N -Glycans of Caenorhabditis elegans Are Specific to Developmental Stages*

We have examined the N -glycans present during the developmental stages of Caenorhabditis elegans using two approaches, 1) a combination of permethylation followed by MALDI-TOF mass spectrometry (MS) and 2) derivatization with 2-aminobenzamide followed by separation by high-performance liquid chromatography and analyses by MALDI-TOF MS, post source decay (PSD) MS, and MALDI-QoTOF MS/MS. The N -glycan profile of each developmental stage (Larva 1, Larva 2, Larva 3, Larva 4, and Dauer and adult) appears to be unique. The pattern of complex N -glycans was stage-specific with the general trend of number and abundance of glycans being Dauer (cid:1) L1 > adult (cid:1) L4 > L3 (cid:1) L2. Dauer larvae contained complex N -glycans with higher molecular masses than those seen in other stages. MALDI-QoTOF MS/MS of Hex 4 HexNAc 4 showed an N -acetyllac- tosamine substitution not previously observed in C. elegans . Phosphorylcholine (Pc)-substituted glycans were also found to be stage-specific. Higher molecular weight Pc-containing glycans, including fucose-containing ones such as difucosyl Pc-glycan (Pc 1 dHex 2 Hex 5 HexNAc 6

Carbohydrate-mediated interactions between cells and their environment are important in differentiation, embryonic development (1), inflammation and immunity (2), and cancer metastasis (3). Errors of glycosylation manifest themselves throughout cell lineages, resultant tissue formation, and cellinteractive processes such as those enumerated above. In humans and mice, mutations that prevent proper formation of N-glycans result in multisystem defects that can be traced to developmental processes and, thus, demonstrate that appropriate glycosylation is essential for development.
Caenorhabditis elegans is a genetically and developmentally well characterized multicellular eukaryote with a short life cycle, invariant cell lineage, and distinct stages of development in which growth, reorganization, and switching between vegetative and developmentally arrested states occur. Knowledge of its sequenced genome and mapped cell fates, as well as accessibility of expression data bases (4) and gene ablation consortia, make this organism attractive for study of the roles of N-glycosylation in development and nematode-pathogen interactions.
In genetic terms, C. elegans has retained the biosynthetic components required for the formation of high mannose and the abbreviated mammalian-type complex glycans. Three Nacetylglucosaminyltransferase I (16 -18) and an N-acetylglucosaminyltransferase V (19) homologues have been expressed and characterized. A homologue of N-acetylglucosaminyltransferase II also appears to exist (17). However, no strong homologies to N-acetylglucosaminyltransferases III, IV, or VI have been identified, although some structural studies suggest that GlcNAc is present in linkages identical to those catalyzed by these enzymes (6,7,9). C. elegans also possesses insect-like pathways to process glycans (20). Insect cell lines have a Golgi N-acetylglucosaminidase, which has been shown to remove N-acetylglucosaminyltransferase I-added GlcNAc, thus preventing formation of complex glycan, and this activity appears to be developmentally related (21). The presence of insect-like pathways suggests that the low abundance of complex glycans seen in C. elegans N-glycans may be related to the activity of this enzyme. Therefore, it is possible that, during some stages of glycoconjugate biosynthesis, enzyme balance is shifted to favor the formation of more complex glycans in some tissues. Here, we present the N-glycan structures correlated to the developmental stages of C. elegans. These findings may provide important clues derived from the glycosylation patterns of these developmentally distinct stages, which should be relevant to differentiation, embryonic development, inflammation, and immunity in this nematode.

MATERIALS AND METHODS
Isolation of Developmental Stages of C. elegans-The temporal progression of life stages was approximated using synchronized cultures by the observation of the cessation of pharyngeal pumping, which precedes each cuticle molt (22). Nematodes were grown in liquid culture and harvested when 90 -95% of individuals were of the desired developmental stage, as observed by light microscopy.
