Enteropeptidase (also known as enterokinase) is a protease of the intestinal brush border that specifically cleaves trypsinogen to yield trypsin, which then cleaves and activates other pancreatic zymogens. This regulatory mechanism confines the activity of digestive hydrolases to the gut. Enteropeptidase is synthesized as a single chain zymogen, whereas active enteropeptidase contains a approximately 47 kDa serine protease domain (light chain) and a disulfide-linked approximately 120 kDa heavy chain. Enteropeptidase is conserved among vertebrates, and congenital deficiency causes intestinal malabsorption. Understanding the targeting and structure-function relationships of this ancient, essential protease is critical to understanding the regulation of digestive enzymes in vivo.
Specific Aim 1 is to characterize the signals that specify apical targeting of enteropeptidase. Preliminary studies have identified two motifs that contain apical targeting information. These signals will be dissected by mutagenesis and expression of chimeric proteins in polarized MDCK cells. In addition, the role of sphingolipid and cholesterol-rich """"""""raft"""""""" domains in enteropeptidase sorting will be defined.
Specific Aim 2 is to define the structural basis for the recognition by enteropeptidase of substrates and inhibitors. Enteropeptidase recognizes substrates that resemble the trypsinogen activation peptide, Val-Asp-Asp-Asp-Asp-Lys, but enteropeptidase cleaves trypsinogen with approximately 500-fold greater catalytic efficiency than it cleaves similar model peptides. This specificity for trypsinogen is determined by """"""""exosites"""""""" on both the light chain and the noncatalytic heavy chain that are distinct from the catalytic center. These exosites will be characterized by targeted mutagenesis and by selection of optimal substrates from phage display libraries.
Specific Aim 3 is to determine the three-dimensional structure of the enteropeptidase catalytic domain complexed with substrate analogs and inhibitors. The crystallographic structure is being refined of the recombinant enteropeptidase catalytic domain (L-BEK) complexed with an analog of the trypsinogen activation peptide, Val-Asp-Asp-Asp-Asp-Lys-chloromethane. This structure provides a framework to understand the specificity of enteropeptidase in atomic detail and to predict the effects of mutagenesis. Additional structures will be determined for mutant enzymes with novel specificity and for selected enzyme-inhibitor complexes, including L-BEK (Lys99A1a), L-BEK-STI, and L-BEK-BPTI. The complementary approaches of mutagenesis, kinetic analysis and structure determination will provide new insight into the range of properties that evolution can confer on trypsin-like serine proteases.
|Zheng, Xinglong; Sadler, J Evan (2002) Mucin-like domain of enteropeptidase directs apical targeting in Madin-Darby canine kidney cells. J Biol Chem 277:6858-63|
|Bodo, I; Katsumi, A; Tuley, E A et al. (2001) Type 1 von Willebrand disease mutation Cys1149Arg causes intracellular retention and degradation of heterodimers: a possible general mechanism for dominant mutations of oligomeric proteins. Blood 98:2973-9|
|Lu, D; Futterer, K; Korolev, S et al. (1999) Crystal structure of enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide. J Mol Biol 292:361-73|
|Zheng, X; Lu, D; Sadler, J E (1999) Apical sorting of bovine enteropeptidase does not involve detergent-resistant association with sphingolipid-cholesterol rafts. J Biol Chem 274:1596-605|
|Yuan, X; Zheng, X; Lu, D et al. (1998) Structure of murine enterokinase (enteropeptidase) and expression in small intestine during development. Am J Physiol 274:G342-9|
|Xue, J; Wu, Q; Westfield, L A et al. (1998) Incomplete embryonic lethality and fatal neonatal hemorrhage caused by prothrombin deficiency in mice. Proc Natl Acad Sci U S A 95:7603-7|
|Kokame, K; Zheng, X; Sadler, J E (1998) Activation of thrombin-activable fibrinolysis inhibitor requires epidermal growth factor-like domain 3 of thrombomodulin and is inhibited competitively by protein C. J Biol Chem 273:12135-9|
|Lu, D; Yuan, X; Zheng, X et al. (1997) Bovine proenteropeptidase is activated by trypsin, and the specificity of enteropeptidase depends on the heavy chain. J Biol Chem 272:31293-300|