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• W2019950831 abstract "The Arabidopsis thaliana constitutive disease resistance 1 (CDR1) gene product is an aspartic proteinase that has been implicated in disease resistance signaling (Xia, Y., Suzuki, H., Borevitz, J., Blount, J., Guo, Z., Patel, K., Dixon, R. A., and Lamb, C. (2004) EMBO J. 23, 980–988). This apoplastic enzyme is a member of the group of “atypical” plant aspartic proteinases. As for other enzymes of this subtype, CDR1 has remained elusive until recently as a result of its unusual properties and localization. Here we report on the heterologous expression and characterization of recombinant CDR1, which displays unique enzymatic properties among plant aspartic proteinases. The highly restricted specificity requirements, insensitivity toward the typical aspartic proteinase inhibitor pepstatin A, an unusually high optimal pH of 6.0–6.5, proteinase activity without irreversible prosegment removal, and dependence of catalytic activity on formation of a homo-dimer are some of the unusual properties observed for recombinant CDR1. These findings unveil a pattern of unprecedented functional complexity for Arabidopsis CDR1 and are consistent with a highly specific and regulated biological function. The Arabidopsis thaliana constitutive disease resistance 1 (CDR1) gene product is an aspartic proteinase that has been implicated in disease resistance signaling (Xia, Y., Suzuki, H., Borevitz, J., Blount, J., Guo, Z., Patel, K., Dixon, R. A., and Lamb, C. (2004) EMBO J. 23, 980–988). This apoplastic enzyme is a member of the group of “atypical” plant aspartic proteinases. As for other enzymes of this subtype, CDR1 has remained elusive until recently as a result of its unusual properties and localization. Here we report on the heterologous expression and characterization of recombinant CDR1, which displays unique enzymatic properties among plant aspartic proteinases. The highly restricted specificity requirements, insensitivity toward the typical aspartic proteinase inhibitor pepstatin A, an unusually high optimal pH of 6.0–6.5, proteinase activity without irreversible prosegment removal, and dependence of catalytic activity on formation of a homo-dimer are some of the unusual properties observed for recombinant CDR1. These findings unveil a pattern of unprecedented functional complexity for Arabidopsis CDR1 and are consistent with a highly specific and regulated biological function. Plant aspartic proteinases (APs) 3The abbreviations used are: AP, aspartic proteinase; CDR1, constitutive disease resistance 1; PCS1, promotion of cell survival 1; CND41, chloroplast nucleoid DNA-binding protein; E-64, l-trans-epoxysuccinylleucylamide-(4-guanidino)butane; CAPS, 3-(cyclohexylamino)propanesulfonic acid; FTC, fluorescein isothiocarbamoyl casein; MCA, (7-methoxycoumarin-4-yl)acetyl; DNP, 2,4-dinitrophenyl.3The abbreviations used are: AP, aspartic proteinase; CDR1, constitutive disease resistance 1; PCS1, promotion of cell survival 1; CND41, chloroplast nucleoid DNA-binding protein; E-64, l-trans-epoxysuccinylleucylamide-(4-guanidino)butane; CAPS, 3-(cyclohexylamino)propanesulfonic acid; FTC, fluorescein isothiocarbamoyl casein; MCA, (7-methoxycoumarin-4-yl)acetyl; DNP, 2,4-dinitrophenyl. are widely distributed in the plant kingdom and have been detected or purified from monocot and dicot species as well as gymnosperms (1Simoes I. Kim C. Eur. J. Biochem. 2004; 271: 2067-2075Crossref PubMed Scopus (211) Google Scholar). APs are synthesized as single chain preproenzymes and, upon activation, converted to mature two-chain enzymes. A characteristic feature of “typical” plant AP precursors is the presence of a protein domain of ∼100 amino acids known as the plant-specific insert, which is highly similar to saposin-like proteins and is removed from the precursors upon activation into the mature form of the enzymes (1Simoes I. Kim C. Eur. J. Biochem. 