Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A dysfunctional C1 inhibitor (C1 INH) from a family in whom the propositus presented with systemic lupus erythematosus but without angioedema previously was shown to have diminished inhibitory activity toward isolated C1r and C1s, and intact C1. The mutation was identified as replacement of Ala443 (P2) with Val. This study further analyzed the reactivity of this mutant and characterized two mutants with Ser or Asp at this position. Ser at P2 does not interfere with binding of target proteases. However, the mutant with Asp at this position is unable to bind C1r and beta factor XIIa, and also has a decreased rate of reaction with C1s and kallikrein. Therefore, alteration of polarity alone had no effect on binding, while a bulky and/or charged side chain was not tolerated. Although defective in inhibition of C1r and C1s, the P2 A-->V mutant had acquired the ability to complex with trypsin. It also completely retained the ability to complex with kallikrein and factor XIIa. None of the 10 individuals expressing this mutant protein has ever had angioedema. This observation, combined with normal inhibition of contact system proteases and defective inhibition of complement proteases, suggests that angioedema is caused by bradykinin generated from contact system activation.
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PMID:Role of the P2 residue of complement 1 inhibitor (Ala443) in determination of target protease specificity: inhibition of complement and contact system proteases. 921 20

C1-inhibitor is a member of the serpin family of proteinase inhibitors and is an important inhibitor of complement and contact system proteinases. The native protein has the characteristic serpin feature of being in a kinetically trapped metastable state rather than in the most stable state it could adopt. A consequence of this is that it readily forms loop-sheet dimers and polymers, by a mechanism believed to be the same as observed with other serpins. An unusual feature of C1-inhibitor is that it has a unique amino-terminal domain, of unknown function, held to the serpin domain by two disulfide bonds not found in other serpins. We report here that reduction of these bonds by DTT, causes a conformational change such that the reactive center loop inserts into beta-sheet A. This form of C1-inhibitor is less stable to heat and urea than the native protein, and is more susceptible to extensive degradation by trypsin. These data show that the disulfide bonds in C1-inhibitor are required for the protein to be stabilized in the metastable state with the reactive center loop expelled from beta-sheet A.
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PMID:The native metastable fold of C1-inhibitor is stabilized by disulfide bonds. 1100 79

Previous analysis of a naturally occurring C1 inhibitor P2 mutant (Ala(443)-->Val) indicated a role for P2 in specificity determination. To define this role and that of other reactive center loop residues, a number of different amino acids were introduced at P2, as well as at P6 (Ala(439)) and P8'/9' (Gln(452)Gln(453)). Ala(439)-->Val is a naturally occurring mutant observed in a patient with hereditary angioedema. Previous data suggested that Gln(452)Gln(453) might be a contact site for C1s. Reactivity of the inhibitors toward target (C1s, C1r, kallikrein, beta factor XIIa, and plasmin) and nontarget proteases (alpha-thrombin and trypsin) were studied. Substitution of P2 with bulky or charged residues resulted in decreased reactivity with all target proteases. Substitution with residues with hydrophobic or polar side chains resulted in decreased reactivity with some proteases, but in unaltered or increased reactivity with others. Second order rate constants for the reaction with C1s were determined for the mutants with activities most similar to the wild-type protein. The three P2 mutants showed reductions in rate from 3.35 x 10(5) M(-1)s(-1) for the wild type to 1.61, 1.29, and 0.63 x 10(5) for the Ser, Thr, and Val mutants, respectively. In contrast, the Ala(439)-->Val and the Gln(452)Gln(453)-->Ala mutants showed little difference in association rates with C1s, in comparison with the wild-type inhibitor. The data confirm the importance of P2 in specificity determination. However, the P6 position appears to be of little, if any, importance. Furthermore, it appears unlikely that Gln(452)Gln(453) comprise a portion of a protease contact site within the inhibitor.
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PMID:C1 inhibitor: analysis of the role of amino acid residues within the reactive center loop in target protease recognition. 1146 70

Mannan-binding lectin-associated serine protease (SP) (MASP)-1 and MASP-2 are modular SP and form complexes with mannan-binding lectin, the recognition molecule of the lectin pathway of the complement system. To characterize the enzymatic properties of these proteases we expressed their catalytic region, the C-terminal three domains, in Escherichia coli. Both enzymes autoactivated and cleaved synthetic oligopeptide substrates. In a competing oligopeptide substrate library assay, MASP-1 showed extreme Arg selectivity, whereas MASP-2 exhibited a less restricted, trypsin-like specificity. The enzymatic assays with complement components showed that cleavage of intact C3 by MASP-1 and MASP-2 was detectable, but was only approximately 0.1% of the previously reported efficiency of C3bBb, the alternative pathway C3-convertase. Both enzymes cleaved C3i 10- to 20-fold faster, but still at only approximately 1% of the efficiency of MASP-2 cleavage of C2. We believe that C3 is not the natural substrate of either enzyme. MASP-2 cleaved C2 and C4 at high rates. To determine the role of the individual domains in the catalytic region of MASP-2, the second complement control protein module together with the SP module and the SP module were also expressed and characterized. We demonstrated that the SP domain alone can autoactivate and cleave C2 as efficiently as the entire catalytic region, while the second complement control protein module is necessary for efficient C4 cleavage. This behavior strongly resembles C1s. Each MASP-1 and MASP-2 fragment reacted with C1-inhibitor, which completely blocked the enzymatic action of the enzymes. Nevertheless, relative rates of reaction with alpha-2-macroglobulin and C1-inhibitor suggest that alpha-2-macroglobulin may be a significant physiological inhibitor of MASP-1.
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PMID:Natural substrates and inhibitors of mannan-binding lectin-associated serine protease-1 and -2: a study on recombinant catalytic fragments. 1253 97

