The N-terminal CUB-Epidermal Growth Factor Module Pair of Human Complement Protease C1r Binds Ca2+ with High Affinity and Mediates Ca2+-dependent Interaction with C1s
1999; Elsevier BV; Volume: 274; Issue: 14 Linguagem: Inglês
10.1074/jbc.274.14.9149
ISSN1083-351X
AutoresNicole M. Thielens, Karine Enrie, Monique Lacroix, Michel Jaquinod, Jean‐François Hernandez, Alfred F. Esser, Gérard J. Arlaud,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoThe Ca2+-dependent interaction between complement serine proteases C1r and C1s is mediated by their α regions, encompassing the major part of their N-terminal CUB-EGF-CUB (where EGF is epidermal growth factor) module array. In order to define the boundaries of the C1r domain(s) responsible for Ca2+ binding and Ca2+-dependent interaction with C1s and to assess the contribution of individual modules to these functions, the CUB, EGF, and CUB-EGF fragments were expressed in eucaryotic systems or synthesized chemically. Gel filtration studies, as well as measurements of intrinsic Tyr fluorescence, provided evidence that the CUB-EGF pair adopts a more compact conformation in the presence of Ca2+. Ca2+-dependent interaction of intact C1r with C1s was studied using surface plasmon resonance spectroscopy, yieldingK D values of 10.9–29.7 nm. The C1r CUB-EGF pair bound immobilized C1s with a higher K D(1.5–1.8 μm), which decreased to 31.4 nmwhen CUB-EGF was used as the immobilized ligand and C1s was free. Half-maximal binding was obtained at comparable Ca2+concentrations ranging from 5 μm with intact C1r to 10–16 μm for C1rα and CUB-EGF. The isolated CUB and EGF fragments or a CUB + EGF mixture did not bind C1s. These data demonstrate that the C1r CUB-EGF module pair (residues 1–175) is the minimal segment required for high affinity Ca2+ binding and Ca2+-dependent interaction with C1s and indicate that Ca2+ binding induces a more compact folding of the CUB-EGF pair. The Ca2+-dependent interaction between complement serine proteases C1r and C1s is mediated by their α regions, encompassing the major part of their N-terminal CUB-EGF-CUB (where EGF is epidermal growth factor) module array. In order to define the boundaries of the C1r domain(s) responsible for Ca2+ binding and Ca2+-dependent interaction with C1s and to assess the contribution of individual modules to these functions, the CUB, EGF, and CUB-EGF fragments were expressed in eucaryotic systems or synthesized chemically. Gel filtration studies, as well as measurements of intrinsic Tyr fluorescence, provided evidence that the CUB-EGF pair adopts a more compact conformation in the presence of Ca2+. Ca2+-dependent interaction of intact C1r with C1s was studied using surface plasmon resonance spectroscopy, yieldingK D values of 10.9–29.7 nm. The C1r CUB-EGF pair bound immobilized C1s with a higher K D(1.5–1.8 μm), which decreased to 31.4 nmwhen CUB-EGF was used as the immobilized ligand and C1s was free. Half-maximal binding was obtained at comparable Ca2+concentrations ranging from 5 μm with intact C1r to 10–16 μm for C1rα and CUB-EGF. The isolated CUB and EGF fragments or a CUB + EGF mixture did not bind C1s. These data demonstrate that the C1r CUB-EGF module pair (residues 1–175) is the minimal segment required for high affinity Ca2+ binding and Ca2+-dependent interaction with C1s and indicate that Ca2+ binding induces a more compact folding of the CUB-EGF pair. activated components are indicated by an overbar,e.g. C1̄r CCP module, complement control protein module module found in complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1 epidermal growth factor γ-carboxyglutamic acid containing module polyacrylamide gel electrophoresis polymerase chain reaction resonance unit(s) resonance unit(s) at equilibrium 2-(N-morpholino)ethanesulfonic