Isolation of N-Glycans-The glycoprotein-rich fraction was isolated from 2-g batches of C. elegans, as previously described (6). Briefly, following treatment of extracted proteins with L-1-tosylamido-2-phenylethyl chloromethyl ketone trypsin, N-glycans were released from 0.5-ml samples with 8000 units/ml of PNGase F 1 (New England Biolabs) overnight at pH 8.5 and 37°C. Free glycans were separated from the tryptic peptides by precipitation of the peptides (23) with 50% methanol at pH 5.5 followed by centrifugation at 3500 ϫ g. The solutions, containing free glycans and some peptides, were subjected to rotary evaporation, and the resulting precipitate was suspended in distilled water and applied to Sep Pak C-18 cartridges (Waters). Glycans were collected by elution with distilled water. Adsorbed peptides were eluted with isopropanol and then combined with those that had precipitated in 50% methanol. Free glycans were quantitated using the phenol sulfuric assay for neutral hexose standardized with mannose (24).
Permethylation of Oligosaccharides-Permethylation was carried out using a slight modification of the method of Ciucanu (25) as previously described (6).
Reductive Amination and Chromatographic Separation-Oligosaccharides were dried in a Savant speed evacuation device and reconstituted in 15 l of Me 2 SO. To the reconstituted samples were added 100 l of 2 M cyanoborohydride and 35 l of 0.5 M 2-amino benzamide, both of which were solubilized in Me 2 SO. Glacial acetic acid (50 l) was added. The reaction was performed at 65°C for 2 h. The aminated glycans were applied to chromatography paper (Whatman), dried, and separated from reaction products by ascent in a chromatography tank containing 100 ml of acetonitrile. The aminated products were visualized using a BLAK-RAY 366 nm UV lamp and eluted into a 4% butanol solution. The glycan products were filtered by centrifugation, dried by speed evacuation, reconstituted in 4% butanol, and chromatographed on a Waters high-performance liquid chromatography system equipped with a fluorescence detector and Breeze TM software. The aminated glycans were separated on a Varian C18 column using a gradient of 10 mM ammonium acetate 0.1 to 1% butanol applied over 72 min.
Mass Spectrometric Analysis-MALDI-TOF MS was performed on a Bruker Reflex IV mass spectrometer in positive reflectron mode. Between 20 and 50 pmol of sample dissolved in 20% acetonitrile was applied to the MALDI target with an equal volume of 2,5-dihydroxybenzoic acid (20 mg/ml) in 20% acetonitrile in 10 mM sodium acetate. The spectrum resulting from 150 and 200 shots from a 337 nm nitrogen laser were summed. The laser pulse was 3 ns. Each analysis was performed in duplicate. The intensities of the molecular ion signals were averaged and the mean Ϯ S.E. was calculated.
Post source decay (PSD) experiments were performed using the Bruker Reflex IV mass spectrometer in positive reflectron mode with the same sample application described above. Between 13 and 17 segments were collected for each experiment depending on the selected m/z value. The signal generated by exactly 150 shots from a 337 nm nitrogen laser was summed for each segment. Concatenation was performed using X-TOF software.
Collision induced dissociation fragmentation data were collected using a MALDI (nitrogen laser, 337 nm) source on a QStar Pulsar i quadrupole orthogonal time-of-flight mass spectrometer (Applied Biosystems Inc., Framingham, MA). The MALDI matrix was 2,5-dihy-droxybenzoic acid, and typically the signal from 50 -200 laser shots was summed for each spectrum. The laser power used was 30 -33 J. Nitrogen (3 psi) was used as the collision gas for MS/MS experiments. The range of operator-controlled collision voltages was 35-90 V. Nomenclature is that of Domon and Costello unless otherwise indicated.

