAEC + MS. Both contain 1,X-Fuc present at 5.33 ppm. Guerardel et al. (3) recently reported a terminal 1,2Fuc substitution in C. elegans mucin-type glycans, and its 5.325 C1-H/3.815 C2-H ppm chemical shift is virtually identical to that observed for the Fuc studied here. We tentatively assigned the Fuc here as terminal 1,2Fuc, an assignment that is supported in the following paragraphs. The presence of 0.15 mol of resonance at 5.41 for 2-O-substituted 7 and its substituting terminal residue 10 (0.15 mol of the 0.27 mol at 5.05 ppm) requires that one of these isomers contain this Man1,2Man1,3- disaccharide moiety. The presence of 0.12 mol each of 2-O-substituted 5 at 5.34 ppm, 2-O-substituted 1,2-linked 8 at 5.31 ppm, and terminal 1,2-linked Man 11 (the balance of 0.27- 0.15 mol of 10 at 5.05 ppm) indicates that ~0.12 mol of lower arm Man1,2Man1,2Man1,3- is present. Assigning the 0.12 mol of the 5 plus 7 Man1,2Man1,3-disaccharide moiety to the core Man₃GlcNAc₂ yields the Man₅GlcNAc₂ partial structure Man1,2Man1,2Man1,3(Man1,6)Man1,4GlcNAc14GlcNAca/b assigned here as B. This uses 0.12 mol of the remaining resonances at 5.05, 5.34, 5.31, and 4.92 ppm leaving 0.15 mol each at 5.41, 5.09, 5.05, and 4.87 ppm and 0.27 mol at 5.33 ppm. Assigning all but the 1,2Fuc resonance at 5.33 ppm to the core Man1,4GlcNAc14GlcNAc/ gives Man1,2Man1,3Man1,6-(Man1,3)Man1,4GlcNAc14GlcNAc/ assigned here as Man₅GlcNAc₂ partial structure B.

We cannot say with absolute certainty where the 1,2Fuc is in B and B from the NMR data presented here. However, both contain complete arms of the ER Man₉GlcNAc₂ with intact 1,2Man residues, and this observation led us to hypothesize the existence of a capping substitution of terminal 1,2Man by 1,2Fuc.

To investigate further the position of Fuc substitution, we performed jack bean -mannosidase digestion of the Bio-Gel P-4 pool B glycans, and the products were permethylated and analyzed by linear mode MALDI-TOF MS. Sodiated molecular ions were seen at m/z 765.0, 1550.3, and 1754.2 (data not shown). The m/z 765.0 ion corresponds to Man₁GlcNAc₂ from complete digestion of pool high mannose glycans. The detection of ions at m/z 1550.3 and 1754.4 is consistent with Fuc₁Man₄GlcNAc₂ and Fuc₁Man₅GlcNAc₂ that are 1,2Fuc substituted at 1,2Man as hypothesized. The assignment of 0.15 mol of 1,2Fuc as a substituent of residue 10 of partial structure B gives isomer B3 and is 15% of the pool. The assignment of 0.12 mol of 1,2Fuc as substituting residue 11 gives isomer B4 and is 12% of pool glycans. These assignments complete the anomeric proton allocations of the pool. B4 has a terminal 1,6Man, which is accessible to jack bean -mannosidase. Isomer B3 has terminal 1,3Man, which is slowly accessible to the enzyme. This situation would produce species that, upon permethylation, would exhibit [M + Na] at both m/z 1550.3 and 1754.4, just as observed. We should note that the predicted products of pool glycans with 1,4GlcNAc and 1,6Fuc core substituents were absent from the MALDI-TOF MS spectra of the digestion product, but their presence in the pool was detected by composition and linkage signatures in the pool B MALDI-TOF MS and NMR spectra, respectively. The pool B isomer distribution and relative abundance assigned by NMR are in close agreement with the relative abundance and compositions detected by HPAEC + MS (see Tables II and IV and Scheme II).

Detailed Analysis of Pool C Oligosaccharides-- Pool C contained high mannose, hybrid, and fuco-pausimannosidic oligosaccharides. The high mannose glycans were Man_5-6GlcNAc₂ and were probably the result of mammalian-type ER and Golgi mannosidase trimming. The hybrid glycans were Man_2-4GlcNAc₃ and contained either lower arm 1,2GlcNAc or core-bisecting 1,4GlcNAc substitutions. The fuco-pausimannosidic oligosaccharides were Fuc₁Man_4-5GlcNAc₂, which contained either the novel terminal 1,2Fuc or core 1,6Fuc additions. In the case of 1,2Fuc substitution, the isomers retained the ER Man₉GlcNAc₂ central or lower arm, which was terminally substituted with Fuc. The 1,6-fucosylated isomer was incompletely trimmed and retained a lower arm 1,2Man residue, which suggested that a C. elegans 1,6-fucosyltransferase acts in the absence of lower arm 1,2GlcNAc, unlike that of mammalian systems (38). The pool isomers are shown in Scheme II. Key NMR proton resonances used in isomer assignments are underlined in Table II. Mass spectrometry, HPAEC + MS, exoglycosidase data, and NMR analyses that resulted in isomer assignments are discussed in detail below.