acid C11 (1Bork P. Bairoch A. Trends Biochem. Sci. 1995; 20 (suppl.): C03Google Scholar) is the complex modular protease that triggers the classical pathway of complement in response to the formation of antigen-antibody complexes and to infection by various microorganisms. The human C1 complex comprises three types of proteins (C1q, C1r, and C1s) that form two distinct entities: the recognition subunit C1q and the catalytic subunit C1s-C1r-C1r-C1s, a Ca2+-dependent tetrameric assembly of the two homologous but distinct serine proteases C1r and C1s. Binding of C1 to the target microorganism is mediated by C1q and triggers autocatalytic activation of C1r into C1̄r, which in turn converts C1s into C1̄s, the protease responsible for enzymic activity of C1̄ (2Cooper N.R. Adv. Immunol. 1985; 37: 151-216Crossref PubMed Scopus (392) Google Scholar, 3Schumaker V.N. Zavodsky P. Poon P.H. Annu. Rev. Immunol. 1987; 5: 21-42Crossref PubMed Scopus (128) Google Scholar, 4Volanakis J.E. Arlaud G.J. Frank M. Volanakis J.E. The Human Complement System in Health and Disease. Marcel-Dekker, Inc., New York1998: 49-81Crossref Google Scholar). Both steps of the C1 activation process occur through cleavage of a single peptide bond in the proenzymes, generating active proteases comprising two disulfide-linked chains. C1r and C1s are modular proteins exhibiting homologous overall structures comprising, from the N terminus, two CUB modules (5Bork P. Beckmann G. J. Mol. Biol. 1993; 231: 539-545Crossref PubMed Scopus (525) Google Scholar) surrounding a single EGF-like module, two CCP modules (6Reid K.B.M. Bentley D.R. Campbell D.R. Chung L.P. Sim R.B. Kristensen T. Tack B.F. Immunol. Today. 1986; 7: 230-234Abstract Full Text PDF PubMed Scopus (187) Google Scholar), a connecting segment homologous to the activation peptide in chymotrypsinogen, and a serine protease domain. In the same way, each monomeric protease is thought to be organized in two functional regions, a C-terminal catalytic region and an N-terminal (α) interaction region. A particular feature of C1r and C1s is that they exert their catalytic activities within the Ca2+-dependent C1s-C1r-C1r-C1s complex. The non-covalent C1r-C1r homodimer forms the core of the tetramer, with its catalytic regions in the center, and each of its distal α regions is connected in a Ca2+-dependent fashion to the homologous region of a C1s molecule. N-terminal fragments corresponding to the α regions of C1r and C1s, each comprising the first CUB module, the EGF-like module, and a small N-terminal portion of the following second CUB module, have been obtained by limited proteolysis with trypsin in the presence of Ca2+ (7Busby T.F. Ingham K.C. Biochemistry. 1987; 26: 5564-5571Crossref PubMed Scopus (20) Google Scholar, 8Thielens N.M. Aude C.A. Lacroix M.B. Gagnon J. Arlaud G.J. J. Biol. Chem. 1990; 265: 14469-14475Abstract Full Text PDF PubMed Google Scholar). Each of these fragments contains a single high affinity Ca2+-binding site and retains the ability to mediate Ca2+-dependent heterologous (C1r/C1s) interaction (8Thielens N.M. Aude C.A. Lacroix M.B. Gagnon J. Arlaud G.J. J. Biol. Chem. 1990; 265: 14469-14475Abstract Full Text PDF PubMed Google Scholar). The α regions of C1r and C1s both exhibit similar low temperature transitions, with midpoints of 26–31 °C, which are shifted upward at high ionic strength or in the presence of Ca2+ ions (7Busby T.F. Ingham K.C. Biochemistry. 1987; 26: 5564-5571Crossref PubMed Scopus (20) Google Scholar, 9Busby T.F. Ingham K.C. Biochemistry. 1988; 27: 6127-6135Crossref PubMed Scopus (25) Google Scholar). The α regions of both C1r and C1s also likely participate in the interaction between the C1s-C1r-C1r-C1s tetramer and C1q and therefore represent key elements of the architecture of the C1 complex (10Busby T.F. Ingham K.C. Biochemistry. 