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• W2023165370 abstract "Blastula protease 10 (BP10) is a metalloenzyme involved in sea urchin embryogenesis, which has been assigned to the astacin family of zinc-dependent endopeptidases. It shows greatest homology with the mammalian tolloid-like genes and contains conserved structural motifs consistent with astacin, tolloid, and bone morphogenetic protein 1. Astacin, a crustacean digestive enzyme, has been proposed to carry out hydrolysis via a metal-centered mechanism that involves a metal-coordinated “tyrosine switch.” It has not been determined if the more structurally complex members of this family involved in eukaryotic development share this mechanism. The recombinant BP10 has been overexpressed in Escherichia coli, its metalloenzyme nature has been confirmed, and its catalytic properties have been characterized through kinetic studies. BP10 shows significant hydrolysis toward gelatin both in its native zinc-containing form and copper derivative. The copper derivative of BP10 shows a remarkable 960% rate acceleration toward the hydrolysis of the synthetic substrate N-benzoyl-arginine-p-nitroanilide when compared with the zinc form. The enzyme also shows calcium-dependent activation. These are the first thorough mechanistic studies reported on BP10 as a representative of the more structurally complex members of astacin-type enzymes in deuterostomes, which can add supporting data to corroborate the metal-centered mechanism proposed for astacin and the role of the coordinated Tyr. We have demonstrated the first mechanistic study of a tolloid-related metalloenzyme involved in sea urchin embryogenesis. Blastula protease 10 (BP10) is a metalloenzyme involved in sea urchin embryogenesis, which has been assigned to the astacin family of zinc-dependent endopeptidases. It shows greatest homology with the mammalian tolloid-like genes and contains conserved structural motifs consistent with astacin, tolloid, and bone morphogenetic protein 1. Astacin, a crustacean digestive enzyme, has been proposed to carry out hydrolysis via a metal-centered mechanism that involves a metal-coordinated “tyrosine switch.” It has not been determined if the more structurally complex members of this family involved in eukaryotic development share this mechanism. The recombinant BP10 has been overexpressed in Escherichia coli, its metalloenzyme nature has been confirmed, and its catalytic properties have been characterized through kinetic studies. BP10 shows significant hydrolysis toward gelatin both in its native zinc-containing form and copper derivative. The copper derivative of BP10 shows a remarkable 960% rate acceleration toward the hydrolysis of the synthetic substrate N-benzoyl-arginine-p-nitroanilide when compared with the zinc form. The enzyme also shows calcium-dependent activation. These are the first thorough mechanistic studies reported on BP10 as a representative of the more structurally complex members of astacin-type enzymes in deuterostomes, which can add supporting data to corroborate the metal-centered mechanism proposed for astacin and the role of the coordinated Tyr. We have demonstrated the first mechanistic study of a tolloid-related metalloenzyme involved in sea urchin embryogenesis. The astacin family of zinc-dependent endopeptidases is ubiquitously distributed across all phyla and part of the superfamily of metzincins (1Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (351) Google Scholar). Approximately 30 members of the astacin family have been characterized at the protein level (2Hooper N.M. Zinc Metalloproteases in Health and Disease. Taylor and Francis, Bristol, PA1996: 23-46Google Scholar), such as meprins, bone morphogenetic protein-1 (BMP-1), 3The abbreviations used are: BMP, bone morphogenetic protein; Arg-NHOH, Arg-hydroxamate; BP10, blastula protease 10; LMCT, ligand-to-metal charge transfer transition(s); BAPNA, N-benzoyl-arginine-p-nitroanilide; TEMED, N,N,N′,N′-tetramethylethylenediamine; EGF, epidermal growth factor; CAPS, 3-(cyclohexylamino)propanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid; NTA, nitrilotriacetic acid; PBS, phosphate-buffered saline. 