The Caenorhabditis elegansbus-2 Mutant Reveals a New Class of O-Glycans Affecting Bacterial Resistance
2010; Elsevier BV; Volume: 285; Issue: 23 Linguagem: Inglês
10.1074/jbc.m109.065433
ISSN1083-351X
AutoresElizabeth Palaima, Nancy Leymarie, Dave Stroud, Md. Mizanur Rahman, Jonathan Hodgkin, Maria J. Gravato‐Nobre, Catherine E. Costello, John F. Cipollo,
Tópico(s)Microbial Metabolites in Food Biotechnology
ResumoMicrobacterium nematophilum causes a deleterious infection of the C. elegans hindgut initiated by adhesion to rectal and anal cuticle. C. elegansbus-2 mutants, which are resistant to M. nematophilum and also to the formation of surface biofilms by Yersinia sp., carry genetic lesions in a putative glycosyltransferase containing conserved domains of core-1 β1,3-galactosyltransferases. bus-2 is predicted to act in the synthesis of core-1 type O-glycans. This observation implies that the infection requires the presence of host core-1 O-glycoconjugates and is therefore carbohydrate-dependent. Chemical analysis reported here reveals that bus-2 is indeed deficient in core-1 O-glycans. These mutants also exhibit a new subclass of O-glycans whose structures were determined by high performance tandem mass spectrometry; these are highly fucosylated and have a novel core that contains internally linked GlcA. Lectin studies showed that core-1 glycans and this novel class of O-glycans are both expressed in the tissue that is infected in the wild type worms. In worms having the bus-2 genetic background, core-1 glycans are decreased, whereas the novel fucosyl O-glycans are increased in abundance in this region. Expression analysis using a red fluorescent protein marker showed that bus-2 is expressed in the posterior gut, cuticle seam cells, and spermatheca, the first two of which are likely to be involved in secreting the carbohydrate-rich surface coat of the cuticle. Therefore, in the bus-2 background of reduced core-1 O-glycans, the novel fucosyl glycans likely replace or mask remaining core-1 ligands, leading to the resistance phenotype. There are more than 35 Microbacterium species, some of which are pathogenic in man. This study is the first to analyze the biochemistry of adhesion to a host tissue by a Microbacterium species. Microbacterium nematophilum causes a deleterious infection of the C. elegans hindgut initiated by adhesion to rectal and anal cuticle. C. elegansbus-2 mutants, which are resistant to M. nematophilum and also to the formation of surface biofilms by Yersinia sp., carry genetic lesions in a putative glycosyltransferase containing conserved domains of core-1 β1,3-galactosyltransferases. bus-2 is predicted to act in the synthesis of core-1 type O-glycans. This observation implies that the infection requires the presence of host core-1 O-glycoconjugates and is therefore carbohydrate-dependent. Chemical analysis reported here reveals that bus-2 is indeed deficient in core-1 O-glycans. These mutants also exhibit a new subclass of O-glycans whose structures were determined by high performance tandem mass spectrometry; these are highly fucosylated and have a novel core that contains internally linked GlcA. Lectin studies showed that core-1 glycans and this novel class of O-glycans are both expressed in the tissue that is infected in the wild type worms. In worms having the bus-2 genetic background, core-1 glycans are decreased, whereas the novel fucosyl O-glycans are increased in abundance in this region. Expression analysis using a red fluorescent protein marker showed that bus-2 is expressed in the posterior gut, cuticle seam cells, and spermatheca, the first two of which are likely to be involved in secreting the carbohydrate-rich surface coat of the cuticle. Therefore, in the bus-2 background of reduced core-1 O-glycans, the novel fucosyl glycans likely replace or mask remaining core-1 ligands, leading to the resistance phenotype. There are more than 35 Microbacterium species, some of which are pathogenic in man. This study is the first to analyze the biochemistry of adhesion to a host tissue by a Microbacterium species. IntroductionCaenorhabditis elegans is a genetically and developmentally well characterized organism that has been used as a model to study host-pathogen interactions. The major habitat for C. elegans is soil, where it feeds on bacteria and may also come into contact with commensal or pathogenic bacteria. More than 40 pathogens are known to cause disease in C. elegans, and many of these (or their close relatives) are human pathogens. These pathogenic interactions have recently been reviewed (1Sifri C.D. Begun J. Ausubel F.M. Trends Microbiol. 