2004; 271: 2067-2075Crossref PubMed Scopus (211) Google Scholar). Recently published results on plant APs with unexpected localizations, unusual sequences, and novel structural arrangements (2Ge X. Kim C. Matsuno M. Li G. Berg H. Xia Y. EMBO Rep. 2005; 6: 282-288Crossref PubMed Scopus (114) Google Scholar, 3Murakami S. Kim Y. Nakano T. Sato F. FEBS Lett. 2000; 468: 15-18Crossref PubMed Scopus (69) Google Scholar, 4Athauda S.B. Kim K. Rajapakshe S. Kuribayashi M. Kojima M. Kubomura-Yoshida N. Iwamatsu A. Shibata C. Inoue H. Takahashi K. Biochem. J. 2004; 381: 295-306Crossref PubMed Scopus (102) Google Scholar, 5Bi X. Kim G.S. Bennett J. Plant Cell Physiol. 2005; 46: 87-98Crossref PubMed Scopus (34) Google Scholar, 6Nakano T. Kim S. Shoji T. Yoshida S. Yamada Y. Sato F. Plant Cell. 1997; 9: 1673-1682Crossref PubMed Scopus (102) Google Scholar, 7Xia Y. Kim H. Borevitz J. Blount J. Guo Z. Patel K. Dixon R.A. Lamb C. EMBO J. 2004; 23: 980-988Crossref PubMed Scopus (269) Google Scholar, 8Chen F. Kim M.R. Plant Mol. Biol. 1997; 35: 821-831Crossref PubMed Scopus (120) Google Scholar, 9Kato Y. Kim Y. Murakami S. Sato F. Planta. 2005; 222: 643-651Crossref PubMed Scopus (70) Google Scholar, 10Kato Y. Kim S. Yamamoto Y. Chatani H. Kondo Y. Nakano T. Yokota A. Sato F. Planta. 2004; 220: 97-104Crossref PubMed Scopus (117) Google Scholar) as well as the identification of a wide variety of AP-like proteins in the Arabidopsis genome (11Faro C. Kim S. Curr. Protein Pept. Sci. 2005; 6: 493-500Crossref PubMed Scopus (63) Google Scholar, 12Beers E.P. Kim A.M. Dickerman A.W. Phytochemistry. 2004; 65: 43-58Crossref PubMed Scopus (163) Google Scholar) have triggered a redefinition of the classification of plant APs. Faro and Gal have recently proposed a new classification that takes into account the diversity of plant APs by grouping them into typical, nucellin-like, and atypical members, with the latter group comprising the majority of the newly identified Arabidopsis APs (11Faro C. Kim S. Curr. Protein Pept. Sci. 2005; 6: 493-500Crossref PubMed Scopus (63) Google Scholar). Despite having low sequence identity, these atypical and nucellin-like APs share several common features such as 1) the absence of the internal segment plant-specific insert in their sequence, 2) an unusually high number of cysteines, 3) the type of amino acids preceding the first catalytic triad, and 4) unexpected localizations, which clearly differentiate them from the well studied typical plant APs. Thus far, only a rather small number of atypical and nucellin-like APs have been studied (2Ge X. Kim C. Matsuno M. Li G. Berg H. Xia Y. EMBO Rep. 2005; 6: 282-288Crossref PubMed Scopus (114) Google Scholar, 3Murakami S. Kim Y. Nakano T. Sato F. FEBS Lett. 2000; 468: 15-18Crossref PubMed Scopus (69) Google Scholar, 4Athauda S.B. Kim K. Rajapakshe S. Kuribayashi M. Kojima M. Kubomura-Yoshida N. Iwamatsu A. Shibata C. Inoue H. Takahashi K. Biochem. J. 2004; 381: 295-306Crossref PubMed Scopus (102) Google Scholar, 5Bi X. Kim G.S. Bennett J. Plant Cell Physiol. 2005; 46: 87-98Crossref PubMed Scopus (34) Google Scholar, 6Nakano T. Kim S. Shoji T. Yoshida S. Yamada Y. Sato F. Plant Cell. 1997; 9: 1673-1682Crossref PubMed Scopus (102) Google Scholar, 7Xia Y. Kim H. Borevitz J. Blount J. Guo Z. Patel K. Dixon R.A. Lamb C. EMBO J. 2004; 23: 980-988Crossref PubMed Scopus (269) Google Scholar, 8Chen F. Kim M.R. Plant Mol. Biol. 1997; 35: 821-831Crossref PubMed Scopus (120) Google Scholar, 9Kato Y. Kim Y. Murakami S. Sato F. Planta. 