3The abbreviations used are: BMP, bone morphogenetic protein; Arg-NHOH, Arg-hydroxamate; BP10, blastula protease 10; LMCT, ligand-to-metal charge transfer transition(s); BAPNA, N-benzoyl-arginine-p-nitroanilide; TEMED, N,N,N′,N′-tetramethylethylenediamine; EGF, epidermal growth factor; CAPS, 3-(cyclohexylamino)propanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid; NTA, nitrilotriacetic acid; PBS, phosphate-buffered saline. and tolloid, whereas several others have been identified through gene sequencing, including those in Caenorhabditis elegans (3Moehrlen F. Hutter H. Zwilling R. Eur. J. Biochem. 2003; 270: 4909-4920Crossref PubMed Scopus (63) Google Scholar). The signature of the sequence of the active site motif for this family of enzymes is HEXXHXXGXXH, where one Zn2+ ion coordinates with the three histidines (boldface type), a tyrosine (Met-His/Ser-Tyr in a loop region downstream from the coordinated His residues), and a water molecule (4Gomis-Rüth F.X. Stöcker W. Huber H. Zwilling R. Bode W. J. Mol. Biol. 1993; 229: 945-968Crossref PubMed Scopus (129) Google Scholar). Most members of this family share common domain structures such as the pre- and proenzyme sequences located immediately N-terminal to the protease domain. Several members contain one or two copies of epidermal growth factor (EGF)-like domains and complement-like domains (Clr, Cls) near the C terminus (2Hooper N.M. Zinc Metalloproteases in Health and Disease. Taylor and Francis, Bristol, PA1996: 23-46Google Scholar). The shuffling of different domains in relation to the catalytic protease domain creates a variety of proteins with different structures and functions.Originally isolated and characterized as a developmentally regulated gene in sea urchin embryos (5Lepage T. Ghiglione C. Gache C. Development. 1992; 114: 147-163Crossref PubMed Google Scholar, 6Lepage T. Ghiglione C. Gache C. Eur. J. Biochem. 1996; 238: 744-751Crossref PubMed Scopus (17) Google Scholar), the BP10 protein has remained uncharacterized. It shares sequence similarity with other members of the astacin family of enzymes (astacin itself being a crayfish digestive enzyme (4Gomis-Rüth F.X. Stöcker W. Huber H. Zwilling R. Bode W. J. Mol. Biol. 1993; 229: 945-968Crossref PubMed Scopus (129) Google Scholar, 7Bode W. Gomis-Rüth F.X. Huber R. Zwilling R. Stöcker W. Nature. 1992; 358: 164-167Crossref PubMed Scopus (293) Google Scholar) and hence a novel prototype in catalytic mechanism) important in development, such as tolloid-like and BMP-1 in vertebrates and tolloid in Drosophila. The BP10 protease is constructed of similar structural domains as BMP-1 and tolloid (i.e. astacin domain, CUB, and EGF) but has different arrangements and numbers of these domains. BP10 is most similar in sequence to the mammalian tolloid-like 2 proteins (8Takahara K. Brevard R. Hoffman G.F. Suzuki N. Greenspan D.S. Genomics. 1996; 34: 157-165Crossref PubMed Scopus (84) Google Scholar). The recent sea urchin genome sequencing project has revealed a cluster of genes homologous to BP10 (available on the World Wide Web at www.ncbi.nlm.nih.gov/Genomes/) as well as several related genes with similar structural domains. One of these, SpAN, has been implicated in regulating BMP-4 signaling (9Wardle F.C. Angerer L.M. Angerer R.C. Dev. Biol. 1999; 206: 63-72Crossref PubMed Scopus (28) Google Scholar). The transcription of the BP10 gene is transiently activated around the 16–32-cell stage, and the accumulation of BP10 mRNA peaks at the blastula stage. Temporarily, the highest BP10 activity is detected ∼1.5 h after expression of the sea urchin hatching enzyme (envelysin) reaches a maximum (5Lepage T. Ghiglione C. Gache C. Development. 