RESULTS
General Workup Strategy-Profiling of permethylated oligosaccharides was initially undertaken to compare amounts of high mannose-, complex-, and C. elegans-specific fucosyl Nglycans of different developmental stages of C. elegans. This was achieved with MALDI-TOF MS analysis. Although this procedure does not distinguish among isomers, it allows relative quantitation of individual isobars based on their spectral intensities. Standard errors of the mean of individual isobar peaks were typically less than 5% of peak intensity.
More detailed analyses of N-glycans, from different developmental stages, were carried out following derivatization of the glycans with 2-aminobenzamide (2AB). This allowed detection of glycans after their separation by C18 high-performance liquid chromatography. Thereafter, samples were pooled and analyzed by MALDI-TOF MS, PSD MS, and MALDI-QoTOF MS/ MS. The combination of these analytical techniques has proven useful for glycan identification (26 -28). This approach led to the detection of a broader range of C. elegans oligosaccharides than had been previously described. In the next section we first present an overview of the N-glycan patterns of the different C. elegans developmental stages and follow this with in-depth analyses of the different glycan species.
Overview of N-Glycan Patterns in C. elegans Developmental Stages-The general patterns of 2-aminobenzamide-labeled Nglycans of different developmental stages can be seen in Fig Tables I-IV, whereas important glycan molecular ions of each developmental stage are discussed under "Results" (see "Detailed Analyses of Complex Oligosaccharides," "Detailed Analyses of Phosphorylcholine Oligosaccharides," and "Detailed Analyses of C. elegans-specific Fucosyl and Methylated Oligosaccharides" below).
Phosphorylcholine oligosaccharides were found in all developmental stages, and selected spectra containing some of these glycans are shown below in Fig. 4. The general trend of the number and abundance of the glycan species eluting between 52 and 54 min of the chromatograms shown in Fig. 1 was Dauer Ϸ L1 Ͼ adult Ϸ L4 Ͼ L3 Ϸ L2. These glycans were observed as both the [MϩH] ϩ and [MϩNa] ϩ forms despite doping of the sample matrix with sodium acetate. This is most likely the result of the zwitterionic nature of phosphorylcholine. Detailed descriptions of the different structures, including novel high molecular mass species and some glycoforms not previously described in any organism, are presented under "Detailed Analyses of Complex Oligosaccharides." Ions corresponding to C. elegans-specific fucosyl and (naturally) methylated oligosaccharides were detected in all developmental stages, with the general trend in number and abundance being Dauer Ͼ L1 Ͼ adult Ϸ L4 Ͼ L3 Ϸ L2. The majority of these glycans eluted between 40 and 46 min of chromatograms shown in Fig. 1 and are indicated with a triangle in Fig.  4. Detailed analyses of these structures are discussed under "Detailed Analyses of C. elegans-specific Fucosyl and Methylated Oligosaccharides." In all developmental stages, permethylation profiling revealed that high mannose glycans were the most abundant class (76 -78%), followed by C. elegans-specific fucosyl (18 -21%) and complex glycans (3-5%). The abundance of each class was expressed as percentage of total N-glycans released. In the Dauer stage the high mannose compounds were decreased to 64%, whereas C. elegans-specific fucosyl was 29% and complex was 7%. High mannose glycans included ions consistent with Man 3-5 GlcNAc 2 and Glc 0 -1 Man 7-9 GlcNAc 2 , C. elegans-specific fucosyl N-glycans included ions consistent with Fuc 1-2 Hex 3-5 -GlcNAc 2 , and complex N-glycans included ions consistent with Man 3 GlcNAc 3-4 .
