*2.1. Analysis of CGL Contacts with Galactose/Galactosamine for Mutagenesis*

The theoretical model of the spatial structure of lectin CGL was previously constructed by us [10] based on the crystal structure of the lectin MytiLec determined at 1.05 Å resolution (Protein Data Bank accession: PDB 3WMV) [13]. Superimposition of all Cα atoms of obtained CGL model and CGL crystal structure (PDB 5F8S) [12] showed that they were almost completely superimposable (values of the root-mean-square deviation (RMSD) were 0.4 Å). Thus, the predicted structure of the lectin CGL was in good agreement with the experimentally established CGL structure and suitable for in silico mutagenesis and molecular docking studies.

The analysis of CGL contacts with α-galactose (Protein Data Bank accession: PDB 5F8W) and galactosamine (PDB 5F8Y) showed that CGL amino acid residues His37 and Asn119 from Site 1; His85 and Asn27 from Site 2; Asp127, His129, and Glu75 from Site 3 formed hydrogen bonds with these monosaccharides (Figure 1). These residues were selected for mutagenesis experiments.

## *2.2. Mutagenesis Studies*

To obtain the recombinant CGL of the wild type and Asn27Ala, His37Ala, Glu75Ala, His85Ala, Asn119Ala, Asp127Ala and His129Ala mutants, expression plasmids were constructed on the basis of pET40/CmAP plasmid described earlier [10,14]. The alkaline phosphatase CmAP in the hybrid CGL-CmAP protein allowed for monitoring recombinant lectins during expression and purification steps [10,14].

CGL was shown to exhibit high affinity to porcine stomach mucin [15]. The mucin-binding activity of the recombinant CGL of the wild and mutant types was evaluated by measuring the alkaline phosphatase activity provided by CmAP domain [10] (Figure 2).

The mucin-binding activity of the obtained mutants varied in a wide range and was from 9% to 73% of the wild lectin (Figure 2). CGL mutants with the alanine substitutions of His37, His129, Glu75, Asp127 and His85, Asn27, Asn119 showed decreased mucin-binding activities in 1.4, 2.3, 3.2, 4.5, 5.0, 5.9 and 11.1 times, respectively.

**Figure 1.** 2D-diagrams of the galactose-binding sites (Site 1, 2, 3) in the wild type CGL. Hydrogen bonds lost in the corresponding mutant are indicated with a cross (**x**).

**Figure 2.** Mucin-binding activity of the wild and mutant types of CGL. The lectin-mucin complexes (axis *X*—mucin concentration) were monitored by measuring the phosphatase activity of CGL/CmAP hybrid (axis *Y*).