1992; 114: 147-163Crossref PubMed Google Scholar). The BP10 transcripts are detected in a limited area of the blastula. The protein is first detected in late cleavage, and its level peaks in the blastula stage, declines abruptly before ingression of primary mesenchyme cells, and remains invariable in late development (5Lepage T. Ghiglione C. Gache C. Development. 1992; 114: 147-163Crossref PubMed Google Scholar). Gache and co-workers (5Lepage T. Ghiglione C. Gache C. Development. 1992; 114: 147-163Crossref PubMed Google Scholar) have proposed that BP10 acts as a zymogen activator, since an EGF domain is a highly conserved motif involved in proteolytic cascades or activation of precursors (5Lepage T. Ghiglione C. Gache C. Development. 1992; 114: 147-163Crossref PubMed Google Scholar, 10Appella E. Weber I.T. Blasi F. FEBS Lett. 1988; 231: 1-4Crossref PubMed Scopus (237) Google Scholar), although its in vivo function is not yet known. Mammalian tolloid-like-2 proteins have been shown to process lysyl oxidase but not procollagen or chordin (11Uzel M.I. Scott I.C. Babakhanlou-Chase H. Palamakumbura A.H. Pappanos W.N. Hong H.-H. Greenspan D.S. Trackman P.C. J. Biol. Chem. 2001; 276: 22537-22543Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). In the sea urchin, blocking lysyl oxidase activity inhibits gastrulation (12Butler E. Hardin J. Benson S. Exp. Cell Res. 1987; 173: 174-182Crossref PubMed Scopus (61) Google Scholar, 13Wessel G.M. McClay D.R. Dev. Biol. 1987; 121: 149-165Crossref PubMed Scopus (107) Google Scholar). Despite the uncertainty about its function, blocking BP10 activity prior to hatching with the use of an antibody resulted in abnormal embryos (5Lepage T. Ghiglione C. Gache C. Development. 1992; 114: 147-163Crossref PubMed Google Scholar), reflecting the significance of this protease in embryo development.BP10 has a unique arrangement of structural features (5Lepage T. Ghiglione C. Gache C. Development. 1992; 114: 147-163Crossref PubMed Google Scholar, 6Lepage T. Ghiglione C. Gache C. Eur. J. Biochem. 1996; 238: 744-751Crossref PubMed Scopus (17) Google Scholar), such as the EGF-Ca2+ binding domain, two adjacent CUB domains, and a catalytic domain that is highly homologous to astacin. In particular, the EGF-Ca2+ domain is located between the catalytic domain and the proposed regulatory CUB sequences. More often, these EGF-Ca2+ domains are located between CUB sequences. CUB domains have been implicated in activity and regulation in BMP-1 (14Hartigan N. Garrigue-Antar L. Kadler K.E. J. Biol. Chem. 2003; 278: 18045-18049Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Astacin family enzymes and serine proteases have been implicated in remodeling the pericellular space in sea urchin embryos, which is composed of the extracellular matrix and transmembrane proteins (15Sternlicht M.D. Werb Z. Cell. Dev. Biol. 2001; 17: 463-516Crossref Scopus (3196) Google Scholar, 16Imai K. Kusakabe M. Sakakura T. Nakanishiki I. Okada Y. FEBS Lett. 1994; 352: 216-218Crossref PubMed Scopus (70) Google Scholar). Moreover, several studies have reported gelatinase and collagenase activities from enzymes located in the sea urchin egg and embryo, which were characterized as metalloenzymes due to inactivation with EDTA and 1,10-phenanthroline (17Vafa O. Nishioka D. Mol. Reprod. Dev. 1995; 40: 36-47Crossref PubMed Scopus (21) Google Scholar, 18Karakiulakis G. Papakonstantinou E. Maragoudakis M.E. Miservic G.N. J. Cell. Biochem. 