Verathamjamras Chris, Sriwitool Tanin-Ek, Netsirisawan Pukkavadee, Chaiyawat Parunya, Chokchaichamnankit Daranee, Prasongsook Naiyarat, Srisomsap Chantragan, Svasti Jisnuson, Champattanachai Voraratt
Laboratory of Biochemistry, Chulabhorn Research Institute, Laksi, Bangkok 10210, Thailand.
Applied Biological Sciences Program, Chulabhorn Graduate Institute, Laksi, Bangkok, 10210, Thailand.
Glycoconj J. 2021 Feb;38(1):55-65. doi: 10.1007/s10719-021-09978-8. Epub 2021 Feb 20.
O-GlcNAcylation, a single attachment of N-acetylglucosamine (GlcNAc) on serine and threonine residues, plays important roles in normal and pathobiological states of many diseases. Aberrant expression of O-GlcNAc modification was found in many types of cancer including colorectal cancer (CRC). This modification mainly occurs in nuclear-cytoplasmic proteins; however, it can exist in some extracellular and secretory proteins. In this study, we investigated whether O-GlcNAc-modified proteins are present in serum of patients with CRC. Serum glycoproteins of CRC patients and healthy controls were enriched by wheat germ agglutinin, a glycan binding protein specifically binds to terminal GlcNAc and sialic acid. Two-dimensional gel electrophoresis, RL2 O-GlcNAc immunoblotting, affinity purification, and mass spectrometry were performed. The results showed that RL2 O-GlcNAc antibody predominantly reacted against serum immunoglobulin A1 (IgA1). The levels of RL2-reacted IgA were significantly increased while total IgA were not different in patients with CRC compared to those of healthy controls. Analyses by ion trap mass spectrometry using collision-induced dissociation and electron-transfer dissociation modes revealed one O-linked N-acetylhexosamine modification site at Ser located in the heavy constant region of IgA1; unfortunately, it cannot be discriminated whether it was N-acetylglucosamine or N-acetylgalactosamine because of their identical molecular mass. Although failed to demonstrate unequivocally it was O-GlcNAc, these data indicated that serum-IgA had an aberrantly increased reactivity against RL2 O-GlcNAc antibody in CRC patients. This specific glycosylated form of serum-IgA1 will expand the spectrum of aberrant glycosylation which provides valuable information to cancer glycobiology.
O-连接的N-乙酰葡糖胺化（O-GlcNAcylation）是指N-乙酰葡糖胺（GlcNAc）在丝氨酸和苏氨酸残基上的单一附着，在多种疾病的正常和病理生物学状态中发挥重要作用。在包括结直肠癌（CRC）在内的多种癌症中发现了O-GlcNAc修饰的异常表达。这种修饰主要发生在核质蛋白中；然而，它也可以存在于一些细胞外和分泌蛋白中。在本研究中，我们调查了CRC患者血清中是否存在O-GlcNAc修饰的蛋白。通过麦胚凝集素（一种特异性结合末端GlcNAc和唾液酸的聚糖结合蛋白）富集CRC患者和健康对照者的血清糖蛋白。进行了二维凝胶电泳、RL2 O-GlcNAc免疫印迹、亲和纯化和质谱分析。结果表明，RL2 O-GlcNAc抗体主要与血清免疫球蛋白A1（IgA1）反应。与健康对照者相比，CRC患者中RL2反应性IgA的水平显著升高，而总IgA水平无差异。使用碰撞诱导解离和电子转移解离模式的离子阱质谱分析显示，在IgA1重链恒定区的Ser处有一个O-连接的N-乙酰己糖胺修饰位点；不幸的是，由于N-乙酰葡糖胺和N-乙酰半乳糖胺分子量相同，无法区分是哪种糖胺。尽管未能明确证明其为O-GlcNAc，但这些数据表明CRC患者血清IgA对RL2 O-GlcNAc抗体的反应性异常增加。血清IgA1的这种特定糖基化形式将扩展异常糖基化的范围，为癌症糖生物学提供有价值的信息。
