Biochemical characterization of prostasin, a channel activating protease

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Abstract

Human prostasin was recently identified as a potential regulator of epithelial sodium channel (ENaC) function. Through the use of positional scanning combinatorial substrate libraries, prostasin was shown to have a preference for poly-basic substrates: in position P4 preference was for arginine or lysine; in P3 preference was for histidine, lysine or arginine; in P2 preference was for basic or large hydrophobic amino acids; and in P1 preference was for arginine and lysine. P1′, P2′, and P3′ displayed broad selectivity with the exception of a lack of activity for isoleucine, and P4′ had a preference for small, unbranched, amino acids such as alanine and serine. A prostasin-preferred poly-basic cleavage site was found in the extracellular domains of the ENaC α- and β-subunits, and may present a mechanism for prostasin activation. The absence of activity seen with substrates containing isoleucine in position P1′ explains the inability of prostasin to autoactivate and suggests that prostasin proteolytic activity is regulated by an upstream protease. Prostasin activity was highly influenced by mono- and divalent metal ions which were potent inhibitors and substrate specific modulators of enzymatic activity. In the presence of sub-inhibitory concentrations of zinc, the activity of prostasin increased several-fold and its substrate specificity was significantly altered in favor of a strong preference for histidine in positions P3 or P4 of the substrate.

Section snippets

Reagents

All reagents were from Sigma–Aldrich unless otherwise stated. α1-antitrypsin, α1-antichymotrypsin, α1-antitrypsinPDX, and soybean trypsin inhibitor (SBTI) were from Calbiochem. Recombinant placental bikunin (HAI-2A) was obtained from R&D Systems.

Cloning

The cDNA sequence of human prostasin (Accession No. NM_002773) was isolated from a human testis cDNA library (Clontech) by PCR with sense primer, 5′-ATGGCCCAGAAGGGGGTC-3′, and antisense primer, 5′-GTGCTCGCTGAGCCATGG-3′. The gene was modified by PCR to

Protein expression

The mature protease domain of prostasin was secreted into the insect culture medium at a level of 0.8–1.2 mg/L over 72 h. The expression of this active form of prostasin was low in comparison to what was achieved with the prostasin pro-enzyme under similar conditions; however, the pro-form was not active without digestion by trypsin in vitro (data not shown). Auto-activation of the pro-enzyme was not observed with prolonged incubation at 4 or 37 °C. The purified active protein migrated as a single

Discussion

The mature epithelial sodium channel (ENaC) is a heterotetramer composed of two α-subunits, one β-subunit, and one γ-subunit [24], [25], [26] although an alternative stoichiometry has been proposed [27]. The α-, β-, and γ-subunits are homologous, and each consists of a large extracellular domain flanked on each side by a transmembrane domain and a short intracellular amino- or carboxy-terminal domain [28]. ENaC is responsible for transepithelial Na+ transport and thereby fluid homeostasis in a

Acknowledgments

We thank Dr. Ian Hunt for advice and Mr. Reynand Pacoma for technical assistance.

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    Abbreviations: acc, 7-amino-4-carbamoylmethyl coumarin; acmc, 7-amino-3-carbamoylmethyl-4-methyl coumarin; amc, 7-amino-4-methyl coumarin; CAP, channel activating protease; DTT, dithiothreitol; ENaC, epithelial sodium channel; hCAP, human channel activating protease; Nle, norleucine; NBS, non-binding surface; PNGaseF, peptide N-glycosidase F; SBTI, soybean trypsin inhibitor.

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