2005; 222: 643-651Crossref PubMed Scopus (70) Google Scholar, 10Kato Y. Kim S. Yamamoto Y. Chatani H. Kondo Y. Nakano T. Yokota A. Sato F. Planta. 2004; 220: 97-104Crossref PubMed Scopus (117) Google Scholar). However, the proposed roles in highly regulated processes like plastid homeostasis (tobacco CND41) (9Kato Y. Kim Y. Murakami S. Sato F. Planta. 2005; 222: 643-651Crossref PubMed Scopus (70) Google Scholar, 10Kato Y. Kim S. Yamamoto Y. Chatani H. Kondo Y. Nakano T. Yokota A. Sato F. Planta. 2004; 220: 97-104Crossref PubMed Scopus (117) Google Scholar), disease resistance (Arabidopsis CDR1) (7Xia Y. Kim H. Borevitz J. Blount J. Guo Z. Patel K. Dixon R.A. Lamb C. EMBO J. 2004; 23: 980-988Crossref PubMed Scopus (269) Google Scholar), or programmed cell death (Barley nucellin and Arabidopsis PCS1) (2Ge X. Kim C. Matsuno M. Li G. Berg H. Xia Y. EMBO Rep. 2005; 6: 282-288Crossref PubMed Scopus (114) Google Scholar, 8Chen F. Kim M.R. Plant Mol. Biol. 1997; 35: 821-831Crossref PubMed Scopus (120) Google Scholar) suggest functional specialization of these particular set of plant APs as found e.g. in mammalian renin as well as tight activity regulation, in contrast to the rather unspecific housekeeping role normally ascribed to typical plant APs. Therefore, there is an increasing interest on the structure and function of atypical and nucellin-like APs. Arabidopsis CDR1 is a member of the larger group of the atypical APs and was first identified by Xia and co-workers (7Xia Y. Kim H. Borevitz J. Blount J. Guo Z. Patel K. Dixon R.A. Lamb C. EMBO J. 2004; 23: 980-988Crossref PubMed Scopus (269) Google Scholar) while screening for gain-of-function mutants displaying enhanced resistance to bacterial pathogens. CDR1 is an extracellular AP, and this was the first time a phenotype was associated with the gain-of-function of a gene coding for a plant AP. The enhanced resistance of the CDR1 gain-of-function mutant is likely due to the proteolytic generation of an endogenous peptide elicitor, which might be involved in the activation of salicylic acid-dependent local and systemic defense responses. The systemic acquired resistance is an inducible plant defense response, conferring long lasting protection against a broad spectrum of microorganisms. Given the controversy on the nature of the systemic signal for systemic acquired resistance (13Durrant W.E. Kim X. Annu. Rev. Phytopathol. 2004; 42: 185-209Crossref PubMed Scopus (2005) Google Scholar, 14Kunkel B.N. Kim D.M. Curr. Opin. Plant Biol. 2002; 5: 325-331Crossref PubMed Scopus (1134) Google Scholar, 15Maldonado A.M. Kim P. Dixon R.A. Lamb C.J. Cameron R.K. Nature. 2002; 419: 399-403Crossref PubMed Scopus (602) Google Scholar), an extensive characterization of CDR1 could give insights into the mechanism by which this atypical AP exerts its function in disease resistance signaling. Here, we report the characterization of recombinant CDR1 overexpressed in Escherichia coli. The recombinant enzyme displays unique enzymatic properties if compared with other plant APs and unprecedented structure-function relationships. The results herein described, in combination with the very specific plant phenotype previously reported, strongly support the concept that CDR1 exerts precise, limited, and highly regulated proteolytic functions. Plant Material—Arabidopsis ecotype Columbia were seeded and grown in a climatic room, under continuous light at 25 °C. Seedlings were collected and frozen immediately in liquid nitrogen and kept at -80 °C until use. cDNA Cloning of Arabidopsis CDR1—Total RNA was isolated from Arabidopsis thaliana seedlings using TRIzol reagent (Invitrogen) according to the manufacturer's protocol, and its quality was checked