Alpha1-antitrypsin inhibits the activity of the matriptase catalytic domain in vitro - PubMed (original) (raw)

Alpha1-antitrypsin inhibits the activity of the matriptase catalytic domain in vitro

Sabina Janciauskiene et al. Am J Respir Cell Mol Biol. 2008 Dec.

Abstract

Matriptase is a type II transmembrane protease that is characterized by an N-terminal transmembrane and multiple extracellular domains, in addition to the conserved extracellular serine protease catalytic domain. The expression pattern of matriptase suggests that this protease may play broad roles in the biology of surface lining epithelial cells. In this study we report that alpha1-antitrypsin (AAT), an endogenous inhibitor of serine proteases, inhibits the catalytic domain of human recombinant matriptase in vitro. Co-incubation of AAT with matriptase (at a molar ratio 1:2) resulted in the formation of heat stable complexes, clearly seen in sodium dodecyl sulfate electrophoresis and Western blots. AAT was found to be a slow, tight-binding inhibitor of the catalytic domain of matriptase with a second order reaction rate constant of 0.31 x 10(3) M(-1)s(-1). Notably, the oxidized form of AAT, which lacks serine protease inhibitor activity, failed to generate matriptase complexes and to inhibit matriptase activity. Since matriptase is involved in a number of physiologic processes, including activation of epithelial sodium channels, our findings offer considerable new insights into new regulatory function of AAT in vivo.

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Figures

**Figure 1.

ClustalW alignment of the sequences of human neutrophil elastase (1B0F_A), porcine pancreatic elastase (1B0E_A), and human matriptase (1EAX_A). Sequence identities are marked with an asterisk underneath and shown in red. The catalytic triad consisting of amino acids His 57, Asp 102, and Ser 195 is shown in yellow. Residues with “:” or “.” underneath denote conserved amino acids.

**Figure 2.

Superimposed crystal structures of human neutrophil elastase (violet) and human matriptase (blue) with the catalytic triad amino acids (His 57, Asp 102, and Ser 195) highlighted in red (for matriptase) and green (for elastase).

**Figure 3.

(A) 7.5% SDS-PAGE and (B) Western blot of native AAT and matriptase catalytic domain mixtures. (A) Native AAT/matriptase mixtures were prepared by incubating native AAT (1 mg/ml, 19.2 μM) with matriptase (0.5 mg/ml, 19.2 μM) at 1:2 molar ratio in PBS, pH 7.4, at room temperature for variable periods of time. AAT alone was incubated under the same experimental conditions. The reaction was stopped by the addition of an equal volume of SDS-PAGE sample buffer and boiling. Representative gel which was repeated seven times with identical results. (B) Native AAT/matriptase complex was prepared by incubating AAT with matriptase at 1:1 molar ratio in PBS, pH 7.4, at room temperature for 18 hours. Native AAT alone or in mixture with matriptase were separated on 7.5% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane using a semi-dry blot electrophoretic transfer system, and were immunostained with an anti-human AAT monoclonal antibody (B9) IgG1 (1:2,000 dilution). Three forms of AAT were detected: AAT in complex with matriptase, unreacted, and cleaved AAT. Representative Western blot, which was repeated three times, is shown.

**Figure 4.

SDS-PAGE (7.5%) of oxidized and native AAT incubated with human elastase. Lane 1, molecular size markers; lane 2, native (n) AAT; lane 3, native AAT + elastase incubated for 30 minutes at a molar ratio 1.2:1; lane 4, oxidized (ox) AAT; lane 5, oxidized AAT + elastase. Figure is characteristic of an experiment that was repeated twice, with identical results.

**Figure 5.

SDS-PAGE (7.5%) of oxidized AAT and matriptase catalytic domain mixtures. Oxidized AAT/matriptase complex was prepared by incubating AAT with matriptase at 1:1 molar ratio in PBS, pH 7.4, for (A) 30 minutes and (B) 18 hours at room temperature. Figure is characteristic of an experiment that was repeated twice, with similar results.

**Figure 6.

Inhibition of matriptase activity by AAT. Effects of native (n) ad oxidized (ox) AAT, as well as bovine serum albumin (0.19 μM each), on the enzymatic activity of matriptase catalytic domain (2.5 nM). Results are shown as percent inhibition of maximum activity. Values are mean ± SD (n = 3); * P < 0.001 compared with nAAT (by t test).

**Figure 7.

Stoichiometry of inhibition. Matriptase (19.2 μM) was pre-incubated with various concentrations of AAT (from 1:0.25 to 1:10, [Matriptase]:[AAT] molar ratio) for 18 hours before assay of matriptase inhibition. Data were fitted with linear regression (r2 = 0.882; P < 0.01; X intercept = 1). Figure is characteristic of two experiments with similar results.

**Figure 8.

Determination of reaction rate constant (k). (A) Kinetic curves of matriptase activity (cleavage of substrate) measured after pre-incubating matriptase catalytic domain (5 nM) with vehicle or with native AAT (960 nM) for 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 18 hours. Three independent experiments were performed, with five repeats each. Figure shows one representative experiment of five repeats. (B) As described in M

aterials and

ethods

, the matriptase activity was calculated from A, and the exponential free enzyme activity was plotted versus time of incubation with AAT. A good linear fit was obtained (R = 0.99812). The k was calculated as 0.31 × 103 M−1s−1 from the slope of the plot (_k_obs = k × [AAT]0 = 0.01794 min−1). This experiment was repeated twice, with identical results.

References

1. Shi YE, Torri J, Yieh L, Wellstein A, Lippman ME, Dickson RB. Identification and characterization of a novel matrix-degrading protease from hormone-dependent human breast cancer cells. Cancer Res 1993;53:1409–1415. - PubMed
1. Lin CY, Anders J, Johnson M, Sang QA, Dickson RB. Molecular cloning of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity. J Biol Chem 1999;274:18231–18236. - PubMed
1. Kim MG, Chen C, Lyu MS, Cho EG, Park D, Kozak C, Schwartz RH. Cloning and chromosomal mapping of a gene isolated from thymic stromal cells encoding a new mouse type II membrane serine protease, epithin, containing four LDL receptor modules and two CUB domains. Immunogenetics 1999;49:420–428. - PubMed
1. Satomi S, Yamasaki Y, Tsuzuki S, Hitomi Y, Iwanaga T, Fushiki T. A role for membrane-type serine protease (MT-SP1) in intestinal epithelial turnover. Biochem Biophys Res Commun 2001;287:995–1002. - PubMed
1. Yamada K, Takabatake T, Takeshima K. Isolation and characterization of three novel serine protease genes from Xenopus laevis. Gene 2000;252:209–216. - PubMed

Alpha1-antitrypsin inhibits the activity of the matriptase catalytic domain in vitro - PubMed (original) (raw)