1990; 29: 4613-4618Crossref PubMed Scopus (58) Google Scholar, 11Thielens N.M. Illy C. Bally I.M. Arlaud G.J. Biochem. J. 1994; 301: 509-516Crossref PubMed Scopus (28) Google Scholar). In addition to their structural role, various studies suggest that the α regions of C1r may control autoactivation of the catalytic regions of the protease (11Thielens N.M. Illy C. Bally I.M. Arlaud G.J. Biochem. J. 1994; 301: 509-516Crossref PubMed Scopus (28) Google Scholar). Previous studies performed on C1s have suggested that the structural determinants required for Ca2+ binding and interaction with C1r are contributed by both the N-terminal CUB and EGF modules (12Illy C. Thielens N.M. Gagnon J. Arlaud G.J. Biochemistry. 1991; 30: 7135-7141Crossref PubMed Scopus (15) Google Scholar,13Thielens N.M. van Dorsselaer A. Gagnon J. Arlaud G.J. Biochemistry. 1990; 29: 3570-3578Crossref PubMed Scopus (42) Google Scholar), an hypothesis that is supported by recent functional studies on the recombinant CUB-EGF module pair (14Tsai S.-W. Poon P.H. Schumaker V.N. Mol. Immunol. 1997; 34: 1273-1280Crossref PubMed Scopus (17) Google Scholar). In the case of C1r, a deletion mutant lacking the first CUB module was shown to lose the ability to bind C1s in the presence of Ca2+ (15Cseh S. Gal P. Sarvari M. Dobo J. Lorincz Z. Schumaker V.N. Zavodsky P. Mol. Immunol. 1996; 33: 351-359Crossref PubMed Scopus (14) Google Scholar). Conversely, the isolated EGF module of C1r, which exhibits the consensus sequence pattern characteristic of the particular subset of EGF modules involved in Ca2+ binding (16Campbell I.D. Bork P. Curr. Opin. Struct. Biol. 1993; 3: 385-392Crossref Scopus (331) Google Scholar), retains the ability to bind Ca2+ ions. However, compared with the whole C1rα fragment, its affinity for Ca2+ is decreased about 300-fold (17Hernandez J.-F. Bersch B. Pétillot Y. Gagnon J. Arlaud G.J. J. Pept. Res. 1997; 49: 221-231Crossref PubMed Scopus (21) Google Scholar, 18Bersch B. Hernandez J.-F. Marion D. Arlaud G.J. Biochemistry. 1998; 37: 1204-1214Crossref PubMed Scopus (44) Google Scholar), strongly suggesting that Ca2+ binding by C1r involves residues located outside the EGF module. The objective of the present study was to measure the ability of the N-terminal CUB-EGF module pair of C1r to bind Ca2+ ions and to mediate Ca2+-dependent interaction with C1s, as well as to assess the contribution of the individual CUB and EGF modules to these functions. For this purpose, the CUB module and CUB-EGF pair were expressed in eucaryotic systems, and the EGF module was synthesized chemically. Comparative analysis of the physicochemical and functional properties of these recombinant molecules indicates that the CUB-EGF module pair binds Ca2+ with high affinity and mediates Ca2+-dependent interaction with C1s and supports the hypothesis that Ca2+ induces a more compact conformation of this domain. Diisopropyl phosphorofluoridate and trypsin from bovine pancreas (treated with 1-chloro-4-phenyl-3-(l-tosylamido)-butan-2-one) were from Sigma. Peptide:N-glycosidase F was purified from cultures ofFlavobacterium meningosepticum according to the method of Tarentino et al. (19Tarentino A.L. Gomez C.M. Plummer Jr., G.H. Biochemistry. 1995; 34: 4665-4671Google Scholar), modified as described by Audeet al. (20Aude C.A. Lacroix M.B. Arlaud G.J. Gagnon J. Colomb M.G. Biochemistry. 