by agarose gel electrophoresis. Reverse transcription-PCR was performed with a first strand cDNA Synthesis Kit for reverse transcription-PCR (Roche Applied Science) using 1 μg of total RNA. The resulting first strand cDNA was then used as a PCR template with A. thaliana CDR1-specific primers for the 5′-ATGGCCTCTCTATTCTCTTCAGT (forward) and 3′-CTACATCTTTGCACAATCTGTTG (reverse) ends of the open reading frame of CDR1. These primers were synthesized according to the mRNA sequence deposited in the EBI Data Bank with the accession number AY243479. Full-length CDR1-amplified cDNA was cloned, and both stands were sequenced by automated DNA sequencing. Expression, Refolding, and Purification of Recombinant CDR1 and Active-site Mutant in E. coli—CDR1 cDNA was amplified by PCR using specific primers that include restriction sites for NheI 5′-GCTAGCAAGCCAAAACTAGGCTTCACCGCGG and BamHI 5′-GGATCCCTACATCTTTGCACAATCTGTTGG. The resulting PCR-amplified product, without the putative signal sequence, was subcloned in pET-23d expression vector (Novagen). The positive clones selected by restriction analysis were confirmed by DNA sequencing. The QuikChange site-directed mutagenesis kit (Stratagene) was used to generate the active-site mutant CDR1(D108A) in the vector pET23d using the primers 5′-ATCATGGCCATCGCCGCCACCGGAAGTGATCTC-3′ (forward primer) and 5′-GAGATCACTTCCGGTGGCGGCGATGGCCATGAT-3′ (reverse primer) (mutation underlined). The positive mutant clones were confirmed by DNA sequencing. Both constructs, CDR1 wild-type (wtCDR1) and the mutant CDR1(D108A), were transformed into the E. coli BL21(DE3) strain. The method of recombinant protein purification was adapted from Castanheira et al. (16Castanheira P. Kim B. Sergeant K. Clemente J.C. Dunn B.M. Pires E. Van B.J. Faro C. J. Biol. Chem. 2005; 280: 13047-13054Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Briefly, after growth of the cells at 37 °C to D600 of 0.6, protein expression was induced by the addition of isopropyl 1-thio-β-d-galactopyranoside (0.5 mm final concentration). After 3 h, cells were harvested by centrifugation, resuspended in 50 mm Tris/50 mm NaCl (pH 7.4), and lysed with lysozyme (100 μg/ml). After freezing and thawing, DNase (100 μg/ml) and MgCl2 (100 mm) were added, and the reaction mixture was incubated at 4 °C for 1 h. The cell lysate was then diluted into 1 liter of 50 mm Tris/50 mm NaCl (pH 7.4) and washed overnight at 4 °C with agitation. Then, the material was centrifuged at 10,000 × g and washed again overnight with 50 mm Tris/50 mm NaCl (pH 7.4) containing 0.1% (v/v) Triton X-100. After centrifugation at 10,000 × g for 20 min at 4 °C, the purified inclusion bodies were dissolved in 8 m urea, with 100 mm 2-mercaptoethanol. The protein was refolded by rapid dilution (20-fold) into 20 mm Tris, and the pH was slowly adjusted to pH 8.0. The recombinant protein was then concentrated in a tangential flow ultrafiltration system (Pellicon 2, Millipore), ultracentrifuged at 50,000 × g for 20 min at 4 °C, and the supernatant applied to an S-300 gel filtration column (Amersham Biosciences) equilibrated in 20 mm Tris/0.4 m urea, pH 8.0, buffer. The protein fractions corresponding to the non-aggregated forms of wild-type or mutant recombinant CDR1 were further purified by ion-exchange chromatography with a High Q (Bio-Rad) column followed by a Mono Q (Amersham Biosciences) column in an fast-protein liquid chromatography system using the same buffer used for S-300 chromatography. Elution was carried out with a linear gradient of NaCl (0–0.5 m) at a flow rate of 2.0 ml/min or 0.75 ml/min, respectively. Protease Activity Screening Assays—Fluorescein isothiocarbamoyl casein (FTC-casein, Pierce) was used as a substrate to