Processing, stability, and kinetic parameters of C5a peptidase from Streptococcus pyogenes Elizabeth T. Anderson 1 , Michael G. Wetherell 1 , Laurie A. Winter 1 , Stephen B. Olmsted 1 , Patrick P. Cleary 2 and Yury V. Matsuka 1 1 Wyeth Research, West Henrietta, NY, USA; 2 Microbiology Department, University of Minnesota, Minneapolis, MN, USA A recombinant streptococcal C5a peptidase was expressed in Escherichia coli and its catalytic properties and thermal sta- bility were subjected to examination. It was shown that the NH 2 -terminal region of C5a peptidase (Asn32–Asp79/ Lys90) forms the pro-sequence segment. Upon maturation the propeptide is hydrolyzed either via an autocatalytic intramolecular cleavage or by exogenous protease strepto- pain. At pH 7.4 the enzyme exhibited maximum activity in the narrow range of temperatures between 40 and 43 °C. The process of heat denaturation of C5a peptidase investi- gated by fluorescence and circular dichroism spectroscopy revealed that the protein undergoes biphasic unfolding transition with T m of 50 and 70 °C suggesting melting of different parts of the molecule with different stability. Unfolding of the less stable structures was accompanied by the loss of proteolytic activity. Using synthetic peptides corresponding to the COOH-terminus of human comple- ment C5a we demonstrated that in vitro peptidase catalyzes hydrolysis of two His67-Lys68 and Ala58-Ser59 peptide bonds. The high catalytic efficiency obtained for the SQLRANISHKDMQLGR extended peptide compared to the poor hydrolysis of its derivative Ac-SQLRANISH-pNA that lacks residues at P2¢–P7¢ positions, suggest the import- ance of C5a peptidase interactions with the P¢ side of the substrate. Keywords: maturation; propeptide; streptopain; denatura- tion; substrate binding. Group A streptococcus (Streptococcus pyogenes)isa common human pathogen causing a wide variety of diseases. These include relatively mild pathological condi- tions such as pharyngitis and impetigo, more serious nonsuppurative sequelae, acute rheumatic fever, glomerulo- nephritis, deadly toxic shock syndrome and necrotizing fasciitis. S. pyogenes has developed complex and sophisti- cated molecular mechanisms that allow it to avoid human defenses. One of the important virulence factors of strep- tococci involved in such activity is an extracellular C5a peptidase [2]. Streptococcal C5a peptidase is a surface- associated subtilisin-like serine protease with an unusually restricted substrate specificity. The only known protein substrate hydrolyzed by C5a peptidase is human comple- ment fragment C5a [3,4]. C5a peptidase-generated cleavage within the COOH-terminal region of human C5a drastically reduces the ability of this anaphylatoxin to bind receptors on the surface of polymorphonuclear neutrophil leukocytes (PMNLs) and therefore abolishes its chemotactic activity [3]. It is believed that C5a peptidase plays an important role in bacterial colonization of the host by inhibiting the influx of PMNLs and impeding initial clearance of the strepto- cocci. C5a peptidase from S. pyogenes is encoded by the chromosomal scpA gene and consists of 1167 amino residues (Fig. 1A). C5a peptidase is first produced as a precursor, with an NH 2 -terminal 31 amino acid residue signal peptide responsible for exporting the protein across the membrane [2]. The length of C5a peptidase pro- sequence segment and the mechanism of its cleavage remain unknown. Homology modeling has shown that the catalytic domain of C5a peptidase contains the structurally con- served core typical of subtilases, but in addition contains a number of extra segments corresponding to various size inserts located in external loops. All inserts found in the C5a peptidase catalytic domain form a total of 216 additional amino acid residues relative to subtilisin BPN¢ [5]. The active site of C5a peptidase is located within the NH 2 -terminal half of its polypeptide chain and formed by catalytic residues Asp130, His193 and Ser512 (corresponding to Asp32, His64, and Ser221 in subtilisin BPN¢). Asn294 (Asn155 in subtilisin BPN¢) is involved in the formation of an oxyanion-hole and is critical for the catalytic activity of C5a peptidase [2,5]. The function of the COOH-terminal region of C5a peptidase, starting at residue 583 and representing half of the total polypeptide, is not known. The COOH-terminal extension of C5a peptidase is involved in association with the surface of S. pyogenes. This segment is comprised of four R1–R4 hydrophilic 17 amino acid Correspondence to Y. V. Matsuka, Department of Protein Chemistry, Wyeth Research, WV, 211 Bailey Road, West Henrietta, NY 14586-9728, USA. Fax: + 1 585 273 7515, Tel.: + 1 585 273 7565, E-mail: matsukay@wyeth.com Abbreviations: PMNL, polymorphonuclear neutrophil leukocytes; pNA, p-nitroanilide; T m , transition midpoint of denaturation; Pn,P2, P1, P1¢,P2¢,Pn¢, protease substrate residues accommodated by cor- responding Sn,S2,S1,S1¢,S2¢,Sn¢ subsites of the enzyme. The scissile peptide bond is located between the P1 and P1¢. Protease substrate residues and subsites of the enzyme substrate-binding site are desig- nated using the nomenclature of Schechter and Berger [1]. (Received 28 May 2002, revised 8 August 2002, accepted 15 August 2002) Eur. J. Biochem. 269, 4839–4851 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03183.x residues cell wall repeats, followed by the LPTTN motif, hydrophobic membrane-spanning region and a cytoplasmic tail [2]. The presence of the LPTTN motif that precedes the hydrophobic membrane-spanning region and cytoplasmic charged tail suggests covalent linkage of C5a peptidase to the peptidoglycan [6,7]. Despite the recognized role of C5a peptidase as an important streptococcal virulence factor [8,9], there is a lack of data on its biochemical and catalytic properties. Studies with synthetic peptides corresponding to the COOH- terminus of human C5a suggested that C5a peptidase generates a single cleavage site between histidine 67 and lysine 68 residues, resulting in the release of the KDMQLGR heptapeptide from the C5a fragment [4]. The identified His67-Lys68 peptide bond within human C5a represented the only known cleavage site for streptococcal C5a peptidase. Several proteins including human comple- ment C5, C3, human serum albumin, myosin, ovalbumin, and cytochrome c were tested as substrates for the C5a peptidase, but none of them underwent hydrolysis [3,4]. Such a highly restricted substrate specificity of C5a pepti- dase is in striking contrast to the broad specificity of well- studied bacterial serine proteases of the subtilisin family. Thus, it is of great interest to investigate the biochemical and catalytic properties of C5a peptidase. In the present study, we