Department für Chemie, Universität für Bodenkultur Wien, Muthgasse 18, Wien, Austria.
Institut für Parasitologie, Veterinärmedizinische Universität, Veterinärplatz 1, Wien, Austria.
Glycobiology. 2018 Jul 1;28(7):474-481. doi: 10.1093/glycob/cwy045.
O-glycosylation is probably one of the most varied sets of post-translational modifications across all organisms, but amongst the most refractory to analyze. In animals, O-xylosylation of serine residues represents the first stage in the synthesis of glycosaminoglycans, whose repeat regions are generally analyzed as fragments resulting from enzymatic or chemical degradation, whereas their core regions can be isolated by β-elimination or endo-β-xylosidase digestion. In the present study, we show that hydrazinolysis can be employed for release of glycosaminoglycan-type oligosaccharides from nematodes prior to fluorescent labeling with 2-aminopyridine. While various [HexNAcHexA]nGal2Xyl oligosaccharides were isolated from the model organism Caenorhabditis elegans, more unusual glycosaminoglycan-type glycans were found to be present in the porcine parasite Oesophagostomum dentatum. In this case, as judged by MS/MS before and after hydrofluoric acid or β-galactosidase digestion, core sequences with extra galactose and phosphorylcholine residues were detected as [(±PC)HexNAcHexA]n(±PC)Galβ3-(±Galβ4)Galβ4Xyl. Thus, hydrazinolysis and fluorescent labeling can be combined to analyze unique forms of O-xylosylation, including new examples of zwitterionic glycan modifications.
O-糖基化可能是所有生物中翻译后修饰最多变的一组，但也是最难分析的一组。在动物中，丝氨酸残基的 O-木糖基化代表了糖胺聚糖合成的第一阶段，其重复区域通常作为酶或化学降解的片段进行分析，而其核心区域可以通过β-消除或内切-β-木糖苷酶消化分离。在本研究中，我们表明肼解可用于在使用 2-氨基吡啶进行荧光标记之前从线虫中释放糖胺聚糖型寡糖。虽然从模式生物秀丽隐杆线虫中分离出了各种[HexNAcHexA]nGal2Xyl 寡糖，但在猪寄生虫食道口线虫中发现了更不寻常的糖胺聚糖型聚糖。在这种情况下，通过 HF 或β-半乳糖苷酶消化前后的 MS/MS 判断，检测到带有额外半乳糖和磷酰胆碱残基的核心序列为[(±PC)HexNAcHexA]n(±PC)Galβ3-(±Galβ4)Galβ4Xyl。因此，肼解和荧光标记可以结合使用来分析 O-木糖基化的独特形式，包括两性离子聚糖修饰的新实例。