measure purified recombinant CDR1 proteolytic activity. Activity was measured according to the manufacturer's protocol. Briefly, a stock solution of FTC-casein was diluted in 25 mm phosphate buffer, 0.15 m NaCl, pH 6.4, and the reaction was started by adding recombinant wtCDR1. The assay mixture was incubated for 5 h at room temperature. Relative fluorescence was measured in a spectrofluorometer, and an excitation wavelength of 485 nm and an emission wavelength of 538 nm were used to monitor proteolytic activity. The effect of the selective inhibitor pepstatin was also tested by preincubating the inhibitor for 15 min at 37 °C with the enzyme before adding the substrate FTC-casein. The activity toward hemoglobin was tested as follows; a solution of acid-denatured hemoglobin in 0.1 m formate buffer, pH 3.0, was incubated with the purified recombinant wtCDR1 for 3 h. The reaction was stopped by the addition of 5% (v/v) trichloroacetic acid. After centrifugation at 10,000 × g for 10 min, the absorbance of the supernatant was measured at 280 nm. Digestion of Oxidized Insulin B Chain—Digestion of oxidized insulin B chain by purified recombinant wtCDR1 was carried out overnight at 37 °C in the following buffers: 0.1 m formate buffer, pH 3.0; 0.1 m citrate buffer, pH 4.0; 0.1 m acetate buffer, pH 5.0; 0.1 m phosphate buffer, pH 6.0 and pH 7.0; 0.1 m Tris-HCl, pH 8.0. Reaction was stopped with 0.6% (v/v) trifluoroacetic acid (final concentration). After centrifugation (12,000 × g, 2 min), the fragments were separated by reversed-phase high performance liquid chromatography in a C18 column, using a Merck Hitachi system with a Hi-Pore RP-318 250 × 4.6 mm column (Bio-Rad). Elution was carried out with a linear gradient (0–80%) of acetonitrile in 0.1% v/v trifluoroacetic acid for 30 min at a flow rate of 1 ml/min. Absorbance was monitored at 220 nm. The isolated peptides were collected, freeze-dried, and submitted to automated amino acid sequencing. Enzyme Assays—Proteolytic activity of purified recombinant wtCDR1 was tested against several model substrates. Activity assays with the fluorogenic substrates (7-methoxycoumarin-4-yl)acetyl (MCA)Lys-Lys-Pro-Ala-Glu-Phe-Phe-Ala-Leu-Lys-2,4-dinitrophenyl (DNP) (2 μm) and (MCA)Lys-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(DNP) (2 μm) were performed at different pH values. The following buffers between pH 3 and 8 containing 0.1 m NaCl and 8% (v/v) Me2SO were used: 0.05 m sodium citrate, pH 3.0; 0.05 m sodium acetate, pH 4.0–5.5; 0.05 m sodium phosphate, pH 5.5–7.0; 0.05 m Tris-HCl, pH 8.0. The enzyme was incubated with each buffer at 37 °C, and the rate of hydrolysis was monitored at an excitation wavelength of 328 nm and an emission wavelength of 393 nm. In the case of the renin substrate tetradecapeptide porcine (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser), activity of purified recombinant wtCDR1 was tested by incubation with 0.1 mg of substrate for 5 h at 37 °C and at different pH values. After digestion, the fragments were separated by reversed-phase high performance liquid chromatography in a C18 column, using a Merck Hitachi system with a Hi-Pore RP-318 250 × 4.6 mm column (Bio-Rad) as described in the previous section. Due to the lack of activity toward the model substrates tested, a new peptide substrate was designed for CDR1. The proteolytic activities of purified recombinant wtCDR1 and CDR1(D108A) were then assayed using this synthetic fluorogenic substrate (2 μm) (MCA)Lys-Leu-His-Pro-Glu-Val-Leu-Phe-Val-Leu-Glu-Lys(DNP) synthesized by Genosphere Biotechnologies (France). The increase of fluorescence intensity produced by substrate hydrolysis was monitored in a spectrofluorometer. An excitation wavelength of 328 