Detailed Analyses of Complex Oligosaccharides-The pattern of complex glycans was stage-specific ( Fig. 1 5D, was subjected to MALDI-QoTOF MS/MS analysis, and the structure deduced for this glycan will be shown to be a structure not previously observed in C. elegans (Fig. 3). Evidence for two structures was obtained: one contained a lacNAc substitution, whereas the other had a core bisecting GlcNAc substitution. Evidence for the former structure is supported by the following fragments: 1) B 3␣ , C 4 /Z 3␣ , and B 4 /Y 3␣ ions at m/z 550.20. These ions cannot originate from Structure II, whose presence is supported by ion B 2␥ at m/z 347.11. Further support for the presence of Structure II comes from the observation that a biantennary Gal 1 Man 3 GlcNAc 4 standard, prepared from bovine IgG, did not give rise to ions of m/z 347.11. Other possible isomers that by composition must contain a chito-, LacdiNAc, or HexNAc-Hex-HexNAc-Hex moiety would produce ions of m/z 429. 16  Detailed Analyses of Phosphorylcholine Oligosaccharides-As mentioned above, phosphorylcholine oligosaccha-rides were observed in all developmental stages with a generalized composition of Pc 1-2 Fuc 0-2 Hex 2-5 HexNAc 2-8 (Table I)   [MϩH] ϩ m/z 1970.8 (Fig. 4, A-D, filled triangles). Higher molecular weight Pc glycans (Fig. 5, filled triangles), including those with Fuc, were most abundant in L1 and Dauer larvae. Difucosyl Pc glycans such as Pc 1 dHex 2 Hex 5 HexNAc 6 -2AB, [MϩNa] ϩ m/z 2646.8 (Fig.  5B), were also observed in the above stages. To our knowledge, these have not been previously observed in any organism. The relatively high molecular weight compounds observed in Dauer larvae (Fig. 5B) such as Pc 1 Hex 4 HexNAc 8 -2-AB, [MϩH] ϩ m/z 2598.6, are highly substituted with HexNAc, suggesting that Lac-diNAc, chito-, and/or GlcNAc trisubstituted Man may be present. As shown in L1, Fig. 5D, ions consistent with Pc glycans lacking Golgi added GlcNAc were also observed such as Pc 1 dHex 1 Hex 3 HexNAc 2 -2AB, [MϩH] ϩ m/z 1364.7. Table I shows the occurrence of the previous compounds, as well as others containing more Pc than Golgi GlcNAc substitutions, and thereby suggests that Pc may substitute residues other than antennary GlcNAc.
Fragmentation studies were performed on those Pc glycans whose quantities were sufficient for structural studies, including determination of the sites of the Pc substitutions. Thus, Pc 2 Hex 4 HexNAc 3 -2AB, [MϩH] ϩ m/z 1748.5, from Dauer larvae was analyzed by PSD as shown in Fig. 6. This glycan was shown to have a structure not previously reported in any organism. The parent ion produced a PSD profile that can be consistent with three isomers where Pc substitutes one core and one antenna-ryGlcNAc or both core GlcNAc residues. The three possible structures are shown in Fig. 6. The assignments of the fragment ions are shown above each ion peak in the spectrum. Regions within the glycans where fragmentation is observed in the spectrum are indicated. Some key fragment ions are discussed here. The fragment at m/z 369.26 is consistent with phosphorylcholine-substituted GlcNAc. The fragment ion at m/z 897.50 is indicative of a sodiated adduct containing two Pc-substituted GlcNAc residues and the 2-AB terminus, and thus, support core substitution with Pc. Fragments at m/z 1061.36 and m/z 1222.92 represent similar fragments that, in addition, contain one or two Hex residues, respectively. Other fragments that contain two Pc substitutions, such as those present at m/z 1017.51 and m/z 1386.45, can be produced by compounds containing only core Pc or those with antennary GlcNAc substitution. The abundance of the Pc-GlcNAc fragment at m/z 369.26 suggests that a significant amount of antennary Pc is present. Thus, these data are consistent with the notion that Pc can substitute both core and terminally linked GlcNAc. Pc 1 Hex 3 HexNAc 3 -2AB, that eluted at 46 -48 min in samples from adult nematodes (Fig. 1), was also analyzed by PSD (Fig.  7). The parent ion at m/z 1421.56 yielded fragments B 1␣ m/z 369.14 and B 2␣ m/z 530.94, whose presence strongly suggests that Pc substitution of antennary GlcNAc has occurred, as previously reported (9).