1988; 27: 8641-8648Crossref PubMed Scopus (10) Google Scholar). Polyclonal anti-C1r antiserum was raised in rabbits according to standard procedures. Restriction enzymes were from Boehringer Mannheim. VentR polymerase was from New England Biolabs. The pHC1r3 plasmid containing full-length C1r cDNA (21Journet A. Tosi M. Biochem. J. 1986; 240: 783-787Crossref PubMed Scopus (75) Google Scholar) was kindly provided by Dr Agnès Journet (Commissariat àl'Energie Atomique, Grenoble, France). Antibiotics and molecular biology reagents were from Appligene Oncor. Oligonucleotides were obtained from Life Technologies, Inc. (Cergy-Pontoise, France). Activated C1r and C1s were purified from human plasma as described previously (22Arlaud G.J. Sim R.B. Duplaa A.M. Colomb M.G. Mol. Immunol. 1979; 16: 445-450Crossref PubMed Scopus (112) Google Scholar). Their C1rα and C1sα fragments were obtained by limited proteolysis with trypsin and purified as described previously (8Thielens N.M. Aude C.A. Lacroix M.B. Gagnon J. Arlaud G.J. J. Biol. Chem. 1990; 265: 14469-14475Abstract Full Text PDF PubMed Google Scholar). The EGF-like module from C1r was synthesized chemically and characterized as described (17Hernandez J.-F. Bersch B. Pétillot Y. Gagnon J. Arlaud G.J. J. Pept. Res. 1997; 49: 221-231Crossref PubMed Scopus (21) Google Scholar). The concentrations of purified proteins were determined using the following absorption coefficients ( A1 cm1% at 280 nm) and molecular weights as follows: dimeric C1r, 12.4 and 172,600 (8Thielens N.M. Aude C.A. Lacroix M.B. Gagnon J. Arlaud G.J. J. Biol. Chem. 1990; 265: 14469-14475Abstract Full Text PDF PubMed Google Scholar); C1s, 14.5 and 79,800 (8Thielens N.M. Aude C.A. Lacroix M.B. Gagnon J. Arlaud G.J. J. Biol. Chem. 1990; 265: 14469-14475Abstract Full Text PDF PubMed Google Scholar, 23Pétillot Y. Thibault P. Thielens N.M. Rossi V. Lacroix M. Coddeville B. Spik G. Schumaker V.N. Gagnon J. Arlaud G.J. FEBS Lett. 1995; 358: 323-328Crossref PubMed Scopus (25) Google Scholar); C1rα, 7.2 and 27,600 (8Thielens N.M. Aude C.A. Lacroix M.B. Gagnon J. Arlaud G.J. J. Biol. Chem. 1990; 265: 14469-14475Abstract Full Text PDF PubMed Google Scholar); and C1sα, 10.0 and 24,200 (8Thielens N.M. Aude C.A. Lacroix M.B. Gagnon J. Arlaud G.J. J. Biol. Chem. 1990; 265: 14469-14475Abstract Full Text PDF PubMed Google Scholar). The absorption coefficients ( A1 cm1% at 280 nm) used for the C1r EGF-like module (7.0), the recombinant fragments CUB (6.4), aglycosylated CUB-EGF (6.7), and glycosylated CUB-EGF (6.2) were calculated from the number of Trp, Tyr, and disulfides by the method of Edelhoch (24Edelhoch H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (3007) Google Scholar), using molecular weights of 5,970 (17Hernandez J.-F. Bersch B. Pétillot Y. Gagnon J. Arlaud G.J. J. Pept. Res. 1997; 49: 221-231Crossref PubMed Scopus (21) Google Scholar), 14,243, 19,790, and 21,500, respectively, as determined by mass spectrometry analysis (see “Results”). The Spodoptera frugiperda insect cells (Ready-Plaque Sf9 cells from Novagen) were routinely grown and maintained in serum-free Sf900 II SFM medium (Life Technologies, Inc.) supplemented with 50 IU/ml penicillin and 50 mg/ml streptomycin (Life Technologies, Inc.). TheTrichoplusia ni (High FiveTM) insect cells (provided by Dr. Jadwiga Chroboczek, Institut de Biologie Structurale, Grenoble) were maintained in TC100 medium (Life Technologies, Inc.) containing 10% fetal calf serum (Dominique Dutscher SA, Brumath, France) supplemented with 50 IU/ml penicillin and 50 mg/ml streptomycin. Pichia pastoris GS115 (his4) yeast cells (Invitrogen) were propagated in buffered minimal glycerol complex medium (BMGY) containing 100 mm potassium phosphate, pH 6.0, 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base (without amino acids), 0.00004% biotin and 1% glycerol. Expression of recombinant protein was induced in methanol complex medium (BMMY, same composition as BMGY, with 0.5% methanol instead of glycerol). For selecting P. pastoris transformants, MD plates containing 1% dextrose, 1.34% yeast nitrogen base, and 0.00004% biotin were