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 Ser₂₆₈ 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 -GlcNAcylation 是N-乙酰氨基葡萄糖 (GlcNAc) 在丝氨酸和苏氨酸残基上的单一连接，在许多疾病的正常和病理状态中起重要作用。在包括结肠直肠癌 (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，它可以存在于一些细胞外和分泌蛋白中。在这项研究中，我们调查了 CRC 患者的血清中是否存在O -GlcNAc 修饰的蛋白质。CRC 患者和健康对照者的血清糖蛋白富含小麦胚芽凝集素，这是一种聚糖结合蛋白，可特异性结合末端 GlcNAc 和唾液酸。二维凝胶电泳，RL2进行了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) 发生反应。与健康对照组相比，CRC 患者的 RL2 反应的 IgA 水平显着增加，而总 IgA 没有差异。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揭示了位于 IgA1 重恒定区的Ser _{268处的一个}O连接的N-乙酰己糖胺修饰位点；不幸的是，无法区分它是N-乙酰氨基葡萄糖还是N-乙酰半乳糖胺，因为它们的分子量相同。尽管未能明确证明它是O -GlcNAc，但这些数据表明血清-IgA 在 CRC 患者中对 RL2 O -GlcNAc 抗体的反应性异常增加。这种特定的糖基化形式的血清-IgA1 将扩大异常糖基化的范围，从而为癌症糖生物学提供有价值的信息。