TWENTY-THIRD ANNUAL EASTERN FISH HEALTH WORKSHOP JOHN CARVER INN, PLYMOUTH, MA 30 MARCH - 2 APRIL, 1998 Ice-Binding Site Of Atlantic Herring Antifreeze Protein Evolved From The Carbohydrate-Binding Site Of A Soluble C-Type Lectin Vanya K. Ewart1,2, Z. Li1, and C. L. Hew1 1Dept. of Laboratory Medicine and Pathobiology and Dept. of Biochemistry, University of Toronto and Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada; 2National Research Council of Canada, Institute for Marine Biosciences, Halifax, Nova Scotia, Canada Soluble C-type lectins have important roles in immune function throughout the animal kingdom. These proteins recognize pathogens as non-self and bind to them by carbohydrate patterns on their cell surfaces. The antifreeze protein of Atlantic herring (Clupea harengus harengus) is homologous to the C-type lectins. Like these lectins, antifreeze proteins recognize non-self but their particular targets are ice crystals instead of pathogens and their role is to stop ice crystal growth. C-type lectins share a specific Ca2+ and carbohydrate-binding site. The herring antifreeze protein was shown by sequence analysis and modeling to contain precise binding site elements that are conserved among the galactose-binding C-type lectins. The antifreeze protein does not bind carbohydrate at the Ca2+-binding site. Therefore, we sought to determine whether the Ca2+/carbohydrate site had adopted an ice-binding role in the herring AFP. To disrupt the site in the herring AFP, without perturbing the Ca2+-dependent protein fold, a double mutant was prepared in which the Ca2+ and carbohydrate-binding site core from the galactose-type of wild-type AFP containing the sequence Gln-Pro-Asp was changed to the mannose-type, Glu-Pro-Asn, that also binds Ca2+. Folding, stability, and Ca2+ binding were unchanged in the mutant AFP. However, the mutant showed no antifreeze activity (thermal hysteresis) and did not alter ice crystal morphology to form bipyramidal crystals like the active wild-type AFP. These results demonstrate that the ice-binding site of the herring AFP corresponds to the carbohydrate-binding site of the C-type lectins and further suggest that ice-binding function evolved from the carbohydrate-binding site of a pre-existing soluble C-type lectin. Work is underway to identify related lectins in fish. This work was funded by the Medical Research Council of Canada and Postdoctoral Fellowship.   Return to 23rd Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page