MALDI reflectron TOF MS analysis of the permethylated Bio-Gel P4 pool C glycans contained monoisotopic ions at m/z 1783.4, 1579.3, 1549.0, 1538.3, 1416.3, and 1171.4 (see Table I and Supplemental Material Fig. 2), which are consistent with the following respective sodiated glycan compositions: Man₆GlcNAc₂, Man₅GlcNAc₂ Fuc₁Man₄GlcNAc₂, Man₆GlcNAc₁, Man₃GlcNAc₃, and Man₃GlcNAc₂ (see Table I). HF hydrolysis of pool C glycans prior to permethylation and MALDI reflectron TOF MS analysis gave a nearly identical natriated monoisotopic ion series except that Man₃GlcNAc₂ was not seen. Judging from the Bio-Gel P-4 elution position and HPAEC + MS analysis (Figs. 1 and 5), it was concluded that the Man₃GlcNAc₂ was produced during the permethylation procedure by hydrolysis of other pool glycans.

HPAEC + MS analysis of pooled and permethylated glycans (Fig. 5) yielded 9 glycans wholly consistent with Bio-Gel P-4 MS detected compositions with the addition of minor glycan species appearing at m/z 1212. 3 and 1754.2. The pool C HPAEC + MS elution position, composition, and relative pool abundance as seen by electrochemical detection and m/z were as follows: 4.6 min, Fuc₁Man₄GlcNAc₂ (6%, m/z 1549.9); 5.3 min, Man₂GlcNAc₃ (6%, m/z 1212.3); 5.7 min, Man₃GlcNAc₃ (4%, m/z 1416.7); 6.5 min, Man₃GlcNAc₃ (13%, m/z 1416.6); 8.6 min, Man₅GlcNAc₂ (4%, m/z 1579.4); 9.1 min, Man₄GlcNAc₃ (2%, m/z 1622.5); 10.3, Man₆GlcNAc₂ (21%, m/z 1783.8), 11.8 min, Man₆GlcNAc₂ (40%, m/z 1783.7); and 13.3 min, Fuc₁Man₅GlcNAc₂ (5%, m/z 1754.21). Note that there are two species of Man₃GlcNAc₃ (eluting at 5.70 and 6.52 min), and two species of Man₆GlcNAc₂ (eluting at 10.29 and 11.83 min). The linkages were derived by NMR as described below.

Total integrated anomeric proton intensity of pool C was 7.46 mol, which was distributed in peaks centered at 5.41 (0.04 mol, residue 7), 5.33-5.34 (0.48 mol, residues 5 and 15), 5.19 (0.41 mol, residue 1), 5.14 (0.26 mol, residue 6), 5.12 (0.18 mol, residue 5), 5.09 (1.09 mol, residues 7 and 5), 5.05 (0.69 mol, residues 8, 9, and 11), 4.92 (0.68 mol, residues 4 and 6), 4.89 (0.06 mol, residue 12), 4.87 (0.75 mol, residue 4), 4.76 (1.0 mol, residue 3, integrated at 318 K), 4.70 0.60 mol, residue 1b), 4.60/4.63 (1.00 mol, residue 2), 4.55 (0.18 mol, residue 13), and 4.46 ppm (0.04 mol, residue 14). The corresponding DQF-COSY C1-H/C2-H cross-peaks for the one-dimensional NMR resonances of the above pool were seen at the following positions: 5.34 C1-H/4.10 C2-H ppm for 2-O-substituted residue 5; 5.19 C1-H/3.83 C2-H ppm for residue 1; 5.14 C1-H/4.02 C2-H for 2-O-substituted 1,6-linked residue 6; 5.12 C1-H/4.06 C2-H ppm for 2-O-substituted (1,2GlcNAc 13 substituted) 1,3-linked residue 5 (39); 5.09 C1-H/4.06 C2-H ppm for terminal 1,3-linked residue 5; 5.05 C1-H/4.06 C2-H ppm for terminal 1,2-linked residues 8 and 9; 4.92 C1-H/3.98 C2-H ppm for terminal 1,6-linked residues 4 and 6; 4.87 C1-H/4.13 C2-H ppm for 3,(±6)-di-O-substituted residue 4; 4.70 C1-H/3.63 C2-H ppm for residue 1; resonance of residue 2 was split between 4.63 C1-H/3.81 C2-H and 4.60 C1-H/3.78 C2-H ppm due to the presence in the pool of compounds containing Fuc residue 12, as in pool B; 4.54 C1-H/3.70 C2-H for 1,2-linked GlcNAc residue 13 (39); and 4.46 C1-H/3.70 C2-H 3.65 ppm for bisecting 1,4GlcNAc (34). The anomeric protons of residues 1-3 are present in all glycans, and their proton allocations will not be discussed further. The 1,6Fuc residue 12 was only 0.06 mol and its DQF-COSY cross-peak was not seen due to low abundance.