2005; 13: 119-127Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar).Host-pathogen interactions require bidirectional recognition factors, and often at least one of these components is a glycoconjugate (2O'Quinn A.L. Wiegand E.M. Jeddeloh J.A. Cell Microbiol. 2001; 3: 381-393Crossref PubMed Scopus (126) Google Scholar). Examples are recognition factors for bacterial toxins such as aerolysin from Aeromonas hydrophila, cholera from Vibrio cholerae, hemolysin from Escherichia coli, and the crystal proteins from Bacillus thuringiensis (3Rajamohan F. Lee M.K. Dean D.H. Prog. Nucleic Acid Res. Mol. Biol. 1998; 60: 1-27Crossref PubMed Google Scholar). The last bacterium can kill C. elegans by means of a crystal toxin that causes destruction of the intestine. The effect is directly analogous to the effect of this and other Bt toxins on Lepidoptera, Diptera, and Coleoptera species, many of which are agricultural pests (4Chungjatupornchai W. Höfte H. Seurinck J. Angsuthanasombat C. Vaeck M. Eur. J. Biochem. 1988; 173: 9-16Crossref PubMed Scopus (61) Google Scholar). C. elegans mutants that are resistant to crystal toxin are defective in the bre gene family (5Griffitts J.S. Whitacre J.L. Stevens D.E. Aroian R.V. Science. 2001; 293: 860-864Crossref PubMed Scopus (175) Google Scholar, 6Griffitts J.S. Huffman D.L. Whitacre J.L. Barrows B.D. Marroquin L.D. Müller R. Brown J.R. Hennet T. Esko J.D. Aroian R.V. J. Biol. Chem. 2003; 278: 45594-45602Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), one of which is a homolog of Drosophila melanogaster egghead (egh) and encodes a GDP-Man:βGlc-Cer-β1,4-mannosyltransferase (bre-3), another encodes a UDP-GalNAc:β1,4N-acetylgalactosaminyltransferase (bre-4), and a third encodes is a homolog of Drosophila brainiac (brn) and encodes a UDP-GlcNAc:βMan N-acetylglucosaminyl transferase (bre-5). Identification of the BRE activities in C. elegans provides striking evidence of the utility of this nematode as a model organism to study the role of glycoconjugates in pathogenesis.C. elegans possesses many attributes that make it an attractive model for the study of carbohydrate-dependent host-pathogen interactions. These include 1) a sequenced genome, 2) the complete mapping of all somatic cells through development, 3) a transparent body, which confers the possibility for visualization of microbial pathogens in situ and the capacity for direct monitoring of the distribution of glycoconjugates in tissues using lectins and antibodies, 4) the existence of many glycoconjugate-deficient strains, 5) sensitivity to more than 40 bacterial pathogens, many of which also infect humans or are their close relatives, and 6) an increasingly well characterized innate immune system. These properties, when combined with the increasing capability of modern mass spectrometry to determine glycan patterns and fine structural detail and the availability of highly sophisticated genetic and molecular techniques, now make it likely that the infection can be placed in the context of its glycoconjugate requirements at the nematode's “bedside.” From that point, the distribution and identity of the required carbohydrate entities can be revealed in a tissue-specific fashion and in fine structural detail. Genetic requirements needed to facilitate the infection and the innate immune components required to combat it can also be determined.There are more than 35 members of the genus Microbacterium, some of which are human pathogens (7Gneiding K. Frodl R. Funke G. J. Clin. Microbiol. 