Enterokinase is a protease of the intestinal brush border that specifically cleaves trypsinogen to yield active trypsin, which then cleaves and activates other pancreatic zymogens. This regulatory mechanism confines the activity of digestive hydrolases to the gut. Enterokinase appears to be conserved among all vertebrates, and congenital deficiency causes intestinal malabsorption. In preliminary studies the structures of bovine, human, and murine enterokinase were determined by cDNA cloning. These clones were employed to localize the enterokinase gene to chromosome 21q, to demonstrate enterokinase expression specifically in small intestine, and to express recombinant enterokinase in E. coli. Rabbit polyclonal antibodies were prepared to recombinant enterokinase. These results provide the means to address the biosynthesis, regulation, and structure-function relationships of this ancient, essential protease.
Specific Aim 1 is to define the biosynthesis and intracellular targeting of enterokinase. Active two-chain enterokinase is derived by proteolysis of a single-chain precursor, and both chains are extensively glycosylated. The amino-terminal heavy chain is a mosaic of domains found in otherwise unrelated proteins. The carboxyl-terminal light chain is homologous to trypsin-like serine proteases. Membrane association may be mediated by a signal-anchor sequence near the amino-terminus, which may be N- myristoylated. The mechanism of enterokinase delivery to the apical brush border of enterocytes has not been defined. The intracellular processing and sorting of enterokinase will be characterized in cell lines transfected with full-length bovine enterokinase, and in cultured mouse duodenal mucosa.
Specific Aim 2 is to determine the mechanism of proenterokinase activation. The identity of the protease that cleaves and activates enterokinase is not known. The sequence surrounding the amino- terminal Ile of the bovine light chain (Pro-Lys-Ile-Val-Gly) suggests that enterokinase may be activated by trypsin, and the structure of the catalytic domain suggests that proenterokinase may have limited intrinsic activity toward trypsinogen. Alternatively, enterokinase activation may require a separate cofactor or cleavage by a novel protease. These models will be distinguished by kinetic characterization of recombinant wild-type and mutant enterokinase. These studies will determine how the activation of enterokinase is initiated and sustained, thereby filling a critical gap in our understanding of how digestive enzymes are regulated in vivo.
Specific Aim 3 is to determine the structural basis of enterokinase substrate specificity. Enterokinase specifically cleaves substrates that resemble the trypsinogen activation peptide, Val-Asp-Asp-Asp-Asp-Lys, but enterokinase binds trypsinogen approximately 100-fold more avidly than similar model peptides. This remarkable specificity for trypsinogen may be determined by both the light chain and the noncatalytic heavy chain. The contribution to substrate recognition of the enterokinase heavy chain, the light chain, and membrane association will be defined by kinetic studies, by selection of optimal substrates from substrate phage display libraries, and by x-ray crystallography.
