Specificity of the medaka enteropeptidase serine protease and its usefulness as a biotechnological tool for fusion-protein cleavage

Abstract

We cloned two distinct cDNAs for enteropeptidase (EP) from the intestine of the medaka, Oryzias latipes, which is a small freshwater teleost. The mRNAs code for EP-1 (1,036 residues) and EP-2 (1,043 residues), both of which have a unique, conserved domain structure of the N-terminal heavy chain and C-terminal catalytic serine protease light chain. When compared with mammalian EP serine proteases, the medaka enzyme exhibited extremely low amidolytic activity for small synthetic peptide substrates. Twelve mutated forms of the medaka EP protease were produced by site-directed mutagenesis. Among them, one mutant protease, E173A, was found to have considerably reduced nonspecific hydrolytic activities both for synthetic and protein substrates without serious reduction of its Asp-Asp-Asp-Asp-Lys (D4K)-cleavage activity. For the cleavage of fusion proteins containing a D4K-cleavage site, the medaka EP proteases were shown to have advantages over their mammalian counterparts. Based on our present data, we propose that the E173A mutant is the most appropriate protease to specifically cleave proteins containing the D4K cleavage sequence.cleavage specificity ͉ medaka fish E nteropeptidase (EP) (enterokinase, EC 3.4.21.9) is a twochain, membrane-bound serine protease of the duodenal mucosa that converts trypsinogen to active trypsin (1). Trypsin thus produced then cleaves and activates other zymogens in pancreatic secretions, including chymotrypsinogen, proelastase, procarboxypeptidases, and some prolipases. Therefore, EP has been recognized to play a critical role in regulating protein digestion within the lumen of the gut. The biological importance of EP is demonstrated by the malabsorption and malnutrition of patients with primary EP deficiency, a genetic disorder resulting in no or little EP activity in the duodenum (2).EP has been intensively studied in the last decade. To date, EP has been molecularly cloned from several sources, including cattle (3, 4), humans (5), pigs (6), rats (7), and mice (8). These studies provided much information on the structural details and organization of EP, and opened a path to further investigation of the molecular properties of this protease. For example, it was demonstrated that the N-terminal heavy chain is required for efficient activation of trypsinogen by the serine protease domain of the C-terminal light chain (9, 10). In addition, a recent study by Lu et al. (11) established the tertiary structure of the bovine EP catalytic domain. The study demonstrated the involvement of Lys-99, which is situated in a unique exosite on the enzyme surface, in the specific cleavage of trypsinogen and similar peptidyl substrates. More recently, a mucin-like domain found in the heavy chain of EP has been shown to be a possible targeting signal for apical sorting of the protein (12).EP is also of biotechnological interest because of the unique substrate specificity of the serine protease domain. The high degree of specificity exhibited by EP makes it a suitable reagent for cleaving bacterially pr...

A fluorogenic method for measuring enteropeptidase activity: spectral shift in the emission of GD4K-conjugated 7-amino-4-methylcoumarin

“…Next, we determined the kinetic parameters of enteropeptidase for GD4K-AMC. As a control experiment, GD4K-NA was used as a substrate for bovine enteropeptidase light chain and the results agreed with those of previous studies (9)(10)(11), with the KM of GD4K-NA being 0.5 to 0.6 mM. The initial reaction rates of GD4K-AMC hydrolysis were measured and plotted on the Lineweaver-Burk plot (Fig.…”

Trypsin is a multifunctional factor in spermatogenesis

“…Trypsinogen is a precursor of trypsin, a member of the serine protease family that is mainly produced in the pancreas as a digestive enzyme. Although it has been reported that serine proteases are (8,9). In our current study, we show that trypsin plays an important role in the regulation of three reproductive events in the male eel; that is, the initiation of meiosis, spermiogenesis, and fertilization.…”

Apparent Involvement of Plasmin in Early-Stage Follicle Rupture During Ovulation in Medaka1

“…Eluted fractions were dialyzed against the buffer and concentrated. Active site titration was performed as previously described [25]. A total of 98 medaka ovaries were collected for analysis of the N-terminal amino acid sequence of the plasmin light chain.…”