have focused on the mode of C5a peptidase maturation and determination of the exact borders of its pro-sequence region. We have also evaluated the thermal stability and kinetic parameters of C5a peptidase and the results are discussed with regard to the structural organization and biological activity. EXPERIMENTAL PROCEDURES Construction of expression vectors Wild-type C5a peptidase. The region of the scpA gene (bases 94–3112) encoding C5a peptidase amino acid residues 32–1038 (Fig. 1A) was produced by PCR ampli- fication using chromosomal DNA from S. pyogenes M1 strain 90–226 as a template. To clone the scpA gene, we designed the following forward 5¢-CCC GAA TTC AAT ACT GTG ACA GAA GAC ACT CCT GC-3¢ and reverse 5¢-CCC GGA TCC TTA TTG TTC TGG TTT ATT AGA GTG GCC-3¢ PCR primers. The forward primer incorpor- ated the EcoRI restriction site, while the reverse primer included a BamHI site (underlined). The reverse primer also incorporated a TAA stop codon immediately after the coding segment. The amplified PCR product was first ligated into TA cloning vector pCR2.1 (Invitrogen Corp.) and then subcloned into pTrc99a expression vector (Amer- sham Pharmacia Biotech) using the EcoRI and BamHI restriction sites. Incorporation of the EcoRI restriction site within the forward primer for subsequent ligation of the amplified scpA gene into pTrc99a expression vector yielded three NH 2 -terminal extra residues MEF that are not part of the natural protein. The resulting plasmid pTrc99a (wild- type C5a peptidase) was transformed into E. coli DH5a host cells for protein expression. S512A C5a peptidase mutant. Site-directed mutagenesis was performed using inverse PCR amplification [10] using expression plasmid pTrc99a (wild-type C5a peptidase) as a template. For this purpose we designed two PCR primers, so that they would abut each other in opposite orientations: 5¢-ACT GCT ATG TCT GCG CCA TTA G-3¢ (forward) and 5¢-TCC AGA AAG TTT GGC ATA CTT GTT GTT AGC C-3¢ (reverse). The forward primer contains GCT codon that replaced AGT to produce the desired SerfiAla mutation at position 512. The GCTfiAGT mutation also resulted in elimination of a SpeI restriction site. The inverse PCR was performed using Expand TM Long Template PCR System (Boehringer Mannheim Corp.). The resulting blunt ended PCR product was self-ligated and transformed into TOP10F¢ E. coli cells (Invitrogen Corp.). Clones were screened and selected for the presence of the desired mutation by loss of a SpeI restriction site. The presence of SerfiAla mutation at position 512 was also confirmed by sequencing the scpA gene. The resultant plasmid was digested with NcoIandBamHI restriction enzymes and isolated scpA gene containing GCTfiAGT mutation was then subcloned into a pTrc99a expression vector where a kanamycin resistance cassette had been inserted into the ampicillin gene. The plasmid pTrc99a (S512A C5a pepti- dase) was transformed into E. coli DH5a host cells for protein expression. Expression and purification of recombinant C5a peptidase proteins DH5a cells were grown overnight at 37 °C in HSY medium (10 m M potassium phosphate, pH 7.2, 20 gÆL )1 HySoy, Fig. 1. Schematic representation of C5a peptidase (panel A) and SDS/ PAGE analysis of purified recombinant C5a peptidase species (panel B). Panel A depicts location of the major regions of C5a peptidase. The signal sequence (presequence), catalytic triad residues, cell wall, membrane, and cytoplasmic segments are indicated. The recombinant C5a peptidase (residues Asn32 through Gln1038) expressed in E. coli is boxed. Panel B shows the relative mobility of isolated recombinant S512A mutant (lane 2) and the wild-type (lane 3) C5a peptidase species on 10–20% gradient gel. The outer lanes 1 and 4 in the gel contain molecular mass standards as indicated. 4840 E. T. Anderson et al. (Eur. J. Biochem. 269) Ó FEBS 2002 5gÆL )1 yeast extract, 10 m M NaCl). For expression of the wild-type C5a peptidase or S512A mutant, 100 lgÆmL )1 ampicillin or 50 lgÆmL )1 kanamycin, respectively, was incorporated into HSY medium. Overnight cultures were diluted 1 : 100 with fresh HSY medium, grown to D 600 ¼ 1.5, and induced with 3 m M isopropyl thio-b- D -galactoside. After 3 h of induction, DH5a cells were harvested by centrifugation and lysed by the freeze-thaw method. Isolated soluble fractions of bacterial lysate were sequentially fractionated with 50% and then 70% of ammonium sulfate. Material precipitated with 70% ammo- nium sulfate was collected by centrifugation, dissolved in 20 m M Tris, pH 8.5, 25 m M NaCl and dialyzed overnight at 4 °C against the same buffer. Dialyzed samples were diluted 1 : 1 (v/v) with 20 m M Tris, pH 8.5, 2 M urea, and applied to a Q-Sepharose ion exchange column (Amersham Phar- macia Biotech), equilibrated with 20 m M Tris, pH 8.5, 1 M urea. Material bound to the anion exchange resin was eluted with a linear gradient of NaCl and pH using 20 m M Tris, pH 7.0, 1 M Urea, 1 M NaCl buffer. Fractions containing C5a peptidase species were collected, pooled, and dialyzed against NaCl/Tris, pH 7.4. Purified recombinant C5a peptidase samples (wild-type enzyme and S512A mutant) were aliquoted and stored frozen at )20 °C. Protein concentration determination Protein concentrations were determined spectrophotomet- rically, using extinction coefficients (E 280, 0.1% )calculated from the amino acid composition. The extinction coefficients were estimated using the equation: E 280, 0.1% ¼ (5690 W + 1280Y + 120S-S)/M, where W, Y, and S-S represent the number of Trp and Tyr residues and disulfide bonds, respectively, and M represents the molecular mass [11,12]. Molecular masses of the proteins were calculated on the basis of their amino acid composition. The following molecularmassesandE 280, 0.1% values were obtained: wild-type C5a peptidase, 104.2 kDa and 0.92; S512A C5a peptidase, 109.9 kDa and 0.87. Alternatively, protein con- centrations were estimated using bicinchoninic acid assay [13] according to BCA protein assay kit instructions (Pierce Chemical Company). Concentration of streptococcal cys- teine protease was also determined using active-site titration with E64 (Roche Molecular Biochemicals) [14]. Titration of the cysteine protease active-sites was performed in NaCl/ Tris, pH 7.4, 10 m M dithiothreitol, using resorufin-labeled casein (Roche Molecular Biochemicals) as a substrate. SDS/PAGE SDS/PAGE was performed with the Bio-Rad electrophor- esis system (Bio-Rad Laboratories) using precast 10–20% gradient gels. All SDS-polyacrylamide gels in this study were stained with Coomassie Brilliant Blue R (Bio-Rad Laboratories) or Coomassie R-350 (Amersham Pharmacia Biotech) solutions. Amino terminal sequence analysis NH 2 -terminal sequence analysis was performed with