Biophysical Characterization of the C-propeptide Trimer from Human Procollagen III Reveals a Tri-lobed Structure
2001; Elsevier BV; Volume: 276; Issue: 52 Linguagem: Inglês
10.1074/jbc.m108611200
ISSN1083-351X
AutoresSimonetta Bernocco, Stéphanie Finet, Christine Ebel, D. Eichenberger, M. Mazzorana, J. Farjanel, David Hulmes,
Tópico(s)Collagen: Extraction and Characterization
ResumoProcollagen C-propeptide domains direct chain association during intracellular assembly of procollagen molecules. In addition, they control collagen solubility during extracellular proteolytic processing and fibril formation and interact with cell surface receptors and extracellular matrix components involved in feedback inhibition, mineralization, cell growth arrest, and chemotaxis. At present, three-dimensional structural information for the C-propeptides, which would help to understand the underlying molecular mechanisms, is lacking. Here we have carried out a biophysical study of the recombinant C-propeptide trimer from human procollagen III using laser light scattering, analytical ultracentrifugation, and small angle x-ray scattering. The results show that the trimer is an elongated molecule, which by modeling of the x-ray scattering data appears to be cruciform in shape with three large lobes and one minor lobe. We speculate that each of the major lobes corresponds to one of the three component polypeptide chains, which come together in a junction region to connect to the rest of the procollagen molecule. Procollagen C-propeptide domains direct chain association during intracellular assembly of procollagen molecules. In addition, they control collagen solubility during extracellular proteolytic processing and fibril formation and interact with cell surface receptors and extracellular matrix components involved in feedback inhibition, mineralization, cell growth arrest, and chemotaxis. At present, three-dimensional structural information for the C-propeptides, which would help to understand the underlying molecular mechanisms, is lacking. Here we have carried out a biophysical study of the recombinant C-propeptide trimer from human procollagen III using laser light scattering, analytical ultracentrifugation, and small angle x-ray scattering. The results show that the trimer is an elongated molecule, which by modeling of the x-ray scattering data appears to be cruciform in shape with three large lobes and one minor lobe. We speculate that each of the major lobes corresponds to one of the three component polypeptide chains, which come together in a junction region to connect to the rest of the procollagen molecule. C-terminal propeptide region of the human type III procollagen molecule small angle x-ray scattering Fibril-forming collagens (types I, II, III, V, and XI) are synthesized and secreted into the extracellular matrix in precursor form, procollagens (∼500 kDa), with large N- and C-terminal propeptide regions (1Kadler K.E. Sheterline P. Protein Profile. 2. Academic Press, Inc., London1995: 491-619Google Scholar). The propeptides increase the solubility of the procollagen molecule, thus preventing premature fibril formation inside the cell (2Prockop D.J. Hulmes D.J.S. Yurchenco P.D. Birk D.E. Mecham R.P. Extracellular Matrix Assembly and Structure. Academic Press, Inc., San Diego1994: 47-90Crossref Google Scholar). After secretion, propeptides are cleaved to varying extents by specific procollagen N- and C-proteinases (3Kessler E. Takahara K. Biniaminov L. Brusel M. Greenspan D.S. Science. 1996; 271: 360-362Crossref PubMed Scopus (458) Google Scholar, 4Prockop D.J. Sieron A.L. Li S.