Secreted expression of pseudozymogen forms of recombinant matriptase in Pichia pastoris

Matriptase is a transmembrane serine protease expressed in vertebrates. This enzyme is synthesized as a zymogen form and is converted to an active form by cleavage at the N-terminus of the serine protease catalytic domain. In a mammalian cell-based expression system, we have produced pseudozymogen forms of recombinant matriptase (r-matriptase) that are activated by cleavage with a recombinant enterokinase (r-EK) in vitro. In the present study, four different pseudozymogen forms of r-matriptase containing a site for activation by r-EK and a hexahistidine tag (His₆-tag) were expressed in and secreted by Pichia pastoris, a methylotrophic yeast. The pseudozymogens with His₆-tag at their C-termini formed multimers linked by intermolecular disulfide bonds. After treatment with r-EK, they exhibited no detectable hydrolytic activity toward a chromogenic substrate. A pseudozymogen form of matriptase catalytic domain with N-terminal His₆-tag (designated His₆t-S-CD) was secreted as a monomer. His₆t-S-CD after r-EK treatment exhibited activity comparable to that of the activated form of an r-matriptase expressed in mammalian cells. His₆t-S-CD could be purified from culture medium in milligram quantities. The expression in the yeast offers an efficient method of producing larger amounts of r-matriptase.     

Expression in Escherichia coli, refolding, and purification of the recombinant mature form of human matrix metalloproteinase 7 (MMP-7)

In the latent pro-form of matrix metalloproteinase 7 (MMP-7), the cysteine residue in the pro-peptide binds the active-site zinc ion. Hence, recombinant active MMP-7 was prepared from pro-MMP-7 by modification of this cysteine residue with a mercuric reagent. In this study, mature MMP-7 was expressed in Escherichia coli as inclusion bodies, solubilized, and refolded with 1 M L-arginine. The purified product was indistinguishable from the one prepared from pro-MMP-7 as assessed by hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2).     

Identification of the Matriptase Second CUB Domain as the Secondary Site for Interaction with Hepatocyte Growth Factor Activator Inhibitor Type-1

Matriptase is a type II transmembrane serine protease comprising 855 amino acid residues. The extracellular region of matriptase comprises a noncatalytic stem domain (containing two tandem repeats of complement proteases C1r/C1s-urchin embryonic growth factor-bone morphogenetic protein (CUB) domain) and a catalytic serine protease domain. The stem domain of matriptase contains site(s) for facilitating the interaction of this protease with the endogenous inhibitor, hepatocyte growth factor activator inhibitor type-1 (HAI-1). The present study aimed to identify these site(s). Analyses using a secreted variant of recombinant matriptase comprising the entire extracellular domain (MAT), its truncated variants, and a recombinant HAI-1 variant with an entire extracellular domain (HAI-1–58K) revealed that the second CUB domain (CUB domain II, Cys³⁴⁰–Pro⁴⁵²) likely contains the site(s) of interest. We also found that MAT undergoes cleavage between Lys³⁷⁹ and Val³⁸⁰ within CUB domain II and that the C-terminal residues after Val³⁸⁰ are responsible for facilitating the interaction with HAI-1–58K. A synthetic peptide corresponding to Val³⁸⁰–Asp³⁹⁰ markedly increased the matriptase-inhibiting activity of HAI-1–58K, whereas the peptides corresponding to Val³⁸⁰–Val³⁸⁹ and Phe³⁸²–Asp³⁹⁰ had no effect. HAI-1–58K precipitated with immobilized streptavidin resins to which a synthetic peptide Val³⁸⁰–Pro³⁹² with a biotinylated lysine residue at its C terminus was bound, suggesting direct interaction between CUB domain II and HAI-1. These results led to the identification of the matriptase CUB domain II, which facilitates the primary inhibitory interaction between this protease and HAI-1.     