an Applied Biosystems model 490 sequenator. The NH 2 - termini of the proteins and peptides were determined by direct sequencing for 10 or more cycles. Synthetic peptides Peptides corresponding to the COOH-terminal region of the human C5a fragment (VVASQLRANISHKDMQLGR, SQLRANISHKDMQLGR, and VVASQLRANISH) and NH 2 -terminal segment of the C5a peptidase (QTPDEAAE ETI and AEETIADDANDL) were synthesized as C-ter- minal amides on a Gilson AMS422 Multiple Peptide Synthesizer using Fmoc chemistry with pentrafluorophenyl amino acid active esters and a polyethylene glycol polysty- rene support with a 5-(4¢-Fmoc-aminomethyl-3¢-5¢-dimeth- oxyphenoxy)valeric acid linker. After synthesis, peptides were purified by reverse-phase HPLC and lyophilized. Homogeneity of synthesized peptides was assessed by NH 2 - terminal sequence and mass spectral analysis. Peptide solutions of known concentration were prepared by weigh- ing and dissolving purified lyophilized peptide in a known volume of distilled water to give concentrated stock solutions. Two chromogenic p-nitroanilide (pNA) peptide derivatives were used in this study: the Ac-SQLRANISH- pNA was custom synthesized by New England Peptide, Inc. and the Suc-AAPF-pNA was obtained from Sigma. To prepare concentrated stock solutions, the Ac-SQLRAN ISH-pNA was dissolved in distilled water and Suc-AAPF- pNA was dissolved in dimethylsulfoxide. Mass spectral analysis Determination of the molecular masses of proteins and peptides was performed using MALDI-TOF mass spectro- meter Voyager DE-STR (Perseptive Biosystems). Ions formed by laser desorption at 337 nm (N 2 laser) were recorded at an acceleration voltage of 20 kV in the linear mode for proteins and 25 kV in the reflector mode for peptides. In general, 200 single spectra were accumulated for improving the signal/noise ratio and analyzed by use of the DATA EXPLORER software supplied with the spectrometer. Sinapinic acid and a-cyano-4-hydroxycinnamic acid were used as ultraviolet-absorbing matrices for proteins and peptides, respectively. 1 lLofa10-mgÆmL )1 solution of the matrix compounds in 70% acetonitrile/0.1% trifluoroacetic acid was mixed with 1 lL analyte solution (5–10 pmolÆ lL )1 ). For MALDI-TOF MS, 1 lL of this mixture was spotted on a stainless steel sample target and dried at room temperature. The mass spectra were calibrated using external standards: serum albumin (bovine), Glu1-fibrino- peptide B (human), angiotensin I (human), and des-Arg1- bradykinin (synthetic). The mass accuracy was in the range of 0.1%. Hydrolysis of protein and peptide substrates Treatment of the S512A C5a peptidase precursor with C5a peptidase was performed at 25 °C in NaCl/Tris, pH 7.4, 5m M CaCl 2 . For this purpose 33 l M of the S512A C5a peptidase precursor was incubated with 3.3 l M of wild-type C5a peptidase resulting in an enzyme/substrate ratio of 1:10( M / M ). Proteolysis of S512A C5a peptidase precursor (50 l M ) with streptococcal cysteine protease (1.8 l M )was performed at 25 °C in NaCl/Tris, pH 7.4, 10 m M dithio- threitol at enzyme/substrate ratio of 1 : 25 ( M / M ). Samples from each reaction mixture were removed at 15, 30, 45, 60, 120, 240, 360, and 1320 min, mixed with SDS, heated and Ó FEBS 2002 Enzymology of C5a peptidase (Eur. J. Biochem. 269) 4841 later analyzed by SDS/PAGE using 10–20% gradient gel. Streptococcal cysteine protease or streptopain (EC 3.4.422.10) was prepared as described elsewhere [15]. Operational molarity of cysteine protease preparations used in this study corresponded to 98% of that expected on a protein concentration basis. The specificity of streptopain catalyzed cleavage was confirmed using the specific cysteine protease inhibitor E64 (Roche Molecular Biochemicals). The NH 2 -terminal truncation of S512A C5a peptidase precursor by streptococcal cysteine protease was completely blocked in the presence of 20 l M E64. Caseinolytic activity of C5a peptidase was evaluated using resorufin-labeled casein (Roche Molecular Biochemi- cals). Briefly, increasing amounts of wild-type C5a pepti- dase, S512A C5a peptidase mutant, and subtilisin from Bacillus subtilis (EC 3.4.21.14) (Fluka) ranging from 0 to 10 lg were incubated for 60 min at 37 °C in the presence of 0.4% resorufin-labeled casein in NaCl/Tris, pH 7.8, 5 m M CaCl 2 . Undigested substrate was removed by 5% trichlo- roacetic acid precipitation, and followed by centrifugation the absorbance of released resorufin-labeled peptides in the supernatant fractions was measured spectrophotometrically at 574 nm. The C5a peptidase-catalyzed hydrolysis of the 19-mer synthetic peptide VVASQLRANISHKDMQLGR was performed at 25 °C in NaCl/Tris, pH 7.4, 5 m M CaCl 2 . Incubation of 345 l M VVASQLRANISHKDMQLGR peptide with 0.28 l M of C5a peptidase was carried out for 5, 10, 15, 20, 30, 40, 60, 80, 100, 120, and 140 min and reactions were terminated by the addition of trifluoroacetic acid to 0.05%. At each time point, the presence of specific peptide in reaction mixture was monitored at 210 nm by reverse-phase HPLC (Hewlett Packard model 1090 Liquid Chromatograph) using C4 reverse-phase column (Vydac). Buffer A was 0.1% trifluoroacetic acid in distilled water, and buffer B was 0.1% trifluoroacetic acid in 100% acetonitrile. Peptides were eluted with 0–40% linear gradi- ent of Buffer B during a 20-min interval. The relative amount of each peptide in the reaction mixture was determined using the area beneath the peak corresponding to this peptide and then plotted as a function of time. The identity of peptides was determined by NH 2 -terminal sequence- and mass spectral analysis. Treatment of the QTPDEAAEETI and AEETIADD ANDL synthetic peptides with C5a peptidase was per- formed either at 25 or 37 °C in NaCl/Tris, pH 7.4, 5 m M CaCl 2 and in 100 m M Tris, pH 8.6, 5 m M CaCl 2 .Eachof these peptides (100 or 200 l M ) was incubated with 0.1 or 1 l M of C5a peptidase for 1, 18, and 71 h. Reaction mixtures were analyzed using reverse-phase HPLC as described above. At tested conditions, cleavage of the QTPDEAAEETI and AEETIADDANDL peptides was not detected. Effect of temperature on the proteolytic activity of C5a peptidase was evaluated using 19-mer synthetic peptide VVASQLRANISHKDMQLGR. At each tested tempera- ture, 0.1 l M of the C5a peptidase in NaCl/Tris, pH 7.4 was preincubated for 3 min. The reactions were started by addition of 200 l M of the 19-mer peptide to the preincu- bated solution of C5a peptidase followed by another 10-min incubation at the same temperature. After termination of hydrolysis with 0.05% trifluoroacetic acid, the reaction mixtures were analyzed using HPLC as described above. The percentage of hydrolyzed peptide substrate (S hydr %) was determined using the equation: S hydr % ¼ P/(P +S), where P represents area of the product peaks and S represents area of the uncleaved substrate peak. Assays revealing the pH dependence of the hydrolysis of VVASQLRANISHKDMQLGR peptide were performed at 25 °Cin100m M NaAc (pH 4.5–5.0), 100 m M Mes (pH 5.5–6.5), 100 m M Hepes (pH 7.0–8.0), 100 m M Tris (pH 7.0–9.0), 100 m M Ampso (pH 8.5–9.5), 100 m M Caps (pH 10.0–11.0). At each tested pH, 0.1 l M of C5a peptidase was incubated with 200 l M of the 19-mer peptide for 10 min followed by HPLC analysis. The percentage of hydrolyzed peptide was determined and plotted as a function of pH. Hydrolysis of the peptide substrate in both temperature and pH dependence experiments did not exceed 25%. The effect of pH on hydrolysis of pNA substrate Ac-SQLRANISH- pNA was determined at 25 °C in 100 m M Mes (pH 6.0–6.5), 100 m M Hepes (pH 7.0–8.0), 100 m M Tris (pH 7.0–9.0), 100 m M Ampso (pH 8.5–9.5), 100 m M Caps (pH 10.0– 11.0). At each tested pH, 200 l M of the pNA peptide substrate was incubated in quartz cell for 60 min either alone or in the presence of 1 l M of C5a peptidase while monitoring hydrolysis by measurement of absorbance at 405 nm using Spectronic Genesis 2 Spectrophotometer (Spectronic Instruments, Inc.). Kinetic measurements All kinetic data were obtained by incubating various concentrations of peptide with a constant enzyme concen- tration to achieve between 5 and 20% cleavage of the substrate in each reaction. The concentration of C5a peptidaseineachreactionwas0.1l M , while peptide concentrations ranged from 50 l M to 600 l M (16-mer SQLRANISHKDMQLGR) and from 50 l M to 2000 l M (12-mer VVASQLRANISH). Concentration of C5a pepti- dase in each reaction was at least 500-fold lower than the lowest substrate concentration. All reactions were per- formed at 25 °C in NaCl/Tris, pH 7.4, 5 m M CaCl 2 . Reactions were carried out for 5 or 100 min with 16-mer and 12-mer peptide, respectively, and stopped by the addition of trifluoroacetic acid to 0.05%. Cleavage of peptides by C5a peptidase was monitored at 210 nm by reverse-phase HPLC and percentage of hydrolyzed peptide was determined as described above. Initial velocities (V) were determined and plotted against substrate concentra- tion [S]. The data were fitted to the Michaelis–Menten equation V ¼ V max [S]/(K m + [S]) with a nonlinear regres- sion analysis program. The best fits of the data produced V max and K m values, where V max represents the maximum rate of hydrolysis and K m is the Michaelis constant. The turnover number (k cat ) values were calculated from V max /[E], where [E] represents enzyme concentration. The identity of hydrolyzed peptide fragments was determined by NH 2 - terminal sequence and mass spectral analysis. Kinetic studies of C5a peptidase using chromogenic pNA substrate Ac-SQLRANISH-pNA were performed with enzyme present at concentrations between 0.01 and 0.59 l M . The concentration of Ac-SQLRANISH-pNA was varied from 50 to 2000 l M . Reactions were performed at 25 °Cin100m M Tris, pH 8.6, 5 m M CaCl 2 buffer. Assays were carried out in 1-cm path length quartz cells and reaction rates were monitored by continuous measurement 4842 E. T. Anderson et al. (Eur. J. Biochem. 269) Ó FEBS 2002 of absorbance at 405 nm for 180–900 min using a Spec- tronic Genesys 2 Spectrophotometer (Spectronic Instru- ments, Inc.). The concentration of released p-nitroaniline product was estimated based on the molar absorption coefficient e 405 ¼ 10500 M )1 Æcm )1 .TheK m value of C5a peptidase hydrolysis of Ac-SQLRANISH-pNA was too high (K m  [S]) for accurate measurement. Therefore, the k cat and K m constants were not individually determined. The specificity constant k cat /K m for hydrolysis of Ac-SQLRAN ISH-pNA was determined using equation: k cat /K m ¼ V/([E]Æ[S]). During extended kinetic runs, no detectable loss of catalytic activity of the C5a peptidase was observed. The background (nonenzymatic) hydrolysis of Ac-SQLRAN ISH-pNA was evaluated by incubating blank substrate solutions. At tested conditions, nonenzymatic hydrolysis was not detectable. Fluorescence measurements Thermal unfolding was monitored by observing the change in ratio of the intrinsic fluorescence intensity at 350 nm to that at 320 nm with excitation at 280 nm [16] in an SLM Aminco-Bowman Series 2 spectrofluorometer. Temperature was controlled with circulating water bath programmed to raise the temperature at 1 °CÆmin )1 andmonitoredwith Omega DP81 thermocouple probe inserted into a dummy cuvette. All fluorescence measurements were performed at protein concentration ranging from 0.04 to 0.05 mgÆmL )1 . Circular dichroism measurements CD spectra were recorded on a Jasco J-810 spectropola- rimeter equipped with a Peltier PTC-423S/L unit for temperature control. CD measurements were performed in 20 m M NaCl/P i , pH 7.4 using protein concentration of 0.2 mgÆmL )1 in 0.1 cm path length cells. Spectra were recorded at 25 and 98 °C. Four scans were accumulated per each spectrum. Spectra were averaged and expressed as mean residue ellipticity [Q], in units of degreesÆcm 2 Ædmol )1 . Thermal denaturation was monitored by changes in ellip- ticity at 205 nm while heating cell at 1 °CÆmin )1 . RESULTS Preparation of recombinant C5a peptidase and analysis of its NH 2 -terminal truncation The recombinant wild-type C5a peptidase comprising residues Asn32 through Gln1038 (Fig. 1A) was produced in DH5a E. coli cells using pTrc99A expression vector as described in Experimental procedures. During isolation of C5a peptidase from E. coli lysate, its mobility on SDS/ PAGE was slightly but progressively increasing, indicating possible proteolytic degradation. Purified recombinant C5a peptidase exhibited a single band on SDS/PAGE with a relative mobility close to its expected molecular mass (Fig. 1B, lane 3). However, all preparations of freshly isolated wild-type C5a peptidase consistently displayed truncated NH 2 -terminus starting at Ala72 and suggesting the loss of 43 amino acid residues. Subsequent analysis of the same protein samples stored either at 4 °Corsamples that underwent freeze-thaw cycle(s) revealed NH 2 -terminal sequence starting at Asp79, indicating the loss of a total of 50 amino acid residues. This observation was reproducible, suggesting that the Asp79 residue represented a final point of progressive NH 2 -terminal truncation of the wild-type C5a peptidase. The NH 2 -terminal cleavage of the wild-type C5a peptidase may be caused by E. coli proteases, or alternatively might be a result of autocatalytic cleavage (maturation) reaction. In order to investigate the nature of the C5a peptidase truncation, we expressed in E. coli a mutated and enzymatically inactive form of C5a peptidase. Based on homology analysis of subtilisin