Carbohydrate-binding properties of goat secretory glycoprotein (SPG-40) and its functional implications: structures of the native glycoprotein and its four complexes with chitin-like oligosaccharides

A 40 kDa glycoprotein (SPG-40) secreted during involution works as a protective signalling factor through its binding to viable cells. The crystal structure of the native protein has been determined at 2.3 Å resolution. This is the first report on the carbohydrate-binding properties of SPG-40; the structure determinations of the complexes of SPG-40 with four oligosaccharides of different lengths at resolutions ranging from 2.2 to 2.8 Å are described. Carbohydrate-binding studies with N-­acetylglucosamines (GlcNAc_n, n = 3-6) using fluorescence spectroscopy revealed poor binding effects with GlcNAc₃ and GlcNAc₄, while GlcNAc₅ and GlcNAc₆ bound to SPG-40 with considerable strength; the dissociation constants (K_d) were estimated to be 260 ± 3 and 18 ± 4 µM, respectively. SPG-40 was cocrystallized with GlcNAc₃, GlcNAc₄, GlcNAc₅ and GlcNAc₆. The overall structure of native SPG-40 was essentially similar to that reported previously at low resolution. The structures of its complexes with GlcNAc₃, GlcNAc₄, GlcNAc₅ and GlcNAc₆ revealed the positions of these oligosaccharides in the carbohydrate-binding groove and provided insights into the mechanism of binding of oligosaccharides to SPG-40, indicating that the preferred subsites in the carbohydrate-binding groove of SPG-­40 were from -4 to -2. The structure of the protein remained unperturbed upon binding of GlcNAc₃ and GlcNAc₄, but the structure changed significantly upon binding of GlcNAc₅ and GlcNAc₆. Significant conformational variations were observed in the sugar-binding groove: Trp78 partially flipped out of the barrel in GlcNAc₅, while in the GlcNAc₆ complex a completely flipped-out Trp78 was observed along with several other conformational changes, including those of Asp186 and Arg242. Such changes upon binding to carbohydrates have not previously been observed in chitin-hydrolyzing chitinases and reflect less favourable binding of carbohydrates to SPG-40. As this appears to essentially be a binding protein, this loss of binding affinity might be compensated by other intermolecular interactions such as protein-protein interactions and also by the binding of its own glycan chain.