Malectin is a highly-conserved animal lectin from the endoplasmic reticulum (ER), with a quality control function in the N-Glycosylation process. It has a β-sandwich core with long loops connecting the β-sheets. Malectin binding-pocket is in the loops region. Several carbohydrate-binding modules (CBMs) discovered in other domains of life that shared sequence homology with the malectin, were classified and grouped as a novel CBM57 family by Carbohydrate-Active Enzymes (CAZy) database. The members of this family are expected to have a highly conserved β-sandwich core, but high variance in the binding-pocket residues.
To investigate if the specificity of these modules is the same as the malectin, a bioinformatic analysis was performed with 315 members of the CBM57 family found in CAZy database. Several programs were used to predict the protein architecture and to analyse the conservation of amino acids sequences, especially in the binding-pocket. Based on this analysis, we predict animal CBM57 modules to have the same specificity as malectin. However, bacterial CBM57 modules in bacteria domain are predicted, after highlighting the modules associated with glycoside hydrolases from family 2, to have various specificities, and thus different biological functions. For verifying these assumptions, a total of 7 CBMs (family 57 and homologous) associated with glycoside hydrolases from family 2 and belonging to the human gut microbiome – Bacteroides ovatus and Bacteroides thetaiotaomicron- were chosen for characterization studies.
A re-cloning was initially performed for the recombinant DNAs, changing the His-tag position. Afterwards, expression tests were realized, in which 2 CBMs of different bacteria were expressed in soluble form. The production of the proteins was then performed at a larger scale, followed by affinity chromatography purification. By the analysis of the gels, the eluted samples had high purity and were suitable for characterization studies.
Glycan microarrays were performed for determining the binding-specificities of the 2 CBM modules. The CBM module from B.thetaiotaomicron revealed high specificity for pectin polysaccharides, possible recognizing α 1-3 linked galacturonic acid and ramnose. For structural characterization by X-ray crystallography, several crystallization trials were performed. Crystals were obtained for the B.thetaiotaomicron CBM module, which diffracted to high resolution. The structure is, yet, to be solved