family of serine proteases, it was reported earlier that Ser residue at position 512 is involved in the formation of catalytic site of C5a peptidase [2,5]. When S512A C5a peptidase mutant was expressed in DH5a E. coli cells using pTrc99A vector and isolated using the same procedure used for purification of the wild-type enzyme, sequence analysis revealed the presence of an intact NH 2 -terminus starting at MEFNTV TEDT. The three NH 2 -terminal extra residues, MEF, are not part of the natural protein and originated from the cloning strategy as described in Experimental procedures. Electrophoretic mobility of S512A C5a peptidase mutant was slightly decreased compared to that of the wild-type enzyme (Fig. 1B, lane 2) consistent with the presence of an extra 50 amino acid residues. Since both proteins were produced using the same expression vectors, host cells, and isolated using the same purification procedure, it is highly unlikely that the NH 2 -terminus of the wild-type form but not that of the S512A mutant was cleaved by E. coli proteases upon protein expression and subsequent isolation. Given the absence of NH 2 -terminal truncation in the S512A mutant, these results indicate that C5a peptidase undergoes autocatalytic processing resulting in cleavage of its 50 amino acid residue propeptide segment. To further investigate the mechanism of NH 2 -terminal autocatalytic processing, we incubated S512A C5a peptidase mutant in the presence of the wild-type enzyme. Upon treatment of the S512A C5a peptidase precursor with the wild-type C5a peptidase, there was no evidence for NH 2 -terminal truncation (Fig. 2A). Similarly, no cleavage was detected upon incubation of the wild-type C5a peptidase with synthetic peptides corres- ponding to its propeptide region. These experiments were performed with QTPDEAAEETI (Gln67–Ile76) and AEETIADDANDL (Ala72–Leu83) overlapping peptides containing Glu71–Ala72 and Asp78–Asp79 autocatalytic cleavage sites, respectively. Peptides were incubated with, or without C5a peptidase and followed by HPLC monitoring using a C4 reverse phase column (not shown). Failure of the C5a peptidase to cleave the propeptide segment of S512A C5a peptidase precursor or synthetic peptides correspond- ing to its propeptide region and containing autocatalytic cleavage sites indicates that autoprocessing proceeds via an intramolecular route. In this study we also investigated the role of secreted streptococcal cysteine protease in the maturation of C5a peptidase precursor. Streptococcal cysteine protease, or streptopain, is an extracellular thiol endopeptidase pro- duced by Streptococcus pyogenes [17,18]. Cysteine protease has been shown to release biologically active fragments from the bacterial surface such as M protein, protein H and C5a peptidase [19,20]. Released by streptopain, the 116 kDa fragment of C5a peptidase inhibited granulocyte migration into the infectious site, and therefore exhibited characteristic peptidase activity [20]. The ability of streptopain to release Ó FEBS 2002 Enzymology of C5a peptidase (Eur. J. Biochem. 269) 4843 an active C5a peptidase fragment from the surface of streptococci makes this secreted protease an interesting candidate for evaluation of its role in processing of C5a peptidase precursor. To test this hypothesis, the S512A C5a peptidase precursor was incubated in the presence of streptococcal cysteine protease and analyzed by SDS/ PAGE (Fig. 2B). Upon incubation, the band corresponding to C5a peptidase precursor steadily increased its mobility on SDS/PAGE resulting in the appearance of higher mobility truncated specie. The truncated form of S512A C5a peptidase was a terminal product of proteolysis, since no further degradation was observed even after prolonged incubation with cysteine protease (Fig. 2B). After treatment with streptococcal cysteine protease, the S512A C5a pep- tidase exhibited an NH 2 -terminal sequence starting at Lys90 (KTADTPATSK) suggesting the cleavage of 61 amino acids from its NH 2 -terminus. The same NH 2 -terminal sequence was found in C5a peptidase released from the surface of Streptococcus pyogenes by the action of secreted streptococcal cysteine protease [20]. These data suggest that in addition to COOH-terminal cleavage of C5a peptidase, resulting in the release of the anchored enzyme, streptococ- cal cysteine protease is involved in the maturation of C5a peptidase precursor. Thus, the NH 2 -terminal segment comprising of 47–58 amino acid residues forms the pro- sequence peptide region of C5a peptidase. Cleavage of the pro-sequence peptide and maturation of C5a peptidase precursor is realized via an intramolecular autoprocessing mechanism. Alternatively, processing of C5a peptidase precursor can be achieved by an exogenous protease streptopain. Proteolytic activity and thermal stability of the C5a peptidase The earlier reported highly restricted substrate specificity of C5a peptidase for human C5a fragment was further investigated in this study. Proteolytic activity of C5a peptidase was tested using resorufin-labeled casein. In the control reaction, treatment of resorufin-casein with increas- ing amounts of subtilisin was accompanied by a dose- dependant increase of absorbance at 574 nm indicating effective cleavage of the substrate. In contrast, incubation of resorufin-labeled casein with increasing amounts of wild- type or S512A C5a peptidase mutant resulted in absence of detectable hydrolysis (Fig. 3A). Cleavage of casein was not detected even upon prolonged incubation with high con- centrations of C5a peptidase. These results suggest that C5a peptidase does not exhibit caseinolytic activity typical for classical subtilisins. The results also indicate the absence of contaminating E. coli proteases in the C5a peptidase preparations. To gain insight into the mode of C5a peptidase catalysis, we examined cleavage of synthetic peptide corresponding to the COOH-terminus of the human complement fragment C5a. First, we tested the 19-mer VVASQLRANISHKDMQLGR synthetic peptide contain- ing previously described His67-Lys68 cleavage site [4]. This peptide was incubated alone and in the presence of either wild-type C5a peptidase or S512A C5a peptidase mutant. Incubation with the S512A C5a peptidase mutant did not result in detectable cleavage of the peptide. In contrast, treatment of the 19-mer peptide with the wild-type C5a peptidase resulted in progressive hydrolysis of the substrate (Fig. 3B). As seen from Fig. 4A and B, incubation of the 19-mer peptide with C5a peptidase produced several smaller peptide products suggesting the presence within the tested substrate of more than one cleavage site. The products of hydrolysis were examined by mass spectroscopy and NH 2 - terminal sequence