1993; 52: 92-106Crossref PubMed Scopus (15) Google Scholar, 19Quigley J.P. Braithwaite R.S. Armstrong P.B. Differentiation. 1993; 54: 19-23Crossref PubMed Scopus (30) Google Scholar, 20Sharpe C. Robinson J.J. Biochem. Cell Biol. 2001; 79: 461-468Crossref PubMed Scopus (7) Google Scholar).Besides the interesting distribution of astacin-like enzymes across phyla and the numerous functional roles of these enzymes in developmental biology, mechanistic questions about these highly conserved hydrolases domains still remain to be answered. Within the metzincin superfamily of enzymes, minor differences of active site function have been observed, which are likely to account for different substrate specificities (21Stöcker W. Grams F. Baumann U. Reinemer P. Gomis-Rüth F.X. McKay D.B. Bode W. Protein Sci. 1995; 4: 823-840Crossref PubMed Scopus (633) Google Scholar). The metal-coordinated tyrosine in this protease family is a rather unusual metal-binding motif, due to possible reduction of Lewis acidity of the metal ion in the active site induced by the coordination of the negatively charged phenolate to the metal center (22Kimura E. Prog. Inorg. Chem. 1994; 41: 443-491Google Scholar, 23Kimura E. Koike T. Adv. Inorg. Chem. 1997; 44: 229-261Crossref Scopus (120) Google Scholar). However, the coordination of the Tyr-phenolate under physiological conditions does not seem to affect astacin catalysis under neutral conditions. The status of the coordinated tyrosine has been proposed to be an inhibitory process in the metal-centered hydrolysis of peptide bonds by astacin and serralysin (a bacterial metalloprotease containing an astacin-like active site) (24Park H.I. Ming L.-J. J. Biol. Inorg. Chem. 2002; 7: 600-610Crossref PubMed Scopus (24) Google Scholar, 25Park H.I. Ming L.-J. J. Inorg. Biochem. 1998; 72: 57-62Crossref Scopus (40) Google Scholar). A metal-centered mechanism has been proposed for astacin and serralysin based on kinetic and spectroscopic studies of the enzymes and their Cu2+ derivatives (24Park H.I. Ming L.-J. J. Biol. Inorg. Chem. 2002; 7: 600-610Crossref PubMed Scopus (24) Google Scholar, 25Park H.I. Ming L.-J. J. Inorg. Biochem. 1998; 72: 57-62Crossref Scopus (40) Google Scholar). In this mechanism, the active site Zn2+ coordinated by three His, a tyrosine, and a water molecule can be activated via detachment of the Tyr-phenolate with a concomitant deprotonation of the coordinated water assisted by a glutamate residue to afford the arrangement Zn2+–OH···Glu– without a coordinated Tyr. The metal-bound OH– is able to hydrolyze the scissile bond with Zn2+, creating electrostatic strain in the peptide bond by interaction with the carbonyl group of the scissile bond. To gain further insight into the mechanism of this family of developmentally significant proteases, we have performed and present herein the overexpression and thorough mechanistic study of recombinant BP10, which can serve as a model system for astacin-type developmentally regulated metalloenzymes. In this study, we show that BP10 is a zinc-dependent mononuclear metalloprotease with catalytic properties similar to astacin. Through the use of a synthetic substrate and porcine gelatin as substrates for the characterization of recombinant BP10, we demonstrate a unique Ca2+-dependent activation probably due to enhanced substrate binding. BP10 also is shown to have significant stability in the presence of chaotropic reagents by the use of circular dichroism. Finally, the copper derivative of BP10 is shown to have considerable catalytic and spectroscopic activities, which serve as evidence of the conserved astacin mechanism in developmentally regulated enzymes with the