2008; 46: 3646-3652Crossref PubMed Scopus (73) Google Scholar). The C. elegans pathogen M. nematophilum adheres to the rectal and anal region of the nematode, causing a distinct swelling in the region and surrounding tissues (8Hodgkin J. Kuwabara P.E. Corneliussen B. Curr. Biol. 2000; 10: 1615-1618Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The infection is usually non-lethal in wild type C. elegans but causes extensive larval mortality in related Caenorhabditis species or in immunocompromised C. elegans mutants (9Partridge F.A. Tearle A.W. Gravato-Nobre M.J. Schafer W.R. Hodgkin J. Dev. Biol. 2008; 317: 549-559Crossref PubMed Scopus (80) Google Scholar, 10Darby C. Chakraborti A. Politz S.M. Daniels C.C. Tan L. Drace K. Genetics. 2007; 176: 221-230Crossref PubMed Scopus (32) Google Scholar). Genetic screens for altered susceptibility to M. nematophilum infection yielded 19 complementation groups characterized by a loss of swelling in the tail region that led to a bacterially unswollen phenotype (bus) (11Gravato-Nobre M.J. Nicholas H.R. Nijland R. O'Rourke D. Whittington D.E. Yook K.J. Hodgkin J. Genetics. 2005; 171: 1033-1045Crossref PubMed Scopus (87) Google Scholar, 12Yook K. Hodgkin J. Genetics. 2007; 175: 681-697Crossref PubMed Scopus (63) Google Scholar). Among these were mutants of srf-2, srf-3, and srf-5, which have previously been isolated on the basis of their altered cuticle properties, as characterized by ectopic lectin binding (13Link C.D. Silverman M.A. Breen M. Watt K.E. Dames S.A. Genetics. 1992; 131: 867-881Crossref PubMed Google Scholar). Although srf-2 and srf-5 have not been cloned, srf-3 encodes a UDP-Gal/UDP-GlcNAc transporter (14Hoeflich J. Berninsone P. Goebel C. Gravato-Nobre M.J. Libby B.J. Darby C. Politz S.M. Hodgkin J. Hirschberg C.B. Baumeister R. J. Biol. Chem. 2004; 279: 30440-30448Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The bus-2, bus-4, bus-8, bus-12, and bus-17 mutants each harbor a genetic lesion in a different component required for glycoconjugate biosynthesis (9Partridge F.A. Tearle A.W. Gravato-Nobre M.J. Schafer W.R. Hodgkin J. Dev. Biol. 2008; 317: 549-559Crossref PubMed Scopus (80) Google Scholar, 10Darby C. Chakraborti A. Politz S.M. Daniels C.C. Tan L. Drace K. Genetics. 2007; 176: 221-230Crossref PubMed Scopus (32) Google Scholar). The bus-2, bus-4, and bus-17 genes encode homologs of galactosyltransferases predicted to act in O-glycan biosynthesis. The bus-12 gene encodes a nucleotide sugar transporter homolog, and bus-8 encodes a mannosyltransferase homolog possibly active in N-glycan biosynthesis. In our previous study srf-3 strains were found to be deficient in both N- and -O-glycans (15Cipollo J.F. Awad A.M. Costello C.E. Hirschberg C.B. J. Biol. Chem. 2004; 279: 52893-52903Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The most dramatic differences were seen in O-glycans, as they were reduced 65% in overall abundance compared with the parental strain. Five genes identified in the bus screen are likely to be involved in O-glycoconjugate synthesis, including three encoding core-1 galactosyltransferases homologs and two nucleotide encoding sugar transporters. Therefore, it is hypothesized that core-1 O-glycans are required for M. nematophilum infection. Here we further investigate this possibility and the molecular basis for the carbohydrate dependence of the C. elegans–M. nematophilum pathogenic relationship by the analysis of the bus-2 mutant in comparison to its parental strain N2 Bristol.RESULTSTo summarize results the nucleic acid stain SYTO Green 13 was used to visually confirm that the bus-2 mutant had no detectable bacterial colonization in the rectal region compared with its parent strain N2 Bristol, as shown previously (11Gravato-Nobre M.J. Nicholas H.R. Nijland R. O'Rourke D. Whittington D.E. Yook K.J. Hodgkin J. Genetics. 