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 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.

High surfactant-tolerant ß-mannanase isolated from Dynastes hercules larvae excrement, and identification of its hotspot using site-directed mutagenesis and molecular dynamics simulations.

The ß-mannanase from Bacillus subtilis HM7 (Man26HM7) isolated from Dynastes hercules larvae excrement was cloned and expressed in Escherichia coli. Biochemical characterization shows that optimal pH and temperature for catalysis are 6.0 and 50 °C, respectively. Man26HM7 displayed excellent surfactant stability by retaining 70% of initial activity in 1%(w/v) SDS, and more than 90% of initial activity in 1%(w/v) Triton X-100 and Tween 80. Results from amino acid sequence alignment and molecular modeling suggest residue 238 of ß-mannanase as a hotspot of SDS-tolerance. Mutagenesis at the equivalent residue of another homolog, ß-mannanase from Bacillus subtilis CAe24 (Man26CAe24), significantly enhanced the SDS stability of this enzyme. Comparative computational analysis, including molecular docking and molecular dynamics simulation, were then performed to compute the binding free energy of SDS to Man26HM7, Man26CAe24, and variant enzymes. The results suggest that residue 238 of Man26HM7 is involved in SDS binding to the hydrophobic surface of ß-mannanase. This study provides not only the promising application of Man26HM7 in detergent and cleaning products but also valuable information for enhancing the surfactant stability of ß-mannanase by enzyme engineering.