participation of the metal-coordinated tyrosine. Serralysin, a bacterial enzyme with similarity to BP10 only in the structure of the active site and in the primary sequence of the metal binding center, has similar substrate specificity.Further analysis of BP10 will add insight into the catalytic mechanism of members of the astacin family and the degree to which the mechanism is conserved among the enzymes found in deuterostomes. Characterization of BP10 will also provide insight into the role of tolloid family zinc endopeptidases in deuterostome development as well as the factors influencing substrate specificity of these enzymes.EXPERIMENTAL PROCEDURESMaterials—The expression vector pQE30Xa, Ni2+-NTA-agarose, and mouse anti-His primary antibody were from Qiagen (Valencia, CA); XL1-Blue chemically competent Escherichia coli was from Invitrogen; Rosetta Blue chemically competent E. coli and Factor Xa removal kit were from Novagen (San Diego, CA); all primers were from Integrated DNA Technologies (Coralville, IA); all modifying and restriction enzymes were from Promega (Madison, WI); Eppendorf Perfect plasmid preparation was from Eppendorf (Westbury, NY); BM purple phosphatase substrate was from Roche Applied Science; EDTA, ZnCl2, Cu(NO3)2, Ca(NO3)2, glycerol, ninhydrin, guanidine hydrochloride, bovine serum albumin, SDS, Triton X-100, Tween 20, imidazole, NaH2PO4, Na2HPO4, Tris-HCl, acrylamide, bisacrylamide, TEMED, ammonium persulfate, NaN3, dimethyl sulfoxide (Me2SO), sodium citrate, acetic acid, guanidine hydrochloride, and propanol were from Fisher; Type A porcine gelatin 300 bloom, N-benzoyl-arginine-p-nitroanilide (BAPNA), urea, isopropyl-β-thiogalactopyronoside, phenylmethylsulfonyl fluoride, benzamidine, urea, lysozyme, bicinchoninic acid, arginine-hydroxamate, and HEPES, CAPS, TAPS, and MES buffers were from Sigma; and 1,10-phenanthroline was from Acros (Fairlawn, NJ). All reagents were of enzyme or molecular biology grade when available, and all others were reagent grade. All glassware and plasticware were extensively rinsed with EDTA to remove metal contaminants and thoroughly washed with 18-megaohm water to remove the chelator. All buffers contained Chelex resin to remove metal contaminants. CD spectra were collected in an AVIV 215 spectropolarimeter (Rheometric Scientific, Inc., Lakewood, NJ) at room temperature. All of the spectrophotometric measurements were performed in a Varian CARY 50 Bio-Spectrophotometer equipped with a PCB-150 water Peltier thermostable cell.Overexpression, Purification, and Refolding of Recombinant BP1—The cDNA coding for Paracentrotus lividus BP10 cloned into the pBluescript plasmid (pBP10) was a generous contribution from Christian Gache and Thierry Lepage (Unité de Biologie Cellulaire, Center National de la Recherche Scientifique et Université de Paris VI, Station Marine, Villefranche-sur-Mer, France). PCR primers coding for both 5′ and 3′ regions were designed according to the proposed full-length BP10 to subclone the cDNA into the pQE30Xa overexpression vector. The 5′ primer (5′-PO4-AAACTAATACTTTCCCTTTCGGGATTG-3′) codes for the first nine codons in the proposed nucleotide sequence in BP10 and is 5′-phosphorylated for blunt end cloning using the StuI restriction site in pQE30Xa; 3′ primer was designed for cloning into the XmaI restriction site 5′-AATTCCCGGGTTAGTTCAGACGAGGATCTCGGGT-3′ (where the boldface type represents extra base pairs for melting temperature optimization, the underline represents the XmaI restriction site, and underlined boldface type represents the stop codon). The PCR product coding for BP10 was digested with XmaI and cloned into the pQE30Xa vector. The BP10 construct was transformed into Rosetta Blue competent cells. The colonies overexpressing