nm and an emission wavelength of 393 nm were used to monitor the hydrolysis of this substrate. The relationship between fluorescence change and peptide concentration was determined by measuring the total fluorescence change that occurred upon complete hydrolysis of the peptide with pepsin. For activity studies at different pH, the following buffers between pH 3 and 8 containing 0.1 m NaCl and 8% (v/v) Me2SO were used: 0.05 m sodium citrate, pH 3.0; 0.05 m sodium acetate, pH 4.0–5.5; 0.05 m sodium phosphate, pH 5.5–7.0; 0.05 m Tris-HCl, pH 8.0. For activity studies at different temperatures, wtCDR1 was incubated in 0.05 m sodium acetate, pH 6.5, 0.1 m NaCl, 8% (v/v) Me2SO buffer at temperatures between 10 ° and 70 °C. The same assay conditions were used to assay the activity of the mutant CDR1(D108A), and the fractions were collected from size-exclusion chromatography as well as to study the effect of various compounds on the proteolytic activity of recombinant wtCDR1. In this case, the enzyme was preincubated with each compound for 15 min at 37 °C before determination of proteolytic activity. The kinetic parameters were calculated from the Lineweaver-Burk plot using appropriate software. Size-exclusion Chromatography—Native molecular weight of purified recombinant wtCDR1 and CDR1(D108A) was estimated by size-exclusion chromatography on a Superose 12 (Amersham Biosciences) column connected to a fast-protein liquid chromatography system. The column was equilibrated in 20 mm Hepes, pH 7.6, 0.2 m NaCl, 5% glycerol and calibrated with Gel Filtration LMW and HMW calibration kits (Amersham Biosciences) according to the manufacturer's instructions. The molecular mass markers used for calibration were aldolase, 158 kDa; albumin, 67 kDa; ovalbumin, 43 kDa; chymotrypsinogen, 25 kDa; and ribonuclease A, 13.7 kDa. Gel Electrophoresis—Protein samples were separated by SDS-PAGE using 12% gels in a Bio-Rad Mini Protean III electrophoresis apparatus. Gels were stained with Coomassie Brilliant Blue R-250 (Sigma). Protein samples for chemical N-terminal sequence analysis were separated by SDS-PAGE and subsequently transferred onto polyvinylidene difluoride membranes by electroblotting in 10 mm CAPS, 10% methanol, pH 11, at 500 mA for 1 h and stained with Coomassie Brilliant Blue R-250. Molecular Modeling—Model building was performed on a structure of Mucor pusillus aspartic proteinase (PDB code: 1mpp). A sequence alignment of CDR1 deduced amino acid sequence (accession number AY243479) and M. pusillus aspartic proteinase (sequence from PDB file) yielded sequence identity of 22%. Modeling calculations were done on an SGI Octane workstation with dual R12000 processors using the program Moloc (17Gerber P.R. J. Comput. Aided Mol. Des. 1998; 12: 37-51Crossref PubMed Scopus (102) Google Scholar). An initial C-alpha model of CDR1 was built by fitting its aligned sequence on the M. pusillus template C-alpha structure followed by optimization of newly introduced loops. A number of new disulfide bridges were introduced between adjacent Cys amino acids in the structure. Subsequently a full atom model was generated, and newly inserted loops were optimized with the rest of the protein kept stationary. Refinement of the full model with manual removal of repulsive van der Waals interactions followed and was divided in three parts. First, only amino acid side chains were allowed to move; in a second step all atoms except alpha carbons were optimized; and finally, positional constraints were put on alpha carbons only. Quality checks were made with Moloc internal programs and with a program by Luthy et al. (18Luthy R. Kim J.U. Eisenberg D. Nature. 