Development of a monomeric recombinant Butea monosperma agglutinin as a diagnostic and prognostic biomarker for cholangiocarcinoma.

Native Butea monosperma agglutinin (nBMA), is a lectin isolated from the seeds of the Butea monosperma plant, which binds specifically to galactose, N-acetylgalactosamine, and lactose. This study developed a recombinant β-chain of BMA (rBMA) expressed in Escherichia coli. The rBMA exists in a monomeric form, retains native structure and sugar-binding capacity without exhibiting hemagglutination activity. The binding activity of rBMA was evaluated through lectin-cytofluorescent staining of CCA cell lines. Similar to nBMA, rBMA exhibited a positive signal to CCA cell lines but displayed a strong signal in different cell lines. Sodium periodate treatment abolished rBMA binding in CCA tissues and serum dot blots, confirming carbohydrate-dependent interactions. The neutralizing activity for sugar binding specificity indicated that rBMA binds to the complex glycosylated glycans rather than mono- and di-saccharides. Elevated levels of rBMA binding glycans in serum dot blots were found to differentiate CCA patients from healthy individuals, achieving a diagnostic sensitivity of 92.9%, specificity of 36%, and overall accuracy of 74%. High levels of serum rBMA-binding glycans were associated with poorer survival in CCA patients, and directly correlated with serum alkaline phosphatase levels. No correlation was found with carcinoembryonic antigen and CA19-9 levels. These findings position serum rBMA-binding glycans as potential biomarkers reflecting CCA progression. The monomeric nature and retained glycan specificity of rBMA, coupled with its absence of hemagglutination activity, make it a superior candidate to nBMA for diagnostic applications and a promising platform for targeted therapeutic development for CCA.