recombinant BP10 were screened using a colony lift protocol according to Qiagen without modifications, where the production of BP10 was monitored using a mouse anti-His tag primary antibody. The active colonies were picked, propagated in liquid medium to A600 = 0.4. Isopropyl-β-thiogalactopyronoside was added to a final concentration of 1.0 mm, and the culture was grown at 30 °C and 300 rpm for 4.5 h. The bacteria containing recombinant BP10 were pelleted at 4000 × g at 4 °C and resuspended in cell wall lysis buffer (50.0 mm NaH2PO4, 100.0 mm NaCl, 10.0 mm imidazole, 2.0 mm benzamidine, 2.0 mm phenylmethylsulfonyl fluoride, pH 8.0) containing 1.7 mg/ml lysozyme, incubated on ice lightly shaking for 60 min, and then sonicated for 6 × 10 s with 10-s intervals. The cultures were pelleted at 10,000 × g at 4 °C for 20 min, and the inclusion bodies were resuspended in a urea buffer (8.0 m urea, 10.0 mm Tris, 100.0 mm NaH2PO4, 1.0% Triton X-100, 2.0 mm benzamidine, 2.0 mm phenylmethylsulfonyl fluoride, pH 8.0) and incubated at 37 °C at 200 rpm for 60 min. The solubilized inclusion bodies were pelleted at 10,000 × g at 4 °C for 20 min, and 1.0 ml of Ni2+-NTA-agarose was added to the supernatant. The Ni2+-NTA slurry was gently shaken at room temperature for 45 min and then added to a gravityfed column, and the recombinant BP10 was eluted using a pH gradient, with buffers containing 6.0 m urea, 10.0 mm Tris, 100.0 mm NaH2PO4, at pH values of 6.3, 5.9, and 4.5. The recombinant BP10 was completely eluted at pH 4.5 and was immediately titrated to pH 7.4 using 0.5 m NaH2PO4. Overexpression and purification were monitored on a time-dependent basis using 12.5% SDS-PAGE according to Laemmli and Western blot techniques using the anti-His tag antibody.The concentration of recombinant BP10 was determined using standard BCA assay with a bovine serum albumin standard curve. The recombinant protein was diluted 40 times by volume using 50.0 mm Tris with 50 mm NaCl and then dialyzed extensively with several changes against phosphate-buffered saline (PBS) containing 50 μm ZnCl2 for 48 h at 4 °C. Recombinant BP10 was concentrated under 18 p.s.i. N2 using either an YM3 Amicon membrane or an Amicon Centricon YM3 spin column. Final BP10 concentrations were checked using BCA. The His tag fusion was removed using a Factor Xa His tag removal kit from Novagen according to the manufacturer's instructions.Preparation of the Copper Derivative of BP1—During the refolding of urea-denatured BP10, 1.0 mm 1,10-phenanthroline was added to the 50.0 mm Tris, 50.0 mm NaCl, pH 7.5 buffer and then extensively dialyzed against PBS containing 0.5 m guanidine·HCl. The guanidine·HCl-containing buffer was exchanged through dialysis with PBS buffer and then with PBS containing 50 μm Cu(NO3)2. Protein concentrations were determined with BCA assay.CD Studies—CD spectra of urea-denatured and folded Zn-BP10 and Cu-BP10 were collected in PBS using a 0.1-cm cell with a resolution of 0.5 nm. All absorbance readings were converted to molar ellipticity, and the α-helical content was calculated according to published methods (26Chen Y.-H. Yang J.T. Martinez H.M. Biochemistry. 1972; 11: 4120-4131Crossref PubMed Scopus (1895) Google Scholar).Gelatin Zymogram—Gelatin was incorporated into a polyacrylamide gel matrix according to standard protocols with modifications to fit current studies as described below (27Murphy G. Crabbe T. Methods Enzymol. 