analysis, and the exact positions of cleavage sites were identified. Results of both mass spectral and NH 2 -terminal sequence analysis suggested that in addition to the earlier reported cleavage site His67-Lys68 [4], C5a peptidase also hydrolyzed the peptide bond between Ala58-Ser59. Time course digestion of the 19-mer peptide monitored by HPLC revealed that the first cleavage occurs between His67-Lys68, resulting in the formation of VVASQLRANISH and KDMQLGR peptides. Gradual depletion of the initial VVASQLRANISHKDMQLGR substrate and accumulation of VVASQLRANISH product was accompanied by detection of the second cleavage between Ala58-Ser59, resulting in production of SQLR ANISH and presumably VVA (Fig. 4C). Upon elution from reverse-phase column; peaks corresponding to VVA peptide product were not detected, probably as a result of the precipitation of the highly hydrophobic VVA product followed by its release from the parent VVASQLRANISH peptide. To assess the thermal stability of C5a peptidase, we investigated the effect of temperature on both proteolytic activity and structural integrity of the enzyme (Fig. 5). All experiments were performed at neutral pH, where C5a peptidase exhibited maximum activity towards peptide substrate (Fig. 5A, inset). As illustrated in Fig. 5A, raising the temperature from 5 °Cto40°Cresultedina10-fold increase of the relative proteolytic activity of C5a peptidase Fig. 2. Incubation of S512A C5a peptidase precursor with wild-type C5a peptidase (panel A) and streptococcal cysteine protease (panel B) ana- lyzed by SDS/PAGE using 10–20% gradient gel. Lanes 0 contain the starting material. Lanes 15, 30, 45, 60, 120, 240, 360, and 1320 repre- sent increasing times of incubation. The outer lanes in the gels contain molecular mass standards as indicated. Arrows show relative mobility of S512A C5a peptidase bands after incubation with wild-type C5a peptidase (panel A) or streptococcal cysteine protease (panel B). 4844 E. T. Anderson et al. (Eur. J. Biochem. 269) Ó FEBS 2002 towards the 19-mer peptide. Maximum activity of C5a peptidase was observed in the narrow range of temperatures between 40 and 43 °C. A further increase in temperature caused a sharp decline in C5a peptidase activity and subsequently its complete inactivation at 60 °C. Heat- induced unfolding of the C5a peptidase was studied using fluorescence and circular dichroism spectroscopy (Fig. 5B,C,D). Figure 5B presents a melting curve obtained by heating C5a peptidase while monitoring the ratio of fluorescence intensity at 350 nm to that at 320 nm as a measure of the spectral shift that accompanies unfolding. At neutral pH, in response to heating, the protein exhibited a high magnitude sigmoidal denaturation transition with a midpoint (T m )of50°C. The midpoint of the less pro- nounced second transition was observed at 70 °C. The biphasic nature of denaturation curve suggested that the compact structure of C5a peptidase is formed by at least two Fig. 3. Treatment of casein-resorufin (panel A) and 19-mer synthetic peptide VVASQLRANISHKDMQLGR (panel B) with recombinant C5a peptidase species. Casein-resorufin or 19-mer peptide substrate were incubated in the presence of the wild-type C5a peptidase (empty squares) and S512A C5a peptidase mutant (filled diamonds). Subtilisin from B. subtilis (filled squares) was used as a positive control in caseinolytic experiments. Fig. 4. HPLC analysis of C5a peptidase-catalyzed cleavage of 19-mer synthetic peptide VVASQLRANISHKDMQLGR. The19-mer peptide (345 l M ) was incubated in the absence (panel A) or presence (panel B) of C5a peptidase (0.28 l M ) in NaCl/Tris, pH 7.4, 5 m M CaCl 2 for 120 min at 25 °C. The products of enzymatic hydrolysis were identified by NH 2 -terminal sequence and mass spectral analysis. Peaks corres- ponding to each peptide are labeled as peak 1 – (filled diamonds) VVASQLRANISHKDMQLGR, peak 2 – (empty triangles) VVASQLRANISH, peak 3 – (empty diamonds) SQLRANISH, and peak 4 – (empty squares) KDMQLGR. The VVA product of hydro- lysis was not recovered from the reaction mixture. Accumulation of peptide products in reaction mixture was monitored and plotted as a function of incubation time (panel C). Ó FEBS 2002 Enzymology of C5a peptidase (Eur. J. Biochem. 269) 4845 domains with different stability. Melting of C5a peptidase in the presence of 5 m M CaCl 2 did not affect denaturation profile and transition midpoints (Fig. 5C, curve a). The addition of 2 m M EDTA and heating under these condi- tions again produced a biphasic denaturation curve with a high amplitude first transition and low amplitude second transition. The T m of the major transition, however, was shifted about 6 °C to lower temperature (Fig. 5C, curve b), suggesting that the compact structure of C5a peptidase in the presence of EDTA was destabilized. Heat induced denaturation data obtained in the presence or absence of EDTA demonstrated that C5a peptidase contains high affinity metal-binding site(s) that is (are) presumably occupied. Circular dichroism spectroscopy measurements revealed that C5a peptidase has a spectrum in the far UV region that exhibits characteristic positive band at 194 nm and negative bands at 210 nm and 215 nm. Heating of C5a peptidase up to 98 °C abolished these features (Fig. 5D inset). Monitoring of the ellipticity at 205 nm during heating produced a sigmoidal biphasic transition curve indicative of cooperative unfolding for a multidomain protein (Fig. 5D). Again, the midpoints for low and high temperature transitions were observed near 50 and 70 °C, respectively. Results of denaturation experiments are consistent with peptidase activity data and suggest that the decrease of activity of C5a peptidase at temperatures above 43 °Cis associated with the beginning of thermal unfolding. Kinetic parameters of C5a peptidase: hydrolysis of peptide and p NA substrates In order to investigate the kinetic parameters for peptide cleavage by C5a peptidase, we synthesized two derivatives of the 19-mer parent peptide. These include the 16-mer SQLRANISHKDMQLGR and the 12-mer VVASQLR ANISH peptides. Based on the data obtained with the 19- mer peptide, each of these peptides contains a single potential cleavage site, and therefore can be easily utilized for kinetic studies. This was confirmed by HPLC assay with subse- quent mass-spectroscopic evaluation of generated products (Figs 6A,B and 7A,B). To determine kinetic constants for the hydrolysis of peptides by C5a peptidase, time course experiments using different substrate concentrations were performed in NaCl/Tris, pH 7.4, 5 m M CaCl 2 at 25 °C. In each case, the initial velocity of hydrolysis of the peptide bond was obtained. Using nonlinear regression analysis, these data were fitted to