Molecular and Sugar-Binding Heterogeneity of C-Type Lectins from Osmerus (Spirinchus) lanceolatus Eggs

Abstract

Two C-type lectins (OLLafs and OLLafl) were isolated from Osmerus (Spirinchus) lanceolatus eggs using asialofetuin-Sepharose column. OLLafs and OLLafl were eluted with 0.2 M sucrose and 0.2 M lactose from the same column, respectively. OLLafl has been estimated to be a heterodimeric protein composed of H- and L-subunit and involved C-type lectin like domain (CTLD). In this study we revealed that OLLafs was a homodimeric protein composed of L-subunit of OLLafl. Although adding EDTA diminished the hemagglutinating activity of OLLafs, the activity of OLLafl was not influenced. Recombinant lectins (rOLLafl-H and -L) and mutant lectins replaced Cys^{123, 131 and 136} with Ala (mOLLafl-L^{123, 131 and 136}) were established. The activity of mOLLafl-L¹³⁶ was comparable to rOLLafl-L, and rOLLafl-H was 15 times lower than rOLLafl-L. On the other hand, the activity of mOLLafl-L¹²³ and mOLLafl-L¹³¹ were lower than that of rOLLafl-H. Therefore, Cys¹³⁶ may not participate in hemagglutinating activity of rOLLafl-L. In contrast, Cys¹²³ and Cys¹³¹ may partially contribute this activity. Although hemagglutination inhibition profiles of rOLLafl-L, rOLLafl-H and mOLLafl-L¹³⁶ were similar, m-OLLafl-L¹³¹-induced hemagglutination was not inhibited by any sugars tested even at a concentration of 150 mM. Then, Cys¹³¹ may directly contribute to the sugar-binding capacity of OLLafl. Affinities of mOLLafl-L¹²³ for these sugars were lower than the others. These results suggest that Cys¹³⁶ might contribute to the intermolecular disulfide bond in the rOLLafl-L dimer, and that the intramolecular disulfide bond concerning Cys¹³¹ might important for lectin activity.