Abstract
Despite years of research, the glycome of the model nematode Caenorhabditis elegans is still not fully understood. Certainly, data over the years have indicated that this organism synthesizes unusual N-glycans with a range of galactose and fucose modifications on the Man2–3GlcNAc2 core region. Previously, up to four fucose residues were detected on its N-glycans, despite these lacking the fucosylated antennae typical of many other eukaryotes; some of these fucose residues are capped with hexose residues as shown by the studies of us and others. There have, though, been contrasting reports regarding the maximal number of fucose substitutions in C. elegans, which in part may be due to different methodological approaches, including use of either peptide:N-glycosidases F and A (PNGase F and A) or anhydrous hydrazine to cleave the N-glycans from glycopeptides. Here we compare the use of hydrazine with that of a new enzyme (rice PNGase Ar) and show that both enable release of glycans with more sugar residues on the proximal GlcNAc than previously resolved. By use of exoglycosidase sequencing, in conjunction with high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), we now reveal that actually up to five fucose residues modify the core region of C. elegans N-glycans and that the α1,3-fucose on the reducing terminus can be substituted by an α-linked galactose. Thus, traditional PNGase F and A release may be insufficient for release of the more highly core-modified N-glycans, especially those occurring in C. elegans, but novel enzymes can compete against chemical methods in terms of safety, ease of cleanup, and quality of resulting glycomic data.
Highlights
Caenorhabditis elegans is a widely used genetically tractable experimental model for, e.g., development and aging biology research and was the first multicellular organism whose genome was sequenced.[1]
It was clear that C. elegans has an unparalleled fucosylation machinery, and up to four fucose residues were proposed to be present on its NH-egxl5y−c7aHnesxwNiAthc2cFoumc4pMosei0t−io1;n3s−7sufcuhrthaesrmHoexre5−, 8oHveexrN2A0c2pFouscs3iboler fucosyltransferase homologues are encoded by its genome, of which only a few are enzymatically characterized and proven to be able to fucosylate N-glycan structures.[6,8−12] In terms of Nglycans, the roles of two α1,3-fucosyltransferases (FUT-1 and FUT-6) and one α1,6-fucosyltransferase (FUT-8) have been defined by us on the basis of analyses of the recombinant enzymes and of the glycomes of the corresponding single, double, and triple f ut-1, f ut-6, and f ut-8 knockout mutants.[13,14]
In order to resolve the contrasting data regarding the maximal degree of core modification of N-glycans in C. elegans, especially whether only chemical release is suitable for isolation of structures carrying a 3-linked HexFuc element,[7] we compared the results obtained after hydrazinolysis with those with different PNGases, including a new commercially available plant-derived enzyme
Summary
Caenorhabditis elegans is a widely used genetically tractable experimental model for, e.g., development and aging biology research and was the first multicellular organism whose genome was sequenced.[1]. The glycomic studies on mutants revealed five positions for fucosylation, even though only maximally four fucoses were present on any verified structure. As hydrazine cleaves amide bonds, it will destroy peptide and N-glycosidic bonds, and deacetylates GlcNAc, GalNAc, and neuraminic acids which must be re-Nacetylated ( affecting the glycan structure in the case of, e.g., N-glycolylneuraminic acid) and may cause some “peeling” of the N-glycan core.[19] Other chemical methods, e.g., under alkaline conditions,[20] have not gained wide acceptance for N-glycomic studies. New enzymes have the promise of replacing chemical methods for safe and effective release of N-glycans with complex core modifications
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