The ions corresponding to the [MϩNa] ϩ of Me 1 dHex 4 Glc-NAc 2 -2AB from Dauer larvae were subjected to PSD analysis. As shown in Fig. 9, the presence of fragment ions consistent with 3,5 A 5 (m/z 906.11) and 2,4 X 3y /Y 5x m/z (1155.43) strongly suggest that the O-methyl group is located in a terminally linked monosaccharide. Taken together, the internal fragment B 5 /Y 4x , m/z 712.81, and cross-ring fragment 0,2 X 3y , m/z 1253.83, place the likely location of the O-methyl group on a terminal Fuc, as shown in Fig. 9, consistent with a previous report (10). Hex 6 HexNAc 2 -2AB, [MϩNa] ϩ m/z 1539.6, (Fig. 8, C-F) was detected in all development stages and, as hypothesized previously, is likely to represent a biosynthetic precursor to the above fucosylated species (13).   a All ions correspond to ͓MϩNa͔ ϩ . b Indicates the presence of a glycan of the same mass in an adjacent pool.
c Indicates the presence of a glycan of the same mass in a nonadjacent pool.
fragments show that the methyl is at the reducing end GlcNAc.
Other glycans detected as 2-AB derivatives could be assigned as having high mannose structures and are seen in Table IV. It is possible that shorter glycans, such as Hex 4 HexNAc 2 -2AB, may contain solely Man or Gal and Man additions to the chitobiose core, because both compositions can be predicted from glycan precursors previously described in C. elegans.

DISCUSSION
This study has shown major differences in the N-glycans of C. elegans developmental stages. Among the principal findings are: (a) the N-glycan profile of each developmental stage appears to be unique (Fig. 1). (b) the pattern of complex N-glycans was stage-specific (Fig. 2). The general trend of the number and The methodology used in this study, consisting of reductive amination of PNGase F-released oligosaccharides followed by separation on reversed phase high-performance liquid chromatography and offline MS analysis allowed us to detect higher order complex and intact Pc-containing glycans. Fragmentation analyses of some of the latter structures showed that Pc can substitute not only antennary but also core N-acetylglucosamines or both. The latter had not been reported previously. In some Pc oligosaccharides up to two Fuc may be present. Although the amounts available were insufficient for further analysis, we hypothesize that Fuc may be antennary or in a novel linkage to the core GlcNAc, as has been found for glycans in Schistosomes (29,30). We can exclude the possibility that Fuc is linked ␣1,3 to the reducing end GlcNAc, because PNGase F is inactive toward ␣1,3-substituted structures. The majority of the Pc oligosaccharides have the composition previously described for other nematodes in which N-acetylglucosamines in GlcNAc␤1,2(GlcNAc␤1,6)Man-and GlcNAc␤1,2(GlcNAc␤1, 4)Man-are substituted with Pc. This result is consistent with our recent observation that the phosphatidylcholine:oligosaccharide phosphorylcholine transferase of C. elegans preferentially uses as its in vitro substrate oligosaccharides containing Man disubstituted with GlcNAc (7). Rare Pc oligosaccharides with up to five phosphorylcholine substitutions have been reported in some nematodes (31). We did not observe any of these structures in the present study. However, we cannot rule out the possibility that these compounds are present in very low quantities.
