Identification of a type II LacNAc specific binding lectin CMRBL from Cordyceps militaris

The Cordyceps militaris gene CCM_03832 encodes a ricin-B like lectin. The gene was cloned and expressed in Escherichia coli, and its protein product, named CMRBL (C. militaris ricin-B like lectin), was purified by galactose affinity chromatography. Of nine different sources of erythrocytes, CMRBL showed only specific hemagglutinating activity against rat and rabbit erythrocytes with titers of 2² and 2⁸, respectively. Glycan array analyses by the Consortium for Functional Glycomics showed that CMRBL possesses very high specific binding activity of glycans terminated with type II LacNAc (non-reducing Galβ1-4GlcNAc). Compared with other well-known Gal-terminated binding lectins such as Erythrina cristagalli agglutinin, Ricinus communis agglutinin, and Jacalin, CMRBL showed better binding specificity to type II LacNAc compared the other lectins. CMRBL showed lowest binding activity to ZR-75-30 and MDA-MB-468 cell lines among five tested cell lines (H22, THP-1, MDA-MB-231, ZR-75-30, and MDA-MB-468 cells). Transfection of type II LacNAc main galactosyltransferase B4GALT3 to ZR-75-30 significantly improved CMRBL binding activity compared with control. CMRBL was also applied for testing the type II LacNAc modification of Etanercept successfully. Our data suggest that CMRBL would be a useful tool to recognize type II LacNAc, especially distinguish type II from other galactose-terminated glycans in glycan biology research.