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Abstract
Caenorhabditis elegans and Drosophila melanogaster are relevant models for studying the roles of glycosaminoglycans (GAG) during the development of multicellular organisms. The genome projects of these organisms have revealed the existence of multiple genes related to GAG-synthesizing enzymes. Although the putative genes encoding the enzymes that synthesize the GAG-protein linkage region have also been identified, there is no direct evidence that the GAG chains bind covalently to core proteins. This study aimed to clarify whether GAG chains in these organisms are linked to core proteins through the conventional linkage region tetrasaccharide sequence found in vertebrates and whether modifications by phosphorylation and sulfation reported for vertebrates are present also in invertebrates. The linkage region oligosaccharides were isolated from C. elegans chondroitin in addition to D. melanogaster heparan and chondroitin sulfate after digestion with the respective bacterial eliminases and were then derivatized with a fluorophore 2-aminobenzamide. Their structures were characterized by gel filtration and anion-exchange high performance liquid chromatography in conjunction with enzymatic digestion and matrix-assisted laser desorption ionization time-of-flight spectrometry, which demonstrated a uniform linkage tetrasaccharide structure of -GlcUA-Gal-Gal-Xyl- or -GlcUA-Gal-Gal-Xyl(2-O-phosphate)- for C. elegans chondroitin and D. melanogaster CS, respectively. In contrast, the unmodified and phosphorylated counterparts were demonstrated in heparan sulfate of adult flies at a molar ratio of 73:27, and in that of the immortalized D. melanogaster S2 cell line at a molar ratio of 7:93, which suggests that the linkage region in the fruit fly first becomes phosphorylated uniformly on the Xyl residue and then dephosphorylated. It has been established here that GAG chains in both C. elegans and D. melanogaster are synthesized on the core protein through the ubiquitous linkage region tetrasaccharide sequence, suggesting that indispensable functions of the linkage region in the GAG synthesis have been well conserved during evolution.
Highlights
The nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster are ideal model organisms for studying a wide range of fundamental biological disciplines in development
The fluorescence intensity of the sample prepared without the LiOH treatment was significantly weaker, compared with that of the LiOH-treated sample, suggesting the presence of C. elegans GAG attached to core proteins via O-glycosidic bonds
Negative and positive ion mode DE MALDI-TOF/Mass Spectrometry (MS) analyses showed molecular ion signals at m/z 1131 (Fig. 4) and 1133, corresponding to the molecular ions [M Ϫ H]Ϫ and [M ϩ H]ϩ of ⌬HexUA1HexUA1HexNAc1Hex2Pen12AB, respectively (HexUA, HexNAc, Hex, and Pen represent hexuronic acid, Nacetylhexosamine, hexose, and pentose, respectively). Together these results indicate that the C. elegans chondroitin is synthesized as proteoglycans, being covalently bound probably to a Ser residue of the core protein through the nonsulfated linkage region hexasaccharide, -GlcUA1–3GalNAc1– 4GlcUA1–3Gal1–3Gal1– 4Xyl1
Summary
The nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster are ideal model organisms for studying a wide range of fundamental biological disciplines in development. GAG chains in D. melanogaster have been detected in the tissue extracts, based on the [35S]sulfate incorporation and the sensitivity of the materials to chondroitinase and nitrous acid treatments [16], whereas those in C. elegans have been detected in cross-sections of all organs using an electron-dense dye in conjunction with GAG lyase digestion [17] It was reported, based on the sensitivity to heparitinase and nitrous acid but not to chondroitinase ABC, that a single syndecan homolog is expressed as an HS proteoglycan in D. melanogaster [18]. We isolated and characterized the protein linkage region of C. elegans chondroitin in addition to D. melanogaster HS and CS to investigate whether these GAGs in invertebrates are attached to core proteins through the conventional linkage region tetrasaccharide and whether the tetrasaccharide is modified by phosphorylation and sulfation as in vertebrates
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