the Michaelis–Menten equation to yield V max and apparent K m values (Figs 6C and 7C). The kinetic parameters for C5a peptidase-catalyzed cleavage of 16- and 12-mer synthetic peptides corresponding to the COOH-terminus of human complement fragment C5a are summarized in Table 1. As can be seen from Table 1, C5a peptidase hydrolyzes the 16-mer peptide with catalytic efficiency (k cat /K m ) about 14-fold higher than the 12-mer. Fig. 5. Effect of temperature on proteolytic activity of C5a peptidase (panel A) and heat-induced unfolding of C5a peptidase detected by fluorescence (panels B, C) and circular dichroism (panel D) spectroscopy. Panel A shows relative proteolytic activity of C5a peptidase at various temperatures in NaCl/Tris, pH 7.4. Reactions were performed using 19-mer synthetic peptide VVASQLRANISHKDMQLGR as a sub- strate. The pH dependence of C5a peptidase relative activity is shown in the inset. Reactions were performed at 25 °C in 100 m M NaAc (pH 4.5–5.0), 100 m M Mes (pH 5.5–6.5), 100 m M Hepes (pH 7.0–8.0), 100 m M Ampso (pH 8.5–9.5), 100 m M Caps (pH 10.0–11.0) (filled squares) and 100 m M Tris (pH 7.0–9.0) (empty squares). Panel B illustrates fluorescence-detected thermal denaturation of C5a peptidase in NaCl/Tris, pH 7.4. Fluorescence-detected melting curves of C5a peptidase in NaCl/Tris, pH 7.4, 5 m M CaCl 2 (a) and NaCl/Tris, pH 7.4, 2 m M EDTA (b) are presented in panel C. Protein solutions were heated while monitoring the ratio of fluorescence at 350 nm to that at 320 nm with excitation at 280 nm. Panel D shows changes in ellipticity at 205 nm upon heating of C5a peptidase sample; the CD spectra of C5a peptidase at 25 and 98 °Carepresentedintheinset.All CD measurements were performed in NaCl/P i ,pH7.4. 4846 E. T. Anderson et al. (Eur. J. Biochem. 269) Ó FEBS 2002 This is consistent with our data generated using 19-mer parent peptide that contains both cleavage sites (Fig. 4C). The low efficiency of hydrolysis of the 12-mer peptide compared to its 16-mer counterpart was a result of a sixfold reduction in k cat value and threefold increase in K m value. When 5 m M EDTA was included into reaction buffer, hydrolysis of both 16-mer and 12-mer peptides was significantly inhibited (Figs 6C and 7C), again suggesting existence of metal-binding site(s) within C5a peptidase. Based on the sequence of 16-mer SQLRANISHKDMQ LGR peptide, we designed a water-soluble chromogenic pNA substrate, Ac-SQLRANISH-pNA. Incubation of Fig. 6. HPLC analysis of C5a peptidase-catalyzed cleavage of 16-mer synthetic peptide SQLRANISHKDMQLGR. The 16-mer peptide (410 l M ) was incubated in the absence (panel A) or presence (panel B) of C5a peptidase (0.28 l M ) for 120 min. The products of enzymatic hydrolysis were identified by NH 2 -terminal sequence and mass spectral analysis. Peaks corresponding to each peptide are labeled as peak 1 – SQLRANISHKDMQLGR, peak 2 – SQLRANISH, and peak 3 – KDMQLGR. Initial rate of hydrolysis V was plotted vs. the concen- tration of the substrate SQLRANISHKDMQLGR [S] (panel C). Experiments were performed in NaCl/Tris, pH 7.4, containing either 5m M CaCl 2 (filled squares) or 5 m M EDTA (empty squares) at 25 °C. Fig. 7. HPLC analysis of C5a peptidase-catalyzed cleavage of 12-mer synthetic peptide VVASQLRANISH. The12-mer peptide (550 l M )was incubated in the absence (panel A) or presence (panel B) of C5a peptidase (0.28 l M ) for 360 min. The products of enzymatic hydrolysis were identified by NH 2 -terminal sequence and mass spectral analysis. Peaks corresponding to each peptide are labeled as peak 1 – VVASQLRANISH, and peak 2 – SQLRANISH. The VVA product of hydrolysis was not recovered from the reaction mixture. Initial rate of hydrolysis V was plotted vs. the concentration of the substrate VVASQLRANISH [S] (panel C). Experiments were performed in NaCl/Tris, pH 7.4, containing either 5 m M CaCl 2 (filled squares) or 5m M EDTA (empty squares) at 25 °C. Ó FEBS 2002 Enzymology of C5a peptidase (Eur. J. Biochem. 269) 4847 Ac-SQLRANISH-pNA in the presence of C5a peptidase was accompanied by increase of absorbance at 405 nm, suggesting enzymatic release of p-nitroaniline. The linear dependence of Ac-SQLRANISH-pNA cleavage with enzyme concentration is demonstrated in Fig. 8. In contrast, upon incubation of C5a peptidase with Suc-AAPF-pNA, a substrate commonly used for kinetic analysis of subtilisins [21,22], hydrolysis was not detected. This observation is consistent with limited substrate specificity of C5a peptidase and further illustrates significant differences in the organ- ization of its substrate-binding site compared to that of the classical subtilisins. The pH-dependence of the hydrolysis of Ac-SQLRANISH-pNA reveals the optimum activity of C5a peptidase in the alkaline region (pH 8.5–9.5) (Fig. 8, inset). At pH 8.6 activity of C5a peptidase towards Ac-SQLRANISH-pNA was about 60% higher than that observed at pH 7.4. Analysis of Ac-SQLRANISH-pNA cleavage by C5a peptidase revealed that estimated Michaelis constant value is too high and exceeds maximum substrate concentration (2 m M ) used in the experiments and therefore prevents accurate determination of individual kinetic parameters. Instead, the specificity constant k cat /K m was determined directly. Specificity of C5a peptidase towards Ac-SQLRANISH-pNA was only 13 M )1 Æs )1 (Table 1). This value is about 230-fold lower than that obtained for the parent SQLRANISHKDMQLGR extended peptide sub- strate. Thus, substitution of the lysine residue at P1¢ position to pNA moiety and the lack of residues at the P2¢ through P7¢ positions, resulting in a drastic reduction of catalytic efficiency, indicate the importance of C5a peptidase inter- actions with the P¢ side of the substrate. DISCUSSION In Gram-positive bacteria, extracellular proteases are syn- thesized initially as inactive precursors containing an amino- terminal extension that is composed of the signal peptide and propeptide. The signal sequence, or prepeptide, is involved in translocation of precursor through the cyto- plasmic membrane. One of the major functions of the pro- sequence region is to prevent unwanted protein degradation and to enable spatial and temporal regulation of proteolytic activity. The pro-sequence region associates to the protease module, thus preventing access of substrate(s) to the active site [23]. Zymogen conversion to the active enzyme occurs by limited proteolysis of the inhibitory pro-sequence segment and may be either autocatalytic or involve acces- sory molecules. The length of propeptide may vary and range from short polypeptide segments to independently folded domains comprising more than 100 residues [24,25]. Often the precise length of the mature, active enzyme is not known due to the fact