2005; 171: 1033-1045Crossref PubMed Scopus (87) Google Scholar) and in Fig. 1, therefore, verifying the bus phenotype. The bus-2 coding region was cloned and sequenced, thereby revealing an error in the earlier sequence prediction. The NCBI Conserved Domain Data base (26Marchler-Bauer A. Bryant S.H. Nucleic Acids Res. 2004; 32: W327-W331Crossref PubMed Scopus (1461) Google Scholar) revealed that this correction led to an improvement in alignment from 63.8 to 98.0% of domain models used within the core-1 galactosyltransferase pfam01762 (Fig. 2). The improved alignment provided a score of 80.7 bits and an E value of 2e-16. Permethylation profiling by MALDI-TOF MS was used to provide robust and reproducible semiquantitative glycan data to compare abundances of these compounds in the bus-2 and parental strains (Fig. 3). Although the profiles of the N-glycans were nearly identical, those from O-glycans showed reduced abundances in Ce core-1-like O-glycans in the mutant. Additionally, a novel series of highly fucosylated O-glycans that contain an internal GlcA were discovered; in the following sections these will be referred to as Ce core-2 O-glycans. However, it must be noted that the linkages within these glycans have not been determined, and this designation is used to refer to C. elegans O-glycans containing a HexA that is proximal to the core reducing-end HexNAc. They are present at only low levels in the wild type nematode, but their abundances are increased substantially in the bus-2 strain. Tandem MS was used to determine the sequences and compositions of the new subclass of O-glycans (Table 1, Fig. 4 and supplemental Figs. S1–S3). Monosaccharide analyses of the bus-2 and parental N2 Bristol strains O-glycan pools yielded similar results; the oligosaccharide samples contained Gal, Glc, Man, GlcNAc, Fuc, and Rib, consistent with C. elegans glycoconjugates (data not shown). The GlcA and reducing end GalNAc-ol are less stable than other monosaccharide components under the conditions of analysis and were detected only in trace amounts. Rib was likely to be a contaminant from residual RNA. The amount of HexA could not be specifically determined due to the limited sample quantities, but it should be noted that C. elegans has been reported to use GlcA as its charged monosaccharide (15Cipollo J.F. Awad A.M. Costello C.E. Hirschberg C.B. J. Biol. Chem. 2004; 279: 52893-52903Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 27Guérardel Y. Balanzino L. Maes E. Leroy Y. Coddeville B. Oriol R. Strecker G. Biochem. J. 2001; 357: 167-182Crossref PubMed Scopus (99) Google Scholar). Localized glycoconjugate expression was analyzed using lectin staining experiments performed on whole mounted delipidated nematodes (FIGURE 5, FIGURE 6 and supplemental Figs. S4–S6). These data indicated that, in comparison to N2 parent, the bus-2 strain was decreased in the abundance of core-1 glycans in the anus and tail region and increased in fucosylated glycans along the alimentary tract including the anal region. These alterations are significant as the tail, rectum, and anal region are normally infected. Expression analysis of bus-2 revealed that it is normally expressed in the posterior intestine as well as the hypodermal seam cells in the cuticle, both tissues that are close to and can affect the region normally infected (Fig. 7). A detailed discussion of these results appears in the following sections.FIGURE 2The bus-2 sequence and alignment with Pfam 01762.A, underlined is a cDNA segment from the bus-2 gene that was incorrectly predicted as an intron by GeneFinder. B, the alignment with Pfam01762 using NCBI Conserved Domain Data base is shown. The corrected region is underlined.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Permethylation profiles of bus-2 and N2 Bristol O-glycans. MALDI-TOF MS spectra of the permethylated O-glycans released from bus-2 and N2 Bristol and eluted under low salt conditions from anion exchange resin are shown in the top and center panels. Ion abundances for each composition are shown in the lower panel. The inset shows total Ce core-1 and Ce core-2 O-glycans. Error bars indicate S.D. over three analyses.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Composition and assigned structures of Ce core-2 O-glycans* Ion is [M + Na]+. Open table in a new tab FIGURE 4Sequence assignment based on the ESI-CID-MS/MS fragmentation of bus-2 [M+Na]+m/z 1130.553. All fragments contain sodium. The Z2α product ion (circled) was isolated for ESI-CID-MS3 sequence analysis of bus-2 [M+Na]+m/z 1130.553 → 690.328 as illustrated in the inset. Unlabeled peaks within the inset were observed in control spectra and correspond to electronic noise.