Aberrant RL2 O-GlcNAc antibody reactivity against serum-IgA1 of patients with colorectal cancer

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 268 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.