that the NH 2 -terminal processing site(s) has not been mapped. Such information was not available for C5a peptidase from pathogenic Streptococcus pyogenes. One of the aims of this study was to map the pro- sequence region of C5a peptidase and to investigate the mechanism(s) of its maturation. Recombinant wild-type C5a peptidase and its S512A mutant, both lacking NH 2 - terminal signal sequence and COOH-terminal membrane anchor sequence (Asn32 – Gln1038), were overexpressed in E. coli and isolated from the soluble fraction of cell lysate. Mobility of purified S512A C5a peptidase mutant was slightly decreased on SDS/PAGE compared to that of the wild-type enzyme, suggesting partial proteolytic degrada- tion of the latter. Sequence analysis of wild-type C5a peptidase confirmed the loss of its NH 2 -terminal 50 amino residues, while the enzymatically inactive S512A mutant Table 1. Kinetic parameters for the hydrolysis of peptide and pNA substrates by C5a peptidase. The arrow (fl) represents location of scissile bond. ND, not determined. The standard errors of the given k cat and K m values did not exceed 20%. Kinetic constants for hydrolysis of peptide substrates were obtained in NaCl/Tris, pH 7.4, 5 m M CaCl 2 . Specificity constant for hydrolysis of pNA substrate was obtained in 100 m M Tris/HCl, pH 8.6, 5m M CaCl 2 . Substrate (Pn…, P2, P1 fl P1¢,P2¢,… Pn¢) k cat (s )1 ) K m (l M ) k cat /K m ( M )1 Æs )1 ) SQLRANISH fl KDMQLGR 1.1 360 3050 VVA fl SQLRANISH 0.2 936 216 Ac-SQLRANISH fl pNA ND >2000 13 Fig. 8. Incubation of Ac-SQLRANISH-pNA and Suc-AAPF-pNA with different amounts of C5a peptidase. Reactions were carried out at 25 °C for 180 min in 100 m M Tris, pH 8.6, 5 m M CaCl 2 in the presence of 220 l M Ac-SQLRANISH-pNA and in 100 m M Tris, pH 8.6, 5 m M CaCl 2 (containing 2% dimethylsulfoxide) in the presence of 400 l M Suc-AAPF-pNA. The pH-activity profile of C5a peptidase for Ac-SQLRANISH-pNA is shown in the inset. Reactions were per- formedin100m M Mes (pH 6.0–6.5), 100 m M Hepes (pH 7.0–8.0), 100 m M Ampso (pH 8.5–9.5), 100 m M Caps (pH 10.0–11.0) in the presence (filled squares) or absence (circles) of the enzyme. Reactions were also carried out in the presence (empty squares) or absence (tri- angles) of C5a peptidase in 100 m M Tris (pH 7.0–9.0). 4848 E. T. Anderson et al. (Eur. J. Biochem. 269) Ó FEBS 2002 [...]... preparations of wild-type C5a peptidase and S512A mutant consistently displayed truncated and intact NH2-termini, respectively The observed truncation of wild-type C5a peptidase suggested the existence of an autocatalytic processing reaction since substitution of the reactive serine at position 512 to alanine abolished not only proteolytic activity of C5a peptidase, but also prevented the loss of its NH2-terminal... released from the surface of S pyogenes by streptopain In this study we established that streptococcal cysteine protease is also involved in the processing of C5a peptidase precursor Treatment of S512A C5a peptidase with streptococcal cysteine protease resulted in NH2terminal truncation of the peptidase This was evident both from the shift in mobility on SDS/PAGE and from NH2terminal sequence of the... status of C5a peptidase Covalent attachment of the carboxyl group of threonine within the LPXTN motif to the peptidoglycan [7] may limit intermolecular contact between C5a peptidase molecules The surface-associated location of C5a peptidase, however, can be affected by secreted cysteine protease streptopain Berge and Bjorck [20] demonstrated that functionally active 116 kDa fragment of C5a peptidase. .. precursor may be more prominent during stages of maximal synthesis of streptococcal cysteine protease Enzymology of C5a peptidase (Eur J Biochem 269) 4849 Fig 9 Propeptide region of recombinant C5a peptidase precursor The positions of identified autocatalytic cleavage sites are indicated as (+) The late intermediate of autocatalytic processing starts at Ala72, while the terminal product of maturation... the beginning of denaturation process of C5a peptidase coincides with the loss of its enzymatic activity Both processes were observed at temperatures above 43 °C, suggesting that inactivation of C5a peptidase is a result of thermal unfolding of its compact structure The temperature range (40–43 °C) of C5a peptidase maximum activity correlates with the maximum temperature of the human body When melting... reduced it (Figs 6C and 7C) Earlier studies demonstrated that incubation of C5a peptidase with complement C5, C3, albumin, myosin, ovalbumin and cytochrome c and subsequent SDS/PAGE analysis of reaction mixtures resulted in the absence of detectable cleavage of these protein substrates [3,4] Highly restricted macromolecular specificity of C5a peptidase was further confirmed in this study using chromogenic... spectroscopic evaluation of reaction mixtures containing increasing amounts of C5a peptidase and resorufin-labeled casein revealed absence of detectable casein cleavage (Fig 3A) When the 19-mer synthetic peptide corresponding to COOH-terminus of human C5a was tested as a substrate, it was effectively hydrolyzed by C5a peptidase (Fig 3B) In addition to the already known cleavage site between His67-Lys68, we identified... that C5a peptidase forms strong metal ion binding site(s) that may belong to either Ca1 or Ca2 Influence of Ca2+ and EDTA on the C5a peptidase thermal stability was also consistent with their effect on its enzymatic activity Incorporation of 5 mM CaCl2 into the reaction buffer did not cause any detectable changes in C5a peptidase activity toward tested peptide substrates, while 5 mM EDTA drastically reduced... novel secondary cleavage site between Ala58Ser59 (Figs 4 and 7) Since identification of secondary cleavage site was performed using synthetic peptides it represents an in vitro observation Accessibility of the Ala58Ser59 peptide bond within human C5a fragment for streptococcal peptidase has to be further investigated Catalytic efficiency of cleavage of the Ala58-Ser59 peptide bond by C5a peptidase was... stabilization of the transitional state for peptide bond hydrolysis [31–33] As a result, hydrolysis of substrates that lack specific P or P¢ can be less efficient than hydrolysis of longer substrates that contain these residues Interestingly, substrates containing pNA aromatic leaving group are considered more chemically labile than the extended peptide substrate [33,34] and yet a poor hydrolysis of Ac-SQLRANISH-pNA . Processing, stability, and kinetic parameters of C5a peptidase from Streptococcus pyogenes Elizabeth T. Anderson 1 , Michael G proteolytic activity of C5a peptidase Fig. 2. Incubation of S512A C5a peptidase precursor with wild-type C5a peptidase (panel A) and streptococcal cysteine