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5UEA I α-l- fucose specific lectin staining of whole mounted delipidated adult C. elegans. The left column shows control differential interference contrast (DIC) micrographs of lectin-treated nematodes with and without prior incubation with inhibitory concentrations (1 mm) of α-l-Fuc. The images in the center column show that vulva staining is background, as it is not affected by preincubation with α-l-Fuc. The right column shows fluorescence micrographs of UEA I. The bus-2 strains contain a higher abundance of fucosyl glycoproteins. FITC, fluorescein isothiocyanate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6ABA Gal (β1,3) GalNAc - specific lectin staining of whole mounted delipidated adult C. elegans. The left column shows control differential interference contrast (DIC) micrographs of lectin-treated nematodes with and without prior incubation with an inhibitory sugar (β-d-galactose). The right column shows fluorescence micrographs of ABA-stained nematodes. FITC, fluorescein isothiocyanate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7Tissue-specific expression of bus-2. A bus-2::RFP reporter construct is expressed in the posterior intestine (A), hypodermal seam cells (B), and the spermatheca (C).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Bus-2 Is Not Colonized by M. nematophilumThe fluorescent nucleic acid dye STYO Green 13 was used to visualize M. nematophilum colonization (see Fig. 1). This stain permeates cells in proportion to their surface areas and, therefore, under the conditions used here, stains the bacterial DNA preferentially. The bus-2 and wild type strains were incubated with a mixed bacterial lawn of M. nematophilum (0.1%) and OP50 for 72 h at room temperature. Nematodes were then extensively washed, incubated for an additional 60 min to assure complete digestion of microbes in the digestive tract, and extensively washed again before being stained. Fluorescent staining showed colonization of M. nematophilum in the anal region of the N2 Bristol strain but not in the bus-2 mutant, confirming that this bacterial infection cannot be detected in the bus-2 mutant.Bus-2 Contains Conserved Domains of Core Type 1 GalactosyltransferasesThe bus-2 gene, one of three bus genes to contain pfam01762 core type-1 galactosyltransferase conserved domains, is located on chromosome IV of the C. elegans genome (8Hodgkin J. Kuwabara P.E. Corneliussen B. Curr. Biol. 2000; 10: 1615-1618Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). bus-2 is absent from the open reading frame library and was, therefore, cloned from a random-primed cDNA library (29Reboul J. Vaglio P. Tzellas N. Thierry-Mieg N. Moore T. Jackson C. Shin-i T. Kohara Y. Thierry-Mieg D. Thierry-Mieg J. Lee H. Hitti J. Doucette-Stamm L. Hartley J.L. Temple G.F. Brasch M.A. Vandenhaute J. Lamesch P.E. Hill D.E. Vidal M. Nat. Genet. 2001; 27: 332-336Crossref PubMed Scopus (138) Google Scholar) 4D. Stroud, M. J. Gravato-Nobre, and J. Hodgkin, manuscript in preparation. ; it was found to have an open reading frame different from that predicted by GeneFinder. After correction of the sequence the bus-2 alignment with the galactosyltransferase domain, pfam01762 was found to be 98.0% of domain models (rather than the earlier calculated value of 63.8%), as determined by the NCBI Conserved Domain Data base (see Fig. 2) (26Marchler-Bauer A. Bryant S.H. Nucleic Acids Res. 2004; 32: W327-W331Crossref PubMed Scopus (1461) Google Scholar). The score in the improved alignment was 80.7 Bits, and the expect value was 2e-16. The nucleotide and corrected protein sequence of bus-2 is deposited in GenBank™ (accession nos. Banklt 1331141; HM012823). In addition, BUS-2 contains a DXD motif (Fig. 2B), which is a hallmark of glycosyltransferases. It is conserved in 13 glycosyltransferase families (31Breton C. Mucha J. Jeanneau C. Biochimie. 