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J. Biol. Chem., Vol. 280, Issue 28, 26063-26072, July 15, 2005

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

John F. Cipollo, Antoine M. Awad, Catherine E. Costello, and Carlos B. Hirschberg¶

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

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 L1 > adult L4 > L3 L2. Dauer larvae contained complex N-glycans with higher molecular masses than those seen in other stages. MALDI-QoTOF MS/MS of Hex₄HexNAc₄ 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 (Pc₁dHex₂Hex₅HexNAc₆) seen in Dauer larvae, have not been observed in any organism. Pc₂Hex₄HexNAc₃, from Dauer larvae, when subjected to PSD MS analyses, showed Pc may substitute both core and terminally linked GlcNAc; no such structure has previously been reported in any organism. C. elegans-specific fucosyl and native methylated glycans were found in all developmental stages. Taken together, the above results demonstrate that in-depth investigation of the role of the above N-glycans during C. elegans development should lead to a better understanding of their significance and the ways that they may govern interactions, both within the organism during development and between the mobile nematode and its pathogens.

Received for publication, April 8, 2005 , and in revised form, May 17, 2005.

^* This work was supported by National Research Science Award F32 GM66486 (to J. F. C.) and National Institutes of Health Grants RO1-GM30365 (to C. B. H.) and P41-RR10888 and S10-RR15942 (to C. E. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, 715 Albany St., Evans 437, Boston, MA 02118. Tel.: 617-414-1040; Fax: 617-414-1041; E-mail: chirschb{at}bu.edu.

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