Synthetic model and bioactive peptides as potential substrates for enteropeptidase

Likhareva, V. V.; Mikhailova, A. G.; Vaskovsky, B. V.; Garanin, S. K.; Румш, Л. Д.

doi:10.1007/bf02576867

Cited by 4 publications

(5 citation statements)

References 16 publications

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“…Typically, only the catalytic subunit is used because it exhibits an activity similar to the full-length protein (28). Enteropeptidase specificity has been investigated previously by comparing natural substrate sequences and by measurement of the hydrolysis rates of synthetic fluorogenic peptides (4,23,29,30). However, substrate specificity has not been characterized in detail by screening combinatorial peptide libraries, despite the obvious importance of identifying unwanted secondary cleavage sites in fusion proteins (23).…”

Section: Discussionmentioning

confidence: 99%

“…Caspase-3 was chosen to validate CLiPS, because specificity has been investigated extensively by using both substrate phage and fluorogenic substrates (21,22). In contrast, enteropeptidase specificity is less well characterized and has been investigated primarily by using individually synthesized, fluorogenic substrate variants (23). For each protease, optimal substrates were identified by performing a two-step screen for hydrolysis (Fig.…”

Section: Determination Of Enteropeptidase and Caspase-3 Specificity Bmentioning

confidence: 99%

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Protease specificity determination by using cellular libraries of peptide substrates (CLiPS)

Boulware¹,

Daugherty

2006

Proc. Natl. Acad. Sci. U.S.A.

111

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We report a general combinatorial approach to identify optimal substrates of a given protease by using quantitative kinetic screening of cellular libraries of peptide substrates (CLiPS). A whole-cell protease activity assay was developed by displaying fluorescent reporter substrates on the surface of Escherichia coli as N-terminal fusions. This approach enabled generation of substrate libraries of arbitrary amino acid composition and length that are self-renewing. Substrate hydrolysis by a target protease was measured quantitatively via changes in whole-cell fluorescence by using FACS. FACS enabled efficient screening to identify optimal substrates for a given protease and characterize their cleavage kinetics. The utility of CLiPS was demonstrated by determining the substrate specificity of two unrelated proteases, caspase-3 and enteropeptidase (or enterokinase). CLiPS unambiguously identified the caspase-3 consensus cleavage sequence DXVDG. Enteropeptidase was unexpectedly promiscuous, but exhibited a preference for substrates with the motif D ͞ERM, which were cleaved substantially faster than the canonical DDDDK recognition sequence, widely used for protein purification. CLiPS provides a straightforward and versatile approach to determine protease specificity and discover optimal substrates on the basis of cleavage kinetics.caspase-3 ͉ enteropeptidase ͉ FACS ͉ peptidase P roteolytic enzymes play central roles in most biological processes. At the same time, proteases are implicated in the pathogenesis of diverse diseases ranging from viral and bacterial infections (1) to cancer and neurodegenerative diseases (2, 3). The biological functions of proteases, or peptidases, are largely determined by the spectrum of biologically occurring substrates. Frequently, substrate specificity has been inferred by comparison and alignment of known, physiological substrates (4). Given this information, individually synthesized fluorogenic peptide substrates have been used extensively to measure protease activity (5). More recently, the substrate specificity of a small group of proteases has been characterized in greater detail by using combinatorial peptide libraries (6, 7). Library-based approaches to characterize specificity have contributed substantially to our understanding of protease function (6, 7). Nevertheless, quantitative characterization of protease specificity and substrate cleavage kinetics remains a bottleneck in basic and applied biological research (6,8).Among combinatorial methods, the ''substrate phage'' approach has been used to define the substrate specificities of Ϸ30 proteases and has been recently reviewed (6). In this approach, a library of peptide substrates is displayed on the surface of filamentous bacteriophage (9) as fusions between a surface-anchoring affinity domain and the display scaffold protein, typically the phage minor coat protein GPIII. The substrate library then is allowed to bind to a solid surface via the affinity domain. Phage liberated from the surface by proteolytic cleavage are rec...

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Section: Discussionmentioning

confidence: 99%

Section: Determination Of Enteropeptidase and Caspase-3 Specificity Bmentioning

confidence: 99%

Protease specificity determination by using cellular libraries of peptide substrates (CLiPS)

Boulware¹,

Daugherty

2006

Proc. Natl. Acad. Sci. U.S.A.

111

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“…The presence of the heavy chain had almost no effect on recognition of the small peptide substrate GD 4 K-␤NA, whereas this chain had profound effects on recognition of the macromolecular substrate trypsinogen (9). Furthermore, bovine EP is able to hydrolyze specifically several biologically active peptides in vitro (19), and substrate length greatly affects the catalytic efficiency of this hydrolysis. Activity dependency on the heavy chain and peptide substrate length remain to be determined for medaka EP.…”

Section: Discussionmentioning

confidence: 99%

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

Ogiwara

Takahashi²

2007

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…The natural enteropeptidase substrate, trypsinogen, is of Lys-type. Our preliminary data revealed no difference in the efficiency of enteropeptidase hydrolysis of low-efficient synthetic substrates containing only two negatively charged amino acid residues at positions P2-P3: WDDKG and WDDRG [10]. However, in the case of highly efficient specific substrates APFD 4 KIVGG and APFD 4 RIVGG the substitution of Lys residue by Arg at P1 leads to 2-fold increase in the efficiency of enteropeptidase hydrolysis ( Table 1).…”

Section: Resultsmentioning

confidence: 76%

“…Enteropeptidase, with its three substrate-binding centers, is also an excellent object to study the structural determinants providing the highly specific and efficient proteolysis. In the frames of this substrate analysis the kinetics of enteropeptidase-catalyzed hydrolysis of a great number of different peptides was quantitatively studied by us with the aid of the convenient and precise HPLC method [7][8][9][10]. The comparative substrate analysis of trypsin and enteropeptidase, the native full-length enzyme and its truncated variants were parts of this study.…”

Section: Introductionmentioning

confidence: 99%

The Ways of Realization of High Specificity and Efficiency of Enteropeptidase

Mikhailova¹,

Likhareva²,

Teich³

et al. 2007

PPL

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Comparative substrate analysis of full-length bovine enteropeptidase and trypsin, bovine and human enteropeptidase light chains was performed using model N-terminal dodecapeptides corresponding to wild-type human trypsinogen and pancreatitis-associated mutant trypsinogens K23R and D22G. The substitution of Lys residue by Arg at P1 leads to 2-fold increase in the efficiency of enteropeptidase hydrolysis; the absence of the negatively charged residue at P2 reduces the efficiency of such hydrolysis by two orders of magnitude. The difference in efficiency of peptide chain hydrolysis after Lys/Arg residues by enteropeptidase compared to trypsin is equal to the difference in hydrolysis by serine proteases of different primary specificity of their specific substrates.

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Synthetic model and bioactive peptides as potential substrates for enteropeptidase

Cited by 4 publications

References 16 publications

Protease specificity determination by using cellular libraries of peptide substrates (CLiPS)

Protease specificity determination by using cellular libraries of peptide substrates (CLiPS)

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

The Ways of Realization of High Specificity and Efficiency of Enteropeptidase

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