2001; 83: 713-718Crossref PubMed Scopus (107) Google Scholar) and is required for nucleotide sugar substrate binding through metal-coordinated binding of phosphate groups (32Liu J. Mushegian A. Protein Sci. 2003; 12: 1418-1431Crossref PubMed Scopus (175) Google Scholar).The bus-2 O-Glycosylation Profile Is AlteredCe core-1 glycans have been previously described (15Cipollo J.F. Awad A.M. Costello C.E. Hirschberg C.B. J. Biol. Chem. 2004; 279: 52893-52903Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 27Guérardel Y. Balanzino L. Maes E. Leroy Y. Coddeville B. Oriol R. Strecker G. Biochem. J. 2001; 357: 167-182Crossref PubMed Scopus (99) Google Scholar, 33Barrows B.D. Haslam S.M. Bischof L.J. Morris H.R. Dell A. Aroian R.V. J. Biol. Chem. 2007; 282: 3302-3311Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 34Costello C.E. Contado-Miller J.M. Cipollo J.F. J. Am. Soc. Mass Spectrom. 2007; 18: 1799-1812Crossref PubMed Scopus (137) Google Scholar). Permethylation renders the response of glycomers in a related series to be nearly identical with respect to detection by mass spectrometry and, thus, enables reproducible and semiquantitative comparison of biologically related samples (19Ciucanu I. Costello C.E. J. Am. Chem. Soc. 2003; 125: 16213-16219Crossref PubMed Scopus (217) Google Scholar). Permethylated glycan samples were analyzed in triplicate using MALDI-TOF MS, and the observed ion abundances were used to determine glycoform distribution. Analysis of peptide N-glycosidase F-released N-glycans did not reveal any significant differences between the two strains (data not shown). Through this analysis the presence of two O-glycan subfamilies was discovered. In addition to Ce core-1 compounds, the O-glycan profiles revealed the presence of a series of higher molecular weight O-glycans that were more abundant in the mutant than in the parent strain. In terms of total O-glycoform distribution, the bus-2 mutant had a significantly lower percentage of the Ce core-1 O-glycans as compared with the wild type and a greater content of the novel higher molecular weight glycans (see Fig. 3). As noted above, we refer to the new glycan series as Ce type-2 glycans. Total Ce core-1 glycans in N2 Bristol were found to constitute 73.5 ± 2.3% of the glycan pool and those from bus-2 represented 61.9 ± 1.4% of its pool, thus indicating that the latter had nearly a 12% lower content of these glycans. The level of Ce type-2 glycans increased in the bus-2 mutant from 26 ± 1.6 to 38 ± 1.5% of total ion abundance compared with the parent (see Fig. 3, inset), demonstrating a 12% higher content of this class. Individual compositions of Ce core-2 glycans were up to three times more abundant in the mutant. It should also be noted that acidic Ce core-1 glycans, which elute under higher salt conditions from the anion exchange resin used here, also had lower abundances in the bus-2 strain, indicating that all core-1 glycans were diminished in the mutant (data not shown).Structural Detail of Novel Ce Type-2 O-GlycansPermethylation renders a 14-atomic mass unit tag on all terminal residues, as all hydroxyls are replaced with O-methyl groups. At least one hydroxyl of internal residues (dependent on the number of its substituents) is involved in a glycosidic linkage and does not contain this mass tag. Because of this mass difference, it is possible to differentiate between terminal and internal substituents; this is an advantage and often key to structural assignment using permethylated oligosaccharide derivatives in contrast to derivatives that are modified only at the reducing end.Tandem MS analysis revealed the presence of a series of related highly fucosylated glycomers whose presence has not been previously described (see Table 1). Although neutral fucosylated O-glycans have been reported in C. elegans (33Barrows B.D. Haslam S.M. Bischof L.J. Morris H.R. Dell A. Aroian R.V. J. Biol. Chem. 2007; 282: 3302-3311Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), tandem MS analysis of the samples examined here revealed the presence of a series of related acidic highly fucosylated glycomers that have not been previously reported. Oligosaccharide sequence information was determined using