used. Mut+/Muts selection was performed on MM plates (same composition as MD with 0.5% methanol instead of dextrose). A DNA fragment encoding the C1r signal peptide plus the N-terminal CUB-EGF module pair (amino acids 1–175 of the mature C1r protein) was amplified by PCR using VentR polymerase and pHC1r3 as a template, according to established procedures. The sequences of the sense (5′-CGGAATTCATGTGGCTCTTGTAC-3′) and antisense (5′-GGGGTACC CTACTCAGCCTGGCAGGA-3′) primers introduced an EcoRI site (underlined) at the 5′ end of the PCR product and a stop codon (boldface) followed by a KpnI site (underlined) at the 3′ end. The amplified DNA was purified using the Geneclean kit (Bio 101) and subcloned into the pCR-Script Amp SK(+) intermediate vector (Stratagene) according to the manufacturer's instructions. The fragment was excised by digestion withEcoRI and KpnI and cloned into theEcoRI/KpnI sites of the pFastBac1 baculovirus transfer vector (Life Technologies, Inc.). The resulting construct was characterized by restriction mapping and checked by double-stranded DNA sequencing (Genome Express, Grenoble, France). The recombinant baculovirus was generated using the Bac-to-BacTM system (Life Technologies, Inc.). The bacmid DNA was purified using the Qiagen midiprep purification system (Qiagen S.A., Courtaboeuf, France) and used to transfect Sf9 insect cells with Cellfectin (Life Technologies, Inc.) in Sf900 II SFM medium as described by the manufacturer. Recombinant virus particles were collected 4 days later, titered by virus plaque assay, and amplified as described by King and Possee (25King L.A. Possee R.D. The Baculovirus Expression System: A Laboratory Guide. Chapman and Hall Ltd., London1992: 111-114Google Scholar). High Five cells (1.75 × 107 cells/175-cm2tissue culture flask) were infected with the recombinant virus at a multiplicity of infection of 2 in Sf900 II SFM medium for 84 h at 28 °C. The supernatant was collected by centrifugation, and diisopropyl phosphorofluoridate was added to a final concentration of 1 mm. A DNA fragment encoding the CUB module of C1r (amino acids 1–124 of the mature protein) was amplified by PCR using the VentR polymerase and pHC1r3 as a template. The sequence of the sense primer (5′-GGTACGTATCCATTCCCATCCCTC-3′) introduced an SnaBI restriction site (underlined) at the 5′ end of the PCR product and allowed in-frame cloning with the α factor yeast secretion signal. The antisense primer (5′-GGCCTAGG CTACACAGCTTGGTAGTAGGC-3′) introduced a stop codon (boldface) followed by an AvrII restriction site (underlined) at the 3′ end of the PCR product. The purified amplified DNA fragment was subcloned into the pCR-Script Amp SK(+) intermediate vector (Stratagene), excised with SnaBI andAvrII, and ligated into the corresponding restriction sites of the pPIC9K yeast expression vector (Invitrogen). The final construct was submitted to double-stranded DNA sequencing. Ten μg of recombinant plasmid DNA were linearized with SacI and used for transformation of P. pastoris GS115 cells by electroporation as described in the user's manual of thePichia Expression kit (Invitrogen). Histidine-independent transformants were selected on MD plates, replicated on MM plates, and allowed to grow for 3 days at 30 °C. Pure methanol (100 μl) was supplied every 24 h inside the lid of the plates. The colonies were then screened for CUB expression by overlaying the plates with a 0.2-μm nitrocellulose disc (Schleicher & Schuell) overnight at 30 °C and probing it with polyclonal anti-C1r antibodies. Screening for Mut+/Muts phenotypes indicated that the clone yielding the strongest CUB immunoreactive signal was Mut+. An optimal methanol induction time of 30 h for liquid medium expression was determined for this clone as described in the Pichia