Conversion of vitamin D3 to 1alpha,25-dihydroxyvitamin D3 by Streptomyces griseolus cytochrome P450SU-1

Streptomyces griseolus cytochrome P450SU-1 (CYP105A1) was expressed in Escherichia coli at a level of 1.0 micromol/L culture and purified with a specific content of 18.0 nmol/mg protein. Enzymatic studies revealed that CYP105A1 had 25-hydroxylation activity towards vitamin D2 and vitamin D3. Surprisingly, CYP105A1 also showed 1alpha-hydroxylation activity towards 25(OH)D3. As mammalian mitochondrial CYP27A1 catalyzes a similar two-step hydroxylation towards vitamin D3, the enzymatic properties of CYP105A1 were compared with those of human CYP27A1. The major metabolite of vitamin D2 by CYP105A1 was 25(OH)D2, while the major metabolites by CYP27A1 were both 24(OH)D2 and 27(OH)D2. These results suggest that CYP105A1 recognizes both vitamin D2 and vitamin D3 in a similar manner, while CYP27A1 does not. The Km values of CYP105A1 for vitamin D2 25-hydroxylation, vitamin D3 25-hydroxylation, and 25-hydroxyvitamin D3 1alpha-hydroxylation were 0.59, 0.54, and 0.91 microM, respectively, suggesting a high affinity of CYP105A1 for these substrates.     

Effect of nitration on the activity of bovine erythrocyte Cu,Zn-superoxide dismutase (BESOD) and a kinetic analysis of its dimerization-dissociation reaction as examined by subunit exchange between the native and nitrated BESODs

Bovine erythrocyte Cu,Zn-superoxide dismutase (BESOD) is a dimeric enzyme composed of identical subunits associated through unusually strong non-covalent interactions. The state of the unique tyrosyl residue (Tyr 108) of BESOD was examined, and the kinetics of subunit exchange was studied using Tyr 108 as a probe. UV-absorption difference spectra demonstrate that Tyr 108 is exposed to the solvent, and that the accessibilities to ethanol, ethylene glycol, and polyethylene glycol 600 are 53.5, 52.0, and 44.6%, respectively. Tyr 108 was fully nitrated by tetranitromethane. The pK(a) values of the phenolic hydroxyl group of native and nitrated Tyr 108 were determined to be 11.3 and 7.9, whereas those of model compounds, L-tyrosine and 3-nitro-L-tyrosine, are 9.8 and 6.8, respectively. When the native and nitrated BESODs of an equal concentration were mixed, the hybrid dimer was formed. From the amount of hybrid dimer formed, the rate constant (k(-1)) of the subunit dissociation at pH 7.8, 25 degrees C was assessed to be (4.17 +/- 0.13) x 10(-6) s(-1). The activation energy of the subunit dissociation at pH 7.8 was determined to be 117 +/- 4 kJ.mol(-1). The k(-1) value remains constant at BESOD concentrations ranging from 0.62 to 6.8 micro M and at pH ranging from 6.0 to 10.0, but increased remarkably with a decrease in the dielectric constant of the reaction mixture. It is suggested that hydrophobic interaction may play a significant role in the subunit interaction.     

Differential scanning calorimetry of the effects of Ca2+ on the thermal unfolding of Pseudomonas cepacia lipase

Thermal unfolding of P. cepacia lipase was observed by adiabatic differential scanning microcalorimetry in the absence and presence of calcium ions at pH 8, and thermodynamic parameters of unfolding were evaluated to analyze the unfolding mechanism of the enzyme. The temperature of unfolding was higher at higher concentrations of Ca2+. From the Ca2+ concentration-dependence of the unfolding temperature, the number of calcium ions that dissociated from the enzyme molecule upon unfolding was estimated to be one. These results confirmed the validity of the unfolding mechanism proposed previously: NCa2+ < = => D + Ca2+, where N and D represent the native and denatured states, respectively, of the enzyme.     

Inhibitory effects of alcohols on the activity of human matrix metalloproteinase 7 (matrilysin)

Aliphatic alcohols inhibited the activity of human matrix metalloproteinase 7 (matrilysin) competitively with K(i) of 6.1-19.4% (v/v) or 0.66-4.80 M. From the relationship between the structures of alcohols and their K(i) values, alcohols are considered to bind the hydrophobic S1' subsite most plausibly, and the size of the pocket was estimated to be large enough to accommodate the length of 1-butanol (4-carbon chain) and the bulk of tertiary alcohols. Alcohols might be suitable probes for exploring the active-site geometry of enzymes.     