It should be noted that no resonance intensity was seen at 4.09 C2H/3.66 C3-H ppm indicating that all pool components contain residue 5 (see above). Man₆GlcNAc₂ accounts for ~60% of pool components by HPAEC + MS analysis and is distributed between two species at ~20 and 40% of total pool components (see Fig. 5). 2-O-Substituted residue abundance is low with only 0.05 mol as 7 and none as 8, which would be observed at 5.41 and 5.31 ppm, respectively. Therefore, any Man₅GlcNAc₂ partial structure must contain residues 3, 4, 6, and 7 (see Scheme I), giving a core of the following linkage sequence Man1,6(Man1,3)Man1,6(Man1,3)Man1,4GlcNAc1,4GlcNAca/b. The difference between the two Man₆GlcNAc₂ isomers is, thus, in the addition of one of the terminal 1,2-linked residues 8 or 9, which substitute residues 5 and 6, respectively.

At 5.14 ppm 0.26 mol of resonance is integrated. Assigning 26% of the pool glycans as isomer C1 consumes all the resonance of residue 6 and 0.26 mol each of residue 4 at 4.87 ppm, terminal 1,2-linked Man 9 resonance at 5.05 ppm, and 0.52 mol at 5.09 ppm for residues 5 and 7. After this isomer assignment, the remaining resonance intensities left to assign are as follows: at 5.41 ppm, 0.05 mol (residue 7); 5.34 ppm, 0.48 mol (residue 5); 5.12 ppm, 0.18 mol (residue 5); 5.09 ppm, 0.57 mol (residues 7 and 5); 5.05 ppm, 0.43 mol (residues 8, 9, and 11); 4.92 ppm, 0.68 mol (residues 4 and 6); 4.89 ppm, 0.06 mol (residue 12), 4.87 ppm, 0.49 mol (residue 4); 4.55 ppm, 0.18 mol (residue 13); and 4.46 ppm, 0.04 mol (residue 14). This leaves 74% of the pool components left to assign.

The 0.06 mol of resonance at 4.89 ppm is from core-linked 1,6-Fuc residue 12 (see Table II). Exactly 0.08 mol of resonance is present at 5.33 ppm from a1,2Fuc residue 15. The presence of these two Fuc residues is supported by the HPAEC + MS analysis of pool C that detected Fuc₁Man₄GlcNAc₂ (6% integrated area, Fig. 4B) and Fuc₁Man₅GlcNAc₂ (5% of integrated area, Fig. 4J). This leads to the partial structural assignment Fuc1,X: Man1,3(Man1,6)Man1,4GlcNAc1, 4GlcNAc/ giving partial structure C as 10% of the pool. The NMR analysis (see Table II) revealed 0.18 mol of 1,2GlcNAc (4.55 ppm, residue 13) and 0.04 mol of core bisecting 1,4GlcNAc (4.46 ppm, residue 14. These assignments allow the following partial structures to be assigned: GlcNAc1,2Man1,3Man1,4GlcNAc1,4GlcNAc/ as partial structure C, and Man1,3(Man1,6)(GlcNAc1,4)Man1,4GlcNAc1,4GlcNAc/ as partial structure C as 18 and 4% of pool components, respectively. The above assignments consume all resonance intensity at 5.12 ppm for 5 when substituted by 13 (39), 4.89 (1,6-Fuc residue 12), 4.55 (1,2GlcNAc residue 13), and 4.46 ppm (1,4GlcNAc residue 14) and 0.05 mol of intensity at 5.34 ppm (1,2Fuc residue 15). The resonance intensity arising from the remaining partial structure's residues 4 and 5 in C, C, and C will be addressed below as they can be affected by their substitution state. The remaining resonance intensity left to assign are from 2-O-substituted Man residues 7 (5.41 ppm, 0.05 mol) and 5 (5.34 ppm, 0.43 mol), terminally linked Man 5 (5.09 ppm, 0.57 mol), terminally 1,2-linked Man residues 8 and 9 (5.05 ppm, 0.43 mol), terminally 1,6-linked residues 4 and 6 (4.92 ppm, 0.68 mol), and 3,(±6)-di-O-substituted residue 4 (4.87 ppm, 0.49 mol).

