Tracing The Origin Of A Lectin-like Antifreeze Protein
Biochemistry , Antifreeze Protein , Gene Duplication and Divergence
Type II antifreeze proteins (AFPs) are C-type lectin homologs found in three lineages of teleost fish (Cottidae, Clupeiformes and Osmeriformes). Previous work demonstrated that the AFP gene was horizontally transferred from the Atlantic herring to the smelt, and also showed that the herring is not the progenitor species. Questions remain regarding the origin of the type II AFPs, and the relationship between Ca2+-dependent and Ca2+-independent subtypes. In this thesis, I explore the hypothesis that the Ca2+-dependent type II AFP originated in the cottid lineage from a lectin and then gave rise to a Ca2+-independent AFP from a gene duplication and divergence event. Protein, expressed sequence tag, and genomic databases were searched for putative type II AFP sequences. Candidate AFP-like proteins were only found in Eupercaria but were not limited to cottids. Proteins were modelled using Modeller and ColabFold, then evaluated based on conservation of residues potentially involved in ice- and Ca2+-binding. The current habitat and distribution of these species was evaluated in terms of the need for AFPs. In several species, genes for predicted Ca2+-dependent and Ca2+-independent proteins were found back-to-back at the same syntenic locus. Numerous type II AFP-like candidate proteins were identified for future research, and patterns in residues of significance may be used to further refine search criteria for AFP discovery. Additionally, I attempted to produce type II AFPs using a mammalian HEK293E expression system and a Drosophila expression system. Secretion of active AFP from HEK293E cells was unsuccessful, possibly due to the stressors of high temperature and protein overproduction. Expression in the Drosophila cell system was attempted at a lower temperature but did not yield secreted AFP for characterization.