Expression kit manual (Invitrogen). The colony expressing the recombinant CUB fragment was grown overnight in 10 ml of BMGY. This culture was inoculated into 150 ml of BMGY and grown at 30 °C in a shaking incubator until theA 600 reached a value of 6.0. The cells were collected by centrifugation at 1500 × g, resuspended in 600 ml of BMMY, and cultured for 30 h, and pure methanol was added to a final concentration of 0.5% after 24 h of induction. The supernatant was separated from the cells by centrifugation, and diisopropyl phosphorofluoridate added to a final concentration of 1 mm. The culture supernatant containing the CUB-EGF fragment was dialyzed against 50 mm NaCl, 1 mmCaCl2, 50 mm triethanolamine hydrochloride, pH 8.5, and loaded at 1.5 ml/min onto a Q-Sepharose-Fast Flow column (Amersham Pharmacia Biotech) (2.8 × 14 cm) equilibrated in the same buffer. Elution was carried out by applying a 1.2-liter linear gradient from 50 to 500 mm NaCl in the same buffer, and fractions containing the recombinant fragments were identified by Western blot analysis. The CUB-containing culture supernatant was dialyzed against 25 mm MES, pH 6.0, and loaded at 1 ml/min onto a carboxymethylcellulose CM52 column (2.8 × 14 cm) (Whatman) equilibrated in the same buffer. Elution was carried out by applying a 1-liter linear gradient from 0 to 150 mm NaCl in the same buffer. Fractions containing the recombinant fragment were identified by Western blot analysis. Fractions containing the CUB-EGF or CUB fragments were pooled and dialyzed against 1.0 m ammonium sulfate, 1 mmCaCl2, 0.1 m triethanolamine hydrochloride, pH 7.4. Further purification of both fragments was achieved by high pressure hydrophobic interaction chromatography on a TSK-phenyl 5PW column (7.5 × 75 mm) (Beckman). Elution was carried out by decreasing the ammonium sulfate concentration from 1.0 m to 0 in 20 min at a flow rate of 1 ml/min. The purified fragments were dialyzed against 145 mm NaCl, 50 mmtriethanolamine hydrochloride, pH 7.4, and concentrated if necessary to 0.1 mg/ml by ultrafiltration on Microsep microconcentrators (molecular weight cut-off = 3,000) (Filtron). N-terminal sequence analyses were performed after SDS-PAGE and electrotransfer, using an Applied Biosystems model 477 A protein sequencer as described previously (26Rossi V. Gaboriaud C. Lacroix M. Ulrich J. Fontecilla-Camps J.C. Gagnon J. Arlaud G.J. Biochemistry. 1995; 34: 7311-7321Crossref PubMed Scopus (41) Google Scholar). Mass spectrometry analysis of the recombinant proteins was performed using the matrix-assisted laser desorption ionization technique on a Voyager Elite XL instrument (PerSeptive Biosystems, Cambridge, MA), under conditions described previously (27Lacroix M. Rossi V. Gaboriaud C. Chevallier S. Jaquinod M. Thielens N.M. Gagnon J. Arlaud G.J. Biochemistry. 1997; 36: 6270-6282Crossref PubMed Scopus (43) Google Scholar). Fifty μl of 10-fold concentrated culture supernatants containing the CUB-EGF or CUB fragments were incubated with 0.5 μg of peptide:N-glycosidase F for 4 h at 25 °C. Deglycosylation of the recombinant fragments was monitored by SDS-PAGE and Western blot analysis of the samples. The purified CUB-EGF fragment (0.1 mg/ml) in 145 mm NaCl, 50 mmtriethanolamine hydrochloride, pH 7.4, was incubated in the presence of 20% (w/w) peptide:N-glycosidase F for 20 h at 25 °C. Protein deglycosylation was monitored by SDS-PAGE analysis of the samples. SDS-PAGE analysis was performed as described previously (8Thielens N.M. Aude C.A. Lacroix M.B. Gagnon J. Arlaud G.J. J. Biol. Chem. 1990; 265: 14469-14475Abstract Full Text PDF PubMed Google Scholar). Western blot analysis and immunodetection of the recombinant proteins were performed as described (28Rossi V. Bally I. Thielens N.M. Esser