View larger version (30K): [in this window] [in a new window]   Fig. 4.   HPAEC + MS analysis of pool C glycans. The HPAEC trace is shown in A. MALDI-TOF MS analysis of permethylated glycans for each peak are shown as follows: B, 4.6 min; C, 5.3 min; D, 5.7 min; E, 6.5 min; F, 8.6 min; G, 9.1 min; H, 10.3 min; I, 11.8 min; and J, 13.3 min.

The assignment of Fuc- and GlcNAc-substituted partial structures C (10%), C (18%), and C (4%) above can be subtracted from the 74% of isomers left to assign, revealing the remaining 42% as the high mannose isomers Man₆GlcNAc₂ plus Man₅GlcNAc₂. By PAD detection Man₅GlcNAc₂ is ~4% of the pool leaving 38% as Man₆GlcNAc₂. As described above this Man₆GlcNAc₂ isomer differs from the C1 Man₆GlcNAc₂ isomer only in the absence of residue 9, and the presence of residue 8, which 2-O-substitutes residue 5 and leads to the assignment of isomer C2 (see Scheme II). This assignment leaves 0.05, 0.05, 0.19, 0.05, 0.30, and 0.11 mol left to assign, respectively, at 5.41, 5.34, 5.09, 5.05, 4.92, and 4.87 ppm.

Assignment of the 4% C as C3 consumes all of this partial structure and 0.04 mol of terminal residues 1,3Man 5 (5.09 ppm) and 1,6Man 4 (4.92 ppm), leaving 0.05, 0.06, 0.15, 0.05, 0.26, and 0.11 mol of resonance at 5.41, 5.34, 5.09, 5.05, 4.92, and 4.87 ppm left to assign. HPAEC + MS analysis detected Man₃GlcNAc₃ (13%), Man₂GlcNAc₃ (6%), and Man₄GlcNAc₃ (2%). To the 0.18 mol of C 0.12 mol of 4.92 ppm resonance (residue 4) is assigned giving C4 (see Scheme II). Of the remaining 0.06 mol of C, 0.04 is assigned without further residue addition yielding isomer C5 (see Scheme II). To the remaining 0.02 mol of C, 0.02 mol of terminal 1,3Man 7 is assigned giving isomer C6, which also necessitates that resonance of residue 4 be present at 4.87 ppm due to its 3-O-substitution (see Scheme I). Thus, 0.02 mol is consumed at 4.87 ppm. Assignment of C4, C5, and C6 completes the allocation of C and leaves 0.05, 0.06, 0.13, 0.05, 0.14, and 0.09 mol of intensity at 5.41, 5.34, 5.09, 5.05, 4.92, and 4.87 ppm left to assign, respectively (see Scheme II for isomer structures). This completes the assignment of 86% of pool components and leaves the linkage assignment of HPAEC + MS Man₅GlcNAc₂, Fuc₁Man₄GlcNAc₂, and Fuc₁Man₅GlcNAc₂ left to assign. The Man₅GlcNAc₂ isomer comigrates with authentic Man 1,6(Man 1,3)Man 1,6(Man 1,3)Man1,4GlcNAc1,4GlcNAc/ generated by exhaustive digestion of Man₉GlcNAc₂ with Aspergillus satoi 1,2-mannosidase (data not shown). Thus, assignment of isomer C7 as this structure as 4% of pool isomers consumes 0.04 mol each of the intensity at 4.92 and 4.87 ppm and 0.08 mol at 5.09 ppm, which leaves 0.05, 0.06, 0.05, 0.05, 0.10, and 0.05 mol left to assign at 5.41, 5.34, 5.09, 5.05, 4.92, and 4.87 ppm, respectively. Assignment of the Fuc₁Man₄GlcNAc₂ and Fuc₁Man₅GlcNAc₂ as 6 and 4% of pool isomers yields isomers C8 and C9 (see Table I and Scheme II), where the former contains core 1,6Fuc and the later contains the novel Fuc substitution on residue 10. These assignments consume the remainder of the resonance intensity.