MS2 and MS3 analyses when ion abundances were sufficient. The dHex1Hex2HexNAc1HexA1, [M+Na]+m/z 1130.55, ion was analyzed, and the spectra are shown in Fig. 4. Assignment of the Hex2 branch was based on the C2α ion at m/z 463.213 and the Y3α and Y2α ions at m/z 912.437 and 708.339, respectively. These ions underwent sequential losses of two Hex residues from a linear sequence (see Table 1 and Fig. 4). The above assignments limit the position of the HexA and dHex to the core proximal HexNAc. To further define the position of these residues, MS3 experiments were performed on the Z2α fragment seen at m/z 690.328 (Fig. 4, inset). The HexA residue was thereby found to be internal and directly linked to the reducing end HexNAc on the basis of the C1β and Z1α fragments at m/z 229.175 and 472.211, respectively. The C1β fragment mass indicates that it is generated from a terminal dHex (since it contains the 14-atomic mass unit terminal tag). The Z1α fragment indicates loss of an internally linked HexA (loss of HexA not containing the 14-atomic mass unit tag) but retention of the terminal dHex linked to the core HexNAc. Together these assignments localize the HexA as internal.The dHex4Hex3HexNAc2HexA1 [M+2Na]2+m/z 1062.52 was analyzed, and the resulting spectrum is shown in supplemental Fig. S1. The presence of a dHex2Hex1HexNAc1 branch was confirmed by the loss of terminal dHex-HexNAc from the Z4α′ fragment at m/z 1272.615 and the Y3α′, B2α′/Z5α′, and Z3α′ fragments at m/z 1290.625, 628.292, and 1272.615, respectively. Assignment of the dHex-Hex branch is substantiated by the C2α″ ion at m/z 433.203. Fragmentation patterns of all the Ce type-2 saccharides studied (Table 1) were consistent with the presence of a conserved dHex1HexNAc1HexA1 core structure, as previously described herein as an internal component in the assignment of the dHex1Hex2HexNAc1HexA1 sequence above and in Fig. 4. The ion corresponding to dHex4Hex4HexNAc2HexA1, [M+2Na]2+m/z 1164.870 (supplemental Fig. S2) was also subjected to CID, and the presence of a dHex1Hex1HexNAc1 branch was supported by observation of the product ions Z3α′, Y3α′, Z4α′, Y4α′, and Z5α′, Y5α′, which indicate the positions of dHex residues. The assignment of the terminal position of the dHexHex motif was further supported by the presence of C2α′ ion at m/z 433.203. The Y3α″ fragment at m/z 1055.508 (2+) indicated the presence of a terminal Hex. The dHex5Hex4HexNAc2HexA1, [M+2Na]2+m/z 1251.61, had a mass shift corresponding to the addition of one fucose and no longer showed the loss of the non-fucosylated terminal Hex (Table 1). This supported the addition of a dHex residue to the Hex as reported here (compare the results obtained for compounds 3 and 4, Table 1). The ion at m/z 1353.67, corresponding to dHex5Hex5HexNAc2HexA1, [M+2Na]2+, was analyzed and found to have the same structure as the dHex5Hex4HexNAc2HexA1 with the addition of a terminal hexose on the HexNAc-containing branch, as supported by the reducing end fragments Y6α′ and Z6α′ and by the terminal fragments B1α′ and C1α′ (supplemental Fig. S3). A summary of the fragmentation data assignments for the above series of the fucosyl Ce type-2 O-glycans is presented in Table 1 and supplemental Figs. S1–S3. Data from all glycomers were consistent with the presence of an internal HexA and excluded the possibility of a terminal HexA indicating that these compounds are members of a biosynthetically related series and that HexA serves as a linkage point for further branch extension. C. elegans has been reported to use GlcA as its charged group (15Cipollo J.F. Awad A.M. Costello C.E. Hirschberg C.B. J. Biol. Chem. 2004; 279: 52893-52903Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 27Guérardel Y. Balanzino L. Maes E. Leroy Y. Coddeville B. Oriol R. Strecker G. Biochem. J. 2001; 357: 167-182Crossref PubMed Scopus (99) Google Scholar), so the HexA residue observed here is likely to be GlcA.Tissue Expression Pattern of bus-2Expression analysis using a bus-2::RFP operon reporter transgene revealed that bus-2 is expressed in the posterior intestine in
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