1995; 248: 470-475Crossref PubMed Scopus (174) Google Scholar). A volume of 1.25 ml of 1.4 m Tris at pH 8.8, 0.50 ml of 5.0 mg/ml gelatin solution in water, 25.0 μl of 10% (w/v) ammonium persulfate, 200 μl of 10% (w/v) SDS, 25.0 μl of TEMED, 2.0 ml of water, and 1.25 ml of 30:1 acrylamide/bisacrylamide were mixed and allowed to polymerize in a minigel minus a stacking gel. Several concentrations of BP10 were mixed with nonreducing gel-loading dye and incubated for 15 min at room temperature (standard Laemmli protocol minus mercaptoethanol or dithiothreitol (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206048) Google Scholar)). These BP10 samples were loaded into each lane of the gel and run at 200 V and 4 °C until the dye front reached the bottom of the plate. The running buffer did not contain SDS. The gel was washed two times in 0.25% Triton X-100 for 15 min with gentle shaking and then incubated for 10 h in 50.0 mm Tris, pH 7.50, 1.0 μm ZnCl2, 0.5% Triton X-100, 0.02% NaN3, and 2.0 mm CaCl2. After 10 h, the gels were stained with 0.1% Coomassie Brilliant Blue in 40% propanol for 1.0 h. The gel was destained in a 7% acetic acid solution to reveal the digestion and then photographed on a light table with a digital camera.Gelatinase Assay—The gelatinase activity of recombinant BP10 was monitored using a detection method for α-amino groups with ninhydrin as an indicator of peptide hydrolysis according to a standard protocol with modifications to fit current studies as described below (29Grubb J.D. Methods Enzymol. 1994; 235: 602-606Crossref PubMed Scopus (6) Google Scholar). A 5.0 mg/ml gelatin solution was prepared in H2O and heated to 55 °C for 15 min until completely dissolved. A ninhydrin detection solution was prepared by mixing 9.0 ml of glycerol, 3.0 ml of 0.5 m sodium citrate at pH 5.50, and 3.75 ml of 1.0% (w/v) ninhydrin solution in 0.5 m sodium citrate buffer. Gelatin and BP10 (1.0 μm final concentration) were mixed in PBS and incubated at room temperature. A 50.0-μl sample was taken from the reaction at several time points and mixed with 950 μl of ninhydrin detection solution and then boiled for 12.0 min; then the absorbance at 570 nm was determined. A sample containing undigested stock gelatin (the same gelatin used for the experiment, incubated under the same conditions without BP10) mixed with the same detection assay was used as the blank. The pseudo-first-order rate constant kobs was determined from an exponential curve fit. The molar absorptivity of Ruhemann's purple (ϵ570 = 22,000 m–1 cm–1) (30Friedman M. J. Agric. Food Chem. 2004; 52: 385-406Crossref PubMed Scopus (425) Google Scholar) was used to determine gelatin concentrations in millimolar and also allowed for monitoring the substrate-dependent hydrolysis of gelatin. Several dilutions of a 10.0 mg/ml stock solution of gelatin incubated with BP10 with time points taken at 0.0, 2.0, 4.0, 8.0, and 16.0 min were used to calculate the initial rate using the ninhydrin detection method described above. Initial rates were fitted as a function of substrate concentration according to the Michaelis-Menten equation, yielding kcat and Km parameters.Hydrolysis of BAPNA by Zn-BP10 and Cu-BP1—BAPNA stock solutions were made in Me2SO and then diluted with 50.0 mm HEPES, 50.0 mm NaCl, pH 7.50. Less than 2% Me2SO by volume was present in each assay and found not to interfere with kinetic measurements. Several concentrations of BAPNA were incubated with 2.17 μm BP10, and rates were determined colorimetrically from the release of the p-nitroaniline product (ϵ405 = 10,150 m–1 cm–1). Kinetic parameters were determined by nonlinear fitting to the Michaelis-Menten equation.Calcium Activation Assays—Gelatin was extensively dialyzed against an EDTA solution and then extensively dialyzed to remove the chelator. Calcium was carefully titrated under substrate saturation conditions to determine its effect on the hydrolysis of gelatin and BAPNA. Once saturating concentrations of calcium were determined, new kinetic parameters were obtained using sufficient (1.0 mm) calcium in