Metabolic pathways of dioxin by CYP1A1: species difference between rat and human CYP1A subfamily in the metabolism of dioxins

Metabolism of polychlorinated dibenzo-p-dioxins by CYP1A subfamily was examined by using the recombinant yeast microsomes. In substrate specificity and reaction specificity, considerable species differences between rats and humans were observed in both CYP1A1- and CYP1A2-dependent metabolism of dioxins. Among four CYPs, rat CYP1A1 showed the highest activity toward dibenzo-p-dioxin (DD) and mono-, di-, and trichloroDDs. To reveal the mechanism of dioxin metabolism, we examined rat CYP1A1-dependent metabolism of 2-chloro-dibenzo-p-dioxin. In addition to hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, hydroxylation with elimination of a chloride substituent, and cleavage of an ether linkage of the dioxin ring were observed. In particular, the cleavage of an ether linkage of the dioxin ring appeared most important for the detoxication of dioxins. Based on these results, the metabolic pathways of 2-chloro-dibenzo-p-dioxin by rat CYP1A1 were proposed. The metabolic pathways contain most of the metabolites observed in vivo using experimental animals, suggesting that P450 monooxygenase systems including CYP1A1 are greatly responsible for dioxin metabolism in vivo.     

Generation of 2,3,7,8-TCDD-metabolizing enzyme by modifying rat CYP1A1 through site-directed mutagenesis

Polychlorinated dibenzo-p-dioxins (PCDDs) are known as g environmental contaminants on account of the extreme toxicity. Among these compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TetraCDD) is regarded as the most toxic one. The extremely high toxicity of 2,3,7,8-TetraCDD is based on its high affinity for Ah receptor and nearly undetectable metabolism in mammalian body. Based on our previous studies, we assumed that enlarging the space of substrate-binding pocket of rat CYP1A1 might generate the catalytic activity toward 2,3,7,8-TetraCDD. Large-sized amino acid residues located at putative substrate-binding sites of rat CYP1A1 were substituted for alanine by site-directed mutagenesis. Among eight mutants examined, the mutant in the putative F-G loop, F240A, showed metabolic activity toward 2,3,7,8-TetraCDD. HPLC and GC-MS analyses strongly suggested that the metabolite was 8-hydroxy-2,3,7-TriCDD. Ah receptor assay revealed that the affinity of 8-hydroxy-2,3,7-TriCDD for Ah receptor was less than 0.01% of 2,3,7,8-TetraCDD, indicating that the F240A-dependent metabolism resulted in remarkable detoxification of 2,3,7,8-TetraCDD. The novel 2,3,7,8-TetraCDD-metabolizing enzyme could be applicable to bioremediation of contaminated soils with dioxin, elimination of dioxin from foods, and clinical treatment for people who accidentally take dioxin into their systems.     

Transition state stabilization by the N-terminal anticodon-binding domain of lysyl-tRNA synthetase

Lysyl-tRNA synthetase from Bacillus stearothermophilus (B.s. LysRS) (EC ) catalyzes aminoacylation of tRNA(Lys) with l-lysine, in which l-lysine was first activated with ATP to yield an enzyme (lysyladenylate complex), and then the lysine molecule was transferred from the complex to tRNA(Lys). B.s. LysRS is a homodimeric enzyme with a subunit that consists of two domains, an N-terminal tRNA anticodon-binding domain (TAB-ND: Ser(1)-Pro(144)) and a C-terminal Class II-specific catalytic domain (CAT-CD: Lys(151)-Lys(493)). CAT-CD alone retained catalytic activity, although at a low level; TAB-ND alone showed no activity. Size exclusion chromatography revealed that CAT-CD exists as a dimer, whereas TAB-ND was a monomer. The formation of a complex consisting of these domains was detected with the guidance of surface plasmon resonance. In accordance with this, the addition of TAB-ND to CAT-CD significantly enhanced both the l-lysine activation and the tRNA aminoacylation reactions. Kinetic analysis showed that deletion of TAB-ND resulted in a significant destabilization of the transition state of CAT-CD in the l-lysine activation reaction but had little effect on the ground state of substrate binding. A significant role of a cross-subunit interaction in the enzyme between TAB-ND and CAT-CD was proposed for the stabilization of the transition state in the l-lysine activation reaction.