As in pool B, jack bean -mannosidase-digested Bio-Gel P4 pool C glycans were permethylated and analyzed by MALDI-TOF MS in linear mode (data not shown) to investigate further the nature of the novel fucosylated glycan determined by NMR analysis. Ions were detected at m/z 765.08, 1213.78, 1550.31, and 1754.59, which are consistent with the sodium adducts of Man₁GlcNAc₂, Man₂GlcNAc₃, Fuc₁Man₄GlcNAc₂, and Fuc₁Man₅GlcNAc₂. Man₁GlcNAc₂ is produced by complete digestion of Man₅GlcNAc₂ and Man₆GlcNAc₂ pool isomers C1, C2, and C7 (68% of pool). Man₂GlcNAc₃ is generated by complete digestion of Man₃GlcNAc₃ and Man₄GlcNAc₃ pool isomers C4, C5, and C6 (20% of pool). The Fuc₁Man₄GlcNAc₂ and Fuc₁Man₅GlcNAc₂ products were produced by the incomplete digestion of Fuc₁Man₅GlcNAc₂ pool isomer C9 (4% of pool). C9 is identical to B3 and contains 1,3Man, which is slowly accessible to the -mannosidase and results in the appearance of both Fuc₁Man₄GlcNAc₂ and Fuc₁Man₅GlcNAc₂ ions. Although product ions from glycans with 1,6Fuc and 1,4GlcNAc core substituents were not detected, their compositions and unique substituent signatures were seen by MALDI-TOF MS and NMR analysis, respectively. These data strongly support the presence of terminally linked 1,2Fuc as well as high mannose, and lower arm GlcNAc-substituted glycans derived here by NMR above. For every NMR-derived structure there was a corresponding pool isomer of the same predicted mass and nearly identical pool abundance as detected by HPAEC + MS (see Table III).

Detailed Analysis of Pool D Oligosaccharides-- Pool D contained high mannose, hybrid, Pc-, and fuco-pausimannosidic glycans. The most abundant isomer was Man₅GlcNAc₂. It was fully trimmed at the central, upper, and lower arm 1,2Man residues, consistent with activities identical to mammalian ER and Golgi 1,2-mannosidases. The hybrid glycan was Man₃GlcNAc₃ and contained core-bisecting 1,4GlcNAc. The Pc glycan was identical to the Man₅GlcNAc₂ isomer of the pool but was substituted with three Pc groups. Fuco-pausimannosidic glycans included Fuc₁Man₅GlcNAc₂ and Fuc₁Man₃GlcNAc₂. The former was identical to the Man₅GlcNAc₂ pool isomer but was substituted with core 1,6Fuc. The later retained the core mannotriose (Man1,3(Man1,6)Man1,4-) and contained core-linked 1,6Fuc. The pool isomers are shown in Scheme II. Key NMR proton resonances used in isomer assignments are underlined in Table II. Mass spectrometry, HPAEC + MS, and NMR analysis that resulted in isomer assignments are discussed in detail below.

MALDI-TOF MS analysis of the Bio-Gel P4 pool D permethylated glycans revealed ions at m/z 1579.2, 1497.2, 1375.3, 1345.3, and 1171.4, which are consistent with the sodium-adducted ions of Man₅GlcNAc₂, Man₇ (see Table I), Man₄GlcNAc₂, Fuc₁Man₃GlcNAc₂, and Man₃GlcNAc₂. MALDI-TOF MS analysis performed on pool glycans with HF hydrolysis prior to permethylation produced ions consistent with the compositions above with the addition of low abundance ions at m/z 1006.5 and 1417.0, which are consistent with Man₁GlcNAc₃ and Man₃GlcNAc₂.

HPAEC analysis of pool glycoconjugates produced four peaks (Fig. 5A). MALDI-TOF MS analysis of the HPAEC pooled and permethylated glycans produced sodium-adducted ions at m/z 1755.8 (4.6 min), 1345.0 (10.3 min), and 1580.2 (18.1 min) consistent with Fuc₁Man₅GlcNAc₂, Fuc₁Man₃GlcNAc₂, Man₅GlcNAc₂, respectively. MALDI-TOF MS of the 22.7 min peak in native form produced ions at m/z 1755.4, 1345.4, 1278.9, and 867.0, which are consistent with Pc₃Man₅GlcNAc₂[Na⁺], Pi₁Pc₂Man₃GlcNAc₂[Na⁺], Man₅GlcNAc₂[2Na-H⁺], and Pc₃Man₅GlcNAc₂[H2+], respectively (Fig. 5E). These