all buffers.Inhibition Studies—The effects of two inhibitors, 1,10-phenanthroline and arginine-hydroxamate (Arg-NHOH), on the hydrolysis of BAPNA were determined by running Michaelis-Menten kinetics under several concentrations of each inhibitor. Inhibition constants were determined according to the inhibition patterns for 1,10-phenanthroline and Arg-NHOH, respectively.pH Profiles—The pH profiles for Zn- and Cu-BP10 catalysis were constructed by monitoring gelatin and BAPNA hydrolysis under several different pH values using 50.0 mm buffers containing 50.0 mm NaCl and 1.0 mm CaCl2. The following buffers were used: acetate (pH 5.0), MES (pH 5.5–6.5), HEPES (pH 7.0–8.0), TAPS (pH 8.5–9.0), and CAPS (pH 9.5–11.0). The pH-dependent kinetic parameters were determined by nonlinear fitting to the Michaelis-Menten equation, and pKa values were obtained from fitting the kinetic parameters to a two-ionization process.Electronic Spectrum of Cu-BP1—The electronic spectrum of 20.0 μm Cu-BP10 was obtained from 350 to 800 nm, and the tyrosine to copper charge transfer transition was observed at 454 nm. The quenching of this ligand-to-metal charge transfer transition (LMCT) was monitored spectrophotometrically upon the addition of Arg-NHOH.RESULTS AND DISCUSSIONOverexpression and Refolding of Recombinant BP1—The recombinant enzyme was efficiently overexpressed, although it was insoluble and contained within inclusion bodies (Fig. 1, lane 3). Urea solubilization proved to be an efficient method for extracting the enzyme from inclusion bodies, coupled with a fusion His tag at the N terminus to BP10 that allows for efficient purification using Ni2+-NTA-agarose. The overexpression and purification yields an average of 0.7 mg/ml total recombinant BP10 after a pH gradient elution from the Ni2+-NTA-agarose column (Fig. 1). The protein overexpression was monitored on a time course using SDS-PAGE and Western blotting (Fig. 2B). The detection of the His tag using an anti-His antibody upon induction with isopropyl-β-thiogalactopyronoside proved a sensitive and consistent method for monitoring the overexpression of the recombinant protein.FIGURE 2A, SDS-PAGE (12.5%) of intact BP10 after refolding. Lane 1, molecular mass marker (200, 97.4, 66.2, 45, and 31 kDa from the top); lane 2, total urea-solubilized protein; lane 3, recombinant BP10 after refolding. B, Western blot for the time course of overexpression. Lane 1, uninduced sample; lanes 2–5, 1.0, 2.0, 3.0, and 4.0 h, respectively. C, gelatin zymogram for concentration-dependent substrate hydrolysis by BP10; lanes 1–5, 0.25, 0.50, 1.0, 1.50, and 1.75 μm BP10, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Upon removal of the chaotropic reagent urea through extensive dialysis, the protein was refolded, and activity could be monitored after removal of the His tag. It was determined empirically that a 40-fold dilution of the denatured protein prior to dialysis was important, since at higher concentrations, the protein coagulates and precipitates out of solution. Due to the large number of cysteine residues in BP10, cysteine and reduced glutathione were included in the refolding process in separate attempts. However, this refolding process" @default.
• W2023165370 created "2016-06-24" @default.
• W2023165370 creator A5019492374 @default.
• W2023165370 creator A5026798513 @default.
• W2023165370 creator A5077198357 @default.
• W2023165370 creator A5089875217 @default.
• W2023165370 date "2006-04-01" @default.
• W2023165370 modified "2023-10-08" @default.
• W2023165370 title "Overexpression and Mechanistic Characterization of Blastula Protease 10, a Metalloprotease Involved in Sea Urchin Embryogenesis and Development" @default.
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