Chitinase-An Evolutionary Duality
Place, Allen R.
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The purpose of this thesis was to characterize the molecular properties, gene copy number and gene organization for the chitinase enzyme in a lower vertebrate. Rainbow trout (Oncorhynchus mykiss) has one of the highest chitinase activities in all the fishes examined. I also aimed to clarify the evolutionary origin and associated regulatory control of the chitinase gene in this species. The cDNAs from two chitinases (Onmy-Chit.01 and Onmy-Chit.02) were successfully cloned and characterized from rainbow trout tissues. Onmy-Chit.01 is predominately expressed in the stomach, with high mRNA expression in the gastric gland. Its protein secretes along mucosal folds to the stomach lumen. Onmy-Chit.02 is primarily expressed in immunity related organs, such as kidney, spleen, and liver, as well as in reproductive organs. From in situ hybridization and flow cytometry analysis, I show that Onmy-Chit.02 is constitutively expressed in the myeloid cell lineage of the rainbow trout immune system. These two enzymes share many similarities with their mammalian orthologs. Their predicted proteins all have classic chitinase protein structures. In addition, they all have O-glycosylation sites but with different pH optimas. Fluorescent in situ hybridization (FISH) shows that both chitinases are located on Chromosome 17 of the rainbow trout genome. Upon full sequencing of two BACs containing Onmy-Chit.01 or .02, I found two copies of Onmy-Chit.01with almost identical coding regions, but with very different promoter regions. The two copies are apporoximately 9Kb apart and sit in a head to tail arrangement. Only one copy of Onmy-Chit.02 was found in the rainbow trout genome, and its distinct promoter regions are distinct from both copies of Onmy-Chit.01. Phylogenetic analyses revealed that the chitinase gene family fits an evolutionary birth and death model with the chitinase genes derived by duplication of an ancestral chitinase gene. Further gene duplication and loss of chitinolytic activity in mammals gave rise to chitolectins. Hence, I postulate that the function of chitinase is two fold: 1) it is a key element in the first line defense of the innate immunity repertoire; and 2) it serves as a gastric digestive enzyme for chitin containing food items.