**1. Introduction**

Lectins are specific carbohydrate-binding proteins, found in animals, plants and microorganisms, and involved in various biological processes including cell adhesion, innate immunity, fertilization, differentiation et al. [1–4]. First, classifications of lectins were based on the glycan structures, to which they exhibited high affinity [5]. Later, lectins were classified into families on the basis of similarity of amino acid sequences of their carbohydrate recognition domains (C-type lectins, L-, M-, P-, R-, F-type lectins, galectins et al.) [1,2,6]. To date, the amino acid sequences of several hundreds of lectins have been determined, and a number of their three-dimensional structures have been elucidated. Recently, a new lectin classification based on their three-dimensional structures was proposed and 48 lectin families were characterized [7].

In the last two decades, many lectins from marine invertebrates were identified, and their functions in various immune events were demonstrated [3]. Earlier, we reported on a novel GalNAc/Gal-specific lectin from the mussel *Crenomytilus grayanus* (CGL), which did not share sequence homology with known lectins and consisted of three tandem-repeat subdomains with high (up to 73%) sequence identity to each other [8,9]. Three-dimensional structure prediction revealed that CGL adopted a ß-trefoil fold and contained three binding sites including conserved HPY(K)G motifs [9,10], which was later confirmed by X-ray analysis [11,12].

CGL was shown to possess anti-cancer activity through binding globotriose Gb3 [12]. The ability of CGL to recognize Gb3 on the surface of breast cancer cells [12] and bind mucin-type glycoproteins [8,9], which are often associated with oncogenic transformation, makes structural studies highly valuable to discern mechanistic details of its function. In our previous study the role of three conserved HPK(Y)G motifs in hemagglutinating and carbohydrate binding activities of CGL was experimentally shown by site-specific mutagenesis studies [10]. To investigate CGL functions and peculiarities of its molecular organization in more detail, in this study we evaluated the contribution of individual amino acid residues from CGL binding sites into the lectin activity using analysis of recombinant CGL mutants and in silico evaluation of mono- and oligosaccharide structures impacts on CGL binding properties.