A.F. Arlaud G.J. J. Biol. Chem. 1998; 273: 1232-1239Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), using rabbit polyclonal anti-C1r antiserum (1:400 dilution). The C1r CUB-EGF and α fragments (190 pmoles each), either alone or in equimolar mixture with C1sα, were analyzed by high pressure gel permeation on a TSK G3000 SWG column (7.5 × 600 mm) (Toso Haas) equilibrated in 145 mm NaCl, 50 mm triethanolamine hydrochloride, pH 7.4, containing EDTA (2.5 mm) or CaCl2 (2.5 or 0.2 mm), and run at 1 ml/min. Sample volumes ranged from 42 to 56 μl. Proteins were detected from their absorbance at 280 nm. Real time analysis of the Ca2+-dependent interaction between C1r or its fragments and C1s or C1sα was performed at 25 °C using an upgraded BIAcoreTM instrument (BIAcore AB). The running buffer for protein immobilization was 145 mm NaCl, 5 mmEDTA, 10 mm HEPES, pH 7.4. Protein ligands were diluted to 10–20 μg/ml either in 10 mm formate, pH 3.0 (C1̄r, C1̄s, and C1sα), or in 10 mm acetate, 10 mm NaCl, pH 4.8 (CUB-EGF and unglycosylated CUB-EGF), and coupled to the carboxymethylated dextran surface of a CM5 sensor chip (BIAcore AB) using the amine coupling chemistry (BIAcore AB amine coupling kit) according to the manufacturer's instructions. Binding of C1r and its fragments was measured over 530 and 3100 resonance units (RU) of immobilized C1s (or 150 and 600 RU of C1sα), at a flow rate of 10 μl/min in 145 mm NaCl, 0.25 mmCaCl2, 50 mm triethanolamine hydrochloride, pH 7.4. Binding of C1s was measured under the same conditions as above, over 1000 RU of immobilized C1r, 150 and 450 RU of CUB-EGF, or 350 RU of unglycosylated CUB-EGF. Binding of C1sα was measured over 3200 RU of immobilized C1r. Equivalent volumes of each protein sample were injected over a surface with immobilized bovine serum albumin (instead of C1s or C1r) or ovalbumin (instead of C1sα or CUB-EGF) to serve as blank sensorgrams for subtraction of bulk refractive index background. Regeneration of the surfaces was achieved by injection of 10 μl of 20 mm EDTA. The effect of Ca2+ concentration on the interaction between C1r or its fragments and C1s was studied in 145 mm NaCl, 50 mm triethanolamine hydrochloride, pH 7.4, containing 1 mm EGTA and varying amounts of CaCl2 calculated to give the desired free calcium concentrations as described (29Vivaudou M.B. Arnoult C. Villaz M. J. Membr. Biol. 1991; 122: 165-175Crossref PubMed Scopus (70) Google Scholar). Sensorgrams were analyzed by non-linear least squares curve fitting using the BIAevaluation 2.1 Software (Amersham Pharmacia Biotech). A single-site binding model was used for kinetic analysis of all interactions. The equation R t =R 0 exp(−k off(t − t 0)) was used for the dissociation phase, where R t is the amount of ligand (in RU) remaining bound at time t, andt 0 is the beginning of the dissociation phase. The final dissociation rate constant k off was calculated from the mean of the values obtained from a series of injections. To analyze the association phase, the equationR t = R eq(1 − exp(−k s(t −t 0))) was employed, whereR eq is the amount of bound ligand (in RU) at equilibrium, t 0 is the starting time of injection, and k s = k on ×C + k off, where C is the concentration of analyte injected over the sensor chip surface. The association rate constant k on was determined from the slope of a plot of k s versus C, based on a series of at least five analyte concentrations. The apparent equilibrium dissociation constant (K D(kin)) was calculated from the ratio of these two kinetic constants (k off/k on). Whenever this was possible, i.e. when the binding reaction reached or approached equilibrium, the association constant (K A(eq)) was also determined from equilibrium levels of the analyte binding to the surface (R eq). The