C1 inhibitor (C1INH) prevents endotoxin shock in mice via a direct interaction with lipopolysaccharide (LPS). This interaction requires the heavily glycosylated amino-terminal domain of C1INH. C1INH in which N-linked carbohydrate was removed by using N-glycosidase F was markedly less effective in protecting mice from LPS-induced lethal septic shock. N-deglycosylated C1INH also failed to suppress fluorescein isothiocyanate (FITC)-LPS binding to and LPS-induced tumor necrosis factor alpha mRNA expression by the murine macrophage-like cell line, RAW 264.7, and cells in human whole blood. In an enzyme linked immunosorbent assay, the N-deglycosylated C1INH bound to LPS very poorly. In addition, C1INH was shown to bind to diphosphoryl lipid A (dLPA) but only weakly to monophosphoryl lipid A (mLPA). As with intact LPS, binding of N-deglycosylated C1INH to dLPA and mLPA was diminished in comparison with the native protein. Removal of O-linked carbohydrate had no effect on any of these activities. Neither detoxified LPS, dLPA, nor mLPA had any effect on the rate or extent of C1INH complex formation with C1s or on cleavage of the reactive center loop by trypsin. These data demonstrate that N-linked glycosylation of C1INH is essential to mediate its interaction with the LPA moiety of LPS and to protect mice from endotoxin shock.
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PMID:N-linked glycosylation is required for c1 inhibitor-mediated protection from endotoxin shock in mice. 1503 14

The StcE zinc metalloprotease is secreted by enterohemorrhagic Escherichia coli (EHEC) O157:H7 and contributes to intimate adherence of this bacterium to host cells, a process essential for mammalian colonization. StcE has also been shown to localize the inflammatory regulator C1 esterase inhibitor (C1-INH) to cell membranes. We tried to more fully characterize StcE activity to better understand its role in EHEC pathogenesis. StcE was active at pH 6.1 to 9.0, in the presence of NaCl concentrations ranging from 0 to 600 mM, and at 4 degrees C to 55 degrees C. Interestingly, antisera against StcE or C1-INH did not eliminate StcE cleavage of C1-INH. Treatment of StcE with the proteases trypsin, chymotrypsin, human neutrophil elastase, and Pseudomonas aeruginosa elastase did not eliminate StcE activity against C1-INH. After StcE was kept at 23 degrees C for 65 days, it exhibited full proteolytic activity, and it retained 30% of its original activity after incubation for 8 days at 37 degrees C. Together, these results show the StcE protease is a stable enzyme that is probably active in the environment of the colon. Additionally, k(cat)/K(m) data showed that StcE proteolytic activity was 2.5-fold more efficient with the secreted mucin MUC7 than with the complement regulator C1-INH. This evidence supports a model which includes two roles for StcE during infection, in which StcE acts first as a mucinase and then as an anti-inflammatory agent by localizing C1-INH to cell membranes.
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PMID:Characterization of the StcE protease activity of Escherichia coli O157:H7. 1678 73

Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 is an abundant component of the lectin pathway of complement. The related enzyme, MASP-2 is capable of activating the complement cascade alone. Though the concentration of MASP-1 far exceeds that of MASP-2, only a supporting role of MASP-1 has been identified regarding lectin pathway activation. Several non-complement substrates, like fibrinogen and factor XIII, have also been reported. MASP-1 belongs to the C1r/C1s/MASP family of modular serine proteases; however, its serine protease domain is evolutionary different. We have determined the crystal structure of the catalytic region of active MASP-1 and refined it to 2.55 A resolution. Unusual features of the structure are an internal salt bridge (similar to one in factor D) between the S1 Asp189 and Arg224, and a very long 60-loop. The functional and evolutionary differences between MASP-1 and the other members of the C1r/C1s/MASP family are reflected in the crystal structure. Structural comparison of the protease domains revealed that the substrate binding groove of MASP-1 is wide and resembles that of trypsin rather than early complement proteases explaining its relaxed specificity. Also, MASP-1's multifunctional behavior as both a complement and a coagulation enzyme is in accordance with our observation that antithrombin in the presence of heparin is a more potent inhibitor of MASP-1 than C1 inhibitor. Overall, MASP-1 behaves as a promiscuous protease. The structure shows that its substrate binding groove is accessible; however, its reactivity could be modulated by an unusually large 60-loop and an internal salt bridge involving the S1 Asp.
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PMID:MASP-1, a promiscuous complement protease: structure of its catalytic region reveals the basis of its broad specificity. 1956 40


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