Molecular and sugar-binding heterogeneity of C-type lectins from Osmerus (Spirinchus) lanceolatus eggs.
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-L(136) was comparable to rOLLafl-L, and rOLLafl-H was 15 times lower than rOLLafl-L. On the other hand, the activity of mOLLafl-L(123) and mOLLafl-L(131) were lower than that of rOLLafl-H. Therefore, Cys(136) may not participate in hemagglutinating activity of rOLLafl-L. In contrast, Cys(123) and Cys(131) may partially contribute this activity. Although hemagglutination inhibition profiles of rOLLafl-L, rOLLafl-H and mOLLafl-L(136) were similar, m-OLLafl-L(131)-induced hemagglutination was not inhibited by any sugars tested even at a concentration of 150 mM. Then, Cys(131) may directly contribute to the sugar-binding capacity of OLLafl. Affinities of mOLLafl-L(123) for these sugars were lower than the others. These results suggest that Cys(136) might contribute to the intermolecular disulfide bond in the rOLLafl-L dimer, and that the intramolecular disulfide bond concerning Cys(131) might important for lectin activity.
Sugawara
Shigeki
S
Cancer Research Institute, Tohoku Pharmaceutical University, Sendai 981-8558, Japan.
Hosono
Masahiro
M
Ogawa
Yukiko
Y
Takayanagi
Motoaki
M
Nitta
Kazuo
K
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D004912:Erythrocytes / Q000378:metabolism; D005260:Female; D006801:Humans; D037181:Lectins, C-Type / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D036441:Osmeriformes / Q000235:genetics / Q000378:metabolism*; D010063:Ovum / Q000378:metabolism*; D012685:Sepharose / Q000378:metabolism*
D037181:Lectins, C-Type; D012685:Sepharose