equationR eq/C =K A(eq) × R max −K A(eq) × R eq was used, where R max is the maximal binding capacity of the immobilized ligand, and the association constantK A(eq) was determined from the slope of a Scatchard plot of R eq/C versus R eq. The dissociation constant derived from K A(eq) is referred to in the text as K D(eq). Intrinsic protein fluorescence was measured at 20 °C using a SLM-Aminco Bowman Series 2 Luminescence Spectrometer at a photomultiplier voltage of 900 V with a 10-mm path length cell. The excitation wavelength was 275 nm, and the band path was 4 nm. Emission spectra were recorded at a scanning rate of 1 nm/s in the 285–355 nm range using a band path of 4 nm. All measurements were corrected for buffer (145 mm NaCl, 50 mm triethanolamine hydrochloride, pH 7.4, containing 0.1 mm EGTA or 0.9 mm CaCl2) background emission. The modular structures of human C1r and of the various fragments used in the present study are depicted in Fig. 1. The recombinant baculovirus for expression of the CUB-EGF fragment of C1r was generated as described under “Experimental Procedures” and used to infect Sf9 and High Five insect cells for various periods at 28 °C. Secretion of the recombinant protein into the culture medium was monitored by SDS-PAGE and Western blot analysis, as illustrated in Fig. 2. The anti-C1r antibody labeled a band of apparent molecular mass 20 kDa in both cell culture supernatants, a major, broader band of about 22 kDa in the case of High Five cells (Fig. 2 B), and two minor bands of 21 and 22 kDa in the case of Sf9 cells (Fig. 2 A). All bands became detectable after 24 h of infection, and their intensity reached a maximum at 96 h. Incubation of the 96-h Sf9 culture medium with peptide:N-glycosidase F led to the disappearance of the 21- and 22-kDa bands, with a concomitant slight increase in the intensity of the 20-kDa band (data not shown), indicating that the latter represented an unglycosylated form of the CUB-EGF fragment. The presence of unglycosylated material in the supernatant was clearly not the result of the release of intracellular material from lysed infected cells, as the relative amounts of the glycosylated and unglycosylated forms was kept constant throughout the course of infection. Analysis of protein production in the cell pellets showed the presence of equivalent amounts of both forms for Sf9 and High Five insect cells. The amount of unglycosylated fragment present in the 96-h culture supernatant was estimated to be 1 μg/ml for both Sf9 and High Five cells. In contrast, the level of expression of the glycosylated CUB-EGF fragment was about 5 times higher in High Five cells (about 2.5 μg/ml) than in Sf9 cells. High Five cells were therefore selected for subsequent production of the recombinant CUB-EGF fragment.Figure 2Time course of the secretion of the recombinant CUB-EGF fragment from infected Sf9 and High Five insect cells. Sf9 (A) and High Five (B) cells were infected with the recombinant baculovirus at a multiplicity of infection of 2. Culture supernatants (A, 250 μl; B, 125 μl) were collected at various times, concentrated 10 times, and submitted to SDS-PAGE and Western blot analysis. Lanes 1–4, supernatants collected at 96, 72, 48, and 24 h, respectively; lane 5, 96-h supernatant of mock- infected cells. All samples were analyzed under non-reducing conditions. Molecular masses of standard proteins (expressed in kDa) are shown on the left side of the blots.View Large Image Figure ViewerDownload (PPT) Expression of the CUB fragment of C1r was carried out in the GS115 strain of the methylotrophic yeast P. pastoris using the pPIC9K expression vector, as described under “Experimental Procedures.” SDS-PAGE and Western blot analysis of the cu
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