In the present study, it was determined that higher order complex glycans, including those containing up to three Fuc, are most abundant in the L1 and Dauer stages. Because PNGase F was used successfully in the workup, it is likely that the Fuc is antennary; this in turn suggests that one of the glycans is fucosylated LacdiNAc. Previous studies showed that C. elegans has a ␤4GalNAc transferase, which catalyzes the addition of GalNAc to terminal GlcNAc to form the LacdiNAc structure GalNAc␤1,4GlcNAc␤1,R in membrane preparations and in Lec 82 and Lec 8 cells in vivo (14). In addition, a C. elegans ␣1,3-fucosyltransferase, CEFT-I, has been shown to synthesize the fucosylated LacdiNAc, GalNAc␤1,4(Fuc␣1,3)-GlcNAc␤1,R (15). These enzymatic activities are consistent with some oligosaccharide compositions detected in this study and also with species previously reported in Hemonchus contortus (32,33) but not in C. elegans. We have also demonstrated that C. elegans produces LacNAc structures, a feature that had not been previously reported. The C. elegans genome has been shown to possess three homologues of UDP-Gal:␤-GlcNAc␤1,4galactosyltransferase II, and this further supports the hypothesis that LacNAc structures exist in this nematode. In vitro, the C. elegans fucosyltransferase CEFT-I, ␣1,3-fucosyltransferase, catalyzes fucose addition to Gal␤1,4GlcNAc␤1,R to form Gal␤1,4(Fuc␣1,3)GlcNAc␤1,R (fucosylated LacNAc), the Lewis X (LeX) epitope. From the compositions seen here, it is possible that LeX structures exist, even though antibodies to LeX that have been tested so far fail to bind to C. elegans extracts. In the future, antibodies raised against the glycans described here will help to pinpoint their specific tissue location As previously reported in studies where oligosaccharide release was performed using either PNGase A or hydrazinolysis, a novel group of C. elegans-specific, Fuc-substituted, glycans occur with both ␣1,3and 1,6-Fuc core substitutions (5,6,9,12,13,16). Fuc may also be terminal, with an ␣1,2-linkage with Man and Gal as the penultimate sugar. Evidence for this was supported by the observation that, in C. elegans srf-3 mutants, which are defective in UDP-Gal transport, the highly fucosylated structures released by PNGase F and A treatments were diminished. This strongly suggests that most of these glycans contain internal Gal. In the same study we identified Gal 1 Man 5 GlcNAc 2 , which may be an intermediate in the biosynthesis of the fucosylated species.
This and previous studies document the occurrence of natural O-methyl substitutions in glycans from C. elegans (5,9). The 3-O-methyl GlcNAc had also been previously reported in the cellulosome of Clostridium thermocellum (34), as well as in these of the Great pond snail Lymnacea stagnalis and the Roman snail Helix pomatia wherein glycans from hemocyanin contain Fuc with up to three O-methyls (35). In Rhizobium etli CE3, repeating O-trisaccharide chains contain mono-and di-O-methyl substitutions, in addition to the capping sequence containing tri-O-methyl Fuc. Methylation is hypothesized to prevent further elongation of O-chains (36).
Why is the N-glycan pattern more abundant and elaborate in the L1 and Dauer stage of C. elegans? Although we do not yet have a definitive answer to this question, we speculate that both stages occur in conjunction with significant lifestyle changes in the worm. L1 larvae emerge at a time of development when the worm has exited embryonic development and enters vegetative growth while, at the Dauer stage the worm leaves vegetative growth to pass into a developmentally ar- rested stage. Changes associated with glycans may be related to changes in the development of the nematode, innate immunity or processes in the secretory system required for the stage status. In this context, it has been reported that the unfolded protein response is highly active in the L1 through L2 stage, which would suggest a high rate of glycoprotein biosynthesis. Ire-1/pek-2 mutants of C. elegans are deficient in the unfolded protein response and arrest in the L2 stage (37).
In summary, we have presented the first account of stagespecific N-glycan expression in C. elegans. Developmental  Table III for a complete list of C. elegans-specific fucosylated glycans detected in this study. stages that are under-represented in mixed stage preparations were shown here to contain novel N-glycans. The stage-specific glycan expression profiles here should facilitate efficient selection of stages to examine for further study of the C. elegans glycosynthetic pathways.