| Summary: | Thesis (Ph.D.)--Memorial University of Newfoundland, 1994. Biology Bibliography: leaves [196]-216 The structural diversity of fish antifreeze proteins is believed to result from their independent origins in separate fish taxonomic groups. Therefore, antifreeze proteins(AFPs) from two fish species distantly related to all others known to produce AFPs were studied in order to gain a better understanding of the diversity and the distribution of the different AFP types. Results of this study suggest that homologous type II fish AFPs have evolved independendy in three separate groups offish but that they originate from members of a single protein family, the calcium-dependent (C-type) lectins. Moreover, the homology among the three AFPs and the C-type lectins has allowed the recently determined structure of a C-type lectin domain to serve as a prototype for the type II AFPs. This will provide the basis for determining the functional mechanism of this AFP type. These findings also suggest that study of the AFP variants present in diverse fish species may lead to the further insight into the origins, the structures, and the mechanisms of action of the different fish AFPs. -- AFPs were isolated from the blood plasma of smelt (Osmerus mordax) and Atlantic herring (Clupea harengus harengus) using gel filtration, ion exchange chromatography, and reverse phase HPLC. Characterization of smelt AFP revealed six isoforms, each with a Mr of 24,000. Amino acid analysis and endoglycosidase digestion revealed that smelt AFP contained N-linked carbohydrate. The herring AFP had two isoforms with a Mr of 14,600 and no carbohydrate. Further analysis of these proteins revealed that, like the type II AFP of sea raven (Hemitripterus americanus) they were both cystine-rich and sensitive to sulfhydryl reducing agents. Antisera raised against heiring and sea raven AFPs cross- reacted with smelt AFP. However, the thermal hysteresis activities of smelt and herring AFPs were lower than that of sea raven AFP. -- cDNA libraries were constructed from livers of smelt and herring. Libraries were screened using an oligonucleotide with a sequence derived from tryptic peptides of smelt and herring AFPs. AFP cDNA clones were isolated and sequenced and the primary structures of smelt and herring AFPs were deduced. Protein sequence database searches, sequence alignments, and statistical analyses showed that the AFPs of smelt, herring, and sea raven were homologous to one another and to the carbohydrate-recognition domains (CRDs) present in a family of Ca2+-dependent (C-type) lectins. Furthermore, all but one of the residues known to define the hydrophobic cores of a C-type lectin CRD were present in these AFPs. One CRD Ca2+-binding site appeared conserved in smelt and herring AFPs and these AFPs did require Ca2+ for thermal hysteresis activity. Alignment of sea raven AFP with C-type CRDs suggested that it had no functional Ca2+-binding sites and the thermal hysteresis activity of this AFP did not require Ca2+. -- Affinity chromatography and agglutination experiments showed that, unlike the C-type lectins, the type II AFPs did not appear to bind strongly to carbohydrates. However, a trace of smelt AFP was retarded on a matrix containing galactose suggesting a slight affinity for this carbohydrate. Two proteins in the C-type lectin family were isolated and tested for thermal hysteresis activity. Neither human pancreatic stone protein nor a C-type lectin from the venom of a snake (Crotalus atrox) showed detectable thennal hysteresis activity. -- Together, these results suggest that the type II AFPs of smelt, herring, and sea raven are related and that all three have evolved from the CRDs of preexisting proteins in the C-type lectin family, the type II AFPs have a fold similar to that of the lectins, and some resemble lectins in requiring Ca2+ for activity. However, unlike the lectins, the AFPs appear to interact primarily with ice crystals rather than with carbohydrate.
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