1992; 356: 83-85Crossref PubMed Scopus (2562) Google Scholar). Expression, Refolding, and Purification of Recombinant CDR1—The atypical AP CDR1 (Fig. 1) was first identified by T-DNA activation tagging, and it was shown that its function in the activation of inducible resistance mechanisms required protease activity (7Xia Y. Kim H. Borevitz J. Blount J. Guo Z. Patel K. Dixon R.A. Lamb C. EMBO J. 2004; 23: 980-988Crossref PubMed Scopus (269) Google Scholar). To characterize the structural and enzymatic properties of this plant AP we have cloned its full-length cDNA (nucleotide sequence available in the EBI Data Bank under the accession number AY243479) from Arabidopsis seedlings. An expression plasmid construct was subsequently generated containing the cDNA encoding CDR1 without the first 25 amino acid residues, which were assumed to correspond to the putative signal peptide (predicted by SignalP (19Bendtsen J.D. Kim H. von H.G. Brunak S. J. Mol. Biol. 2004; 340: 783-795Crossref PubMed Scopus (5634) Google Scholar)). The CDR1-pET-23d (wtCDR1) construct was expressed in E. coli BL21(DE3) strain and resulted in accumulation of the protein in inclusion bodies. They were purified by a freeze/thaw and detergent-washing procedure and subsequently dissolved in urea. Protein refolding was induced by a rapid dilution step followed by pH adjustment. After refolding the protein was purified by size-exclusion chromatography on a Sephacryl S-300 column, and the protein fractions corresponding to the non-aggregated forms of the enzyme were further purified by ion-exchange chromatography with High Q and Mono Q columns. SDS-PAGE analysis of the purified protein fractions confirmed the presence of a protein with an apparent molecular mass of 50 kDa (Fig. 2). Because the predicted mass of the non-glycosylated form of wtCDR1 is 45 kDa, the identity of the purified protein was subsequently determined by Edman degradation. The results on N-terminal sequencing of the electroblotted purified protein confirmed the expected N-terminal sequence of wtCDR1 (underlined), ASKPKLG, with AS being encoded by the pET-23d sequence. Proteolytic Activity of Recombinant wtCDR1—Despite a relatively low amino acid sequence identity with typical plant APs, the CDR1 sequence contains the typical hallmarks of aspartic proteinases. This includes the active site consensus sequence motifs (DTG/DSG) and the two hydrophobic-hydrophobic-Gly motifs of the Psi-loops. Moreover, the fact that the active-site mutant failed to recapitulate the CDR1-D-like phenotype (7Xia Y. Kim H. Borevitz J. Blount J. Guo Z. Patel K. Dixon R.A. Lamb C. EMBO J. 2004; 23: 980-988Crossref PubMed Scopus (269) Google Scholar) further support the assignment of CDR1 as an aspartic protease with novel features. The purified recombinant wtCDR1 was tested for proteolytic activity against several model substrates (Table 1). The synthetic fluorogenic substrate (MCA)Lys-Lys-Pro-Ala-Glu-Phe-Phe-Ala-Leu-Lys(DNP), whose chromogenic version with a p-nitrophenylalanine group in the P1 position (20Dunn B.M. Kim M. Parten B.F. Valler M.J. Rolph C.E. Kay J. Biochem. J. 1986; 237: 899-906Crossref PubMed Scopus (163) Google Scholar) has been successfully hydrolyzed by a wide variety of aspartic proteinases from animal, microbial, or plant origins, was not cleaved by recombinant wtCDR1, and therefore no activity was observed at a pH range from 3 to 8. This result provided the first evidence that wtCDR1 prefers different substrates from those cleaved by plant APs studied thus far. Moreover, neither the fluorogenic BACE1 substrate (MCA)Lys-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(DNP) (21Ermolieff J. Kim J.A. Koelsch G. Tang J. Biochemistry. 2000; 39: 12450-12456Crossref PubMed Scopus (134) Google Scholar) nor the known renin substrate Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser (Sigma-Aldrich) were cleaved by wtCDR1 in any of our activity assays. When hemoglobin, bovine serum albumin, or FTC-casein were probed as substrates, wtCDR1 processed only the latter. In fact, purified recombinant wtCDR1 displayed a narrow pH tolerance for this substrate with a pH optimum at 6.4. Moreover, pepstatin A, a widely used inhibitor of aspartic proteinases, inhibited this FTC-casein processing very weakly (data not shown).TABLE 1Proteolytic activity of recombinant wtCDR1 Proteolytic activity of purified recombinant wtCDR1 was tested against several model substrates. The assays were performed as described under “Experimental Procedures.” No activity was observed for the majority of peptides or protein substrates used. The exceptions were oxidized insulin B chain (cleaved at Leu15-Tyr16) and FTC-casein.SubstrateSequenceEfficiency of cleavagePeptide substrates Typical AP substrate(MCA) KKPAEFFALK (DNP)n.c.an.c., not cleaved BACE1 substrate(MCA) KSEVNLDAEFK (DNP)n.c. Renin substrate tetradecapeptideDRVYIHPFHLLVYSn.c. Oxidized insulin B chainFVNQHLCGSHLVEALYLVCGER GFFYTPKACleaved at L15-Y16Protein substrates Hemoglobinn.c. BSAn.c. FTC-caseinPoorly cleaveda n.c., not cleaved Open table in a new tab This narrow substrate specificity together with the observed low sensitivity toward pepstatin A and an unusually high optimal pH prompted us to further investigate the catalytic properties of CDR1. Rather unexpectedly, and in contrast to other aspartic proteinases, wtCDR1 cleaved the Leu15-Phe16 bond of the insulin B chain in a selective manner at an optimal pH of 6.0. Under these conditions, addition of pepstatin A only partially inhibited this cleavage activity. Design of a New Fluorogenic Substrate for CDR1 Characterization—Due to the unusual specificity requirements displayed by wtCDR1 a new substrate was required. Therefore, a three-dimensional model of CDR1 was generated (for details see “Experimental Procedures”). Scrutinizing the active site yielded no surprises revealing mostly hydrophobic residues forming binding pockets S2′ to S3. However, two rather interesting features are reminiscent of human BACE (22Hong L. Kim G. Lin X. Wu S. Terzyan S. Ghosh A.K. Zhang X.C. Tang J. Science. 2000; 290: 150-153Crossref PubMed Scopus (700) Google Scholar). These are the short proline-rich loop and Arg400 (Fig. 3), which reaches into the active site and might be capable of forming a salt bridge with a negatively charged glutamate residue in S4. A striking feature of wtCDR1 is its rather high pH optimum of 6.25–6.5 that is even higher than the value (5.0) found for human BACE (23Yan R. Kim M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1337) Google Scholar). At such a pH a glutamate should be deprotonated even at the interface between bulk water and protein interior. Considering size, shape, and physicochemical properties of the individual active site subpockets in modeled CDR1, we have designed a new peptide substrate, (MCA)Lys-Leu-His-Pro-Glu-Val-Leu-Phe-Val-Leu-Glu-Lys(DNP), which turned out to be efficiently cleaved by recombinant wtCDR1 and was subsequently used for enzyme characterization. The ionizable residues at each end of the peptide were introduced for solubility reasons. Mass spectrometry analysis of the peptide digestion products yielded a major fragment (m/z 1220.60) which originates from the sequence (MCA)Lys-Leu-His-Pro-Glu-Val-Leu-Phe, therefore suggesting that the peptide is cleaved at the Phe-Val bond. The pH and temperature dependence of the recombinant enzyme are shown in Fig. 4 and some obvious differences betw" @default.
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