-W. Matrix Biol. 1998; 16: 399-408Crossref PubMed Scopus (151) Google Scholar, 5Moschcovich L. Bernocco S. Font B. Rivkin H. Eichenberger D. Chejanovsky N. Hulmes D.J.S. Kessler E. Eur. J. Biochem. 2001; 268: 2991-2996Crossref PubMed Scopus (34) Google Scholar) and also other proteinases (6Imamura Y. Steiglitz B.M. Greenspan D.S. J. Biol. Chem. 1998; 273: 27511-27517Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 7Kofford M.W. Schwartz L.B. Schechter N.M. Yager D.R. Diegelmann R.F. Graham M.F. J. Biol. Chem. 1997; 272: 7127-7131Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar), thereby triggering fibril assembly of collagen. Once cleaved, both N- and C-propeptides are thought to control further collagen synthesis by a process of feedback inhibition (8Katayama K. Seyer J.M. Raghow R. Kang A.H. Biochemistry. 1991; 30: 7097-7104Crossref PubMed Scopus (54) Google Scholar, 9Wu C.H. Walton C.M. Wu G.Y. J. Biol. Chem. 1991; 266: 2983-2987Abstract Full Text PDF PubMed Google Scholar, 10Nakata K. Miyamoto S. Bernier S. Tanaka M. Utani A. Krebsbach P. Rhodes C. Yamada Y. Ann. N. Y. Acad. Sci. 1996; 785: 307-308Crossref PubMed Scopus (11) Google Scholar, 11Mizuno M. Fujisawa R. Kuboki Y. FEBS Lett. 2000; 479: 123-126Crossref PubMed Scopus (17) Google Scholar). Also, in the extracellular matrix, the N-propeptides are involved in growth factor signaling (12Zhu Y. Oganesian A. Keene D.R. Sandell L.J. J. Cell Biol. 1999; 144: 1069-1080Crossref PubMed Scopus (230) Google Scholar), whereas the C-propeptides have been implicated in interactions with procollagen C-proteinase enhancer (13Kessler E. Adar R. Eur. J. Biochem. 1989; 186: 115-121Crossref PubMed Scopus (83) Google Scholar), mineralization (14Kirsch T. Pfaffle M. FEBS Lett. 1992; 310: 143-147Crossref PubMed Scopus (71) Google Scholar, 15Lee E.R. Smith C.E. Poole A.R. J. Histochem. Cytochem. 1996; 44: 433-443Crossref PubMed Scopus (9) Google Scholar, 16Choglay A.A. Purdom I.F. Hulmes D.J.S. J. Biol. Chem. 1993; 268: 6107-6114Abstract Full Text PDF PubMed Google Scholar), integrin receptor binding (17Davies D. Tuckwell D.S. Calderwood D.A. Weston S.A. Takigawa M. Humphries M.J. Eur. J. Biochem. 1997; 246: 274-282Crossref PubMed Scopus (26) Google Scholar,18Bhattacharyya-Pakrasi M. Dickeson S.K. Santoro S.A. Matrix Biol. 1998; 17: 223-232Crossref PubMed Scopus (9) Google Scholar), cell growth arrest (19Rushton J.A. Schmitz S. Gunn-Moore F. Sherman D. Pappas C.A. Ritchie J.M. Haynes L.W. J. Neurochem. 1999; 73: 1816-1827PubMed Google Scholar), and chemotaxis (20Palmieri D. Camardella L. Ulivi V. Guasco G. Manduca P. J. Biol. Chem. 2000; 275: 32658-32663Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In addition to their extracellular roles, numerous observations demonstrate the importance of the C-propeptide domain in chaperone-assisted chain association during intracellular assembly of procollagen molecules (21Chessler S.D. Byers P.H. J. Biol. Chem. 1993; 268: 18226-18233Abstract Full Text PDF PubMed Google Scholar, 22Mclaughlin S.H. Bulleid N.J. Matrix Biol. 1998; 16: 369-377Crossref PubMed Scopus (98) Google Scholar, 23Lamande S.R. Bateman J.F. Semin. Cell Dev. Biol. 1999; 10: 455-464Crossref PubMed Scopus (163) Google Scholar, 24Bottomley M.J. Batten M.R. Lumb R.A. Bulleid N.J. Curr. Biol. 2001; 11: 1114-1118Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Each procollagen molecule consists of three polypeptide chains encoded by one or more genes, giving rise to homotrimers or heterotrimers, respectively, with specific chain stoichiometries. For example, procollagen III molecules are homotrimers with the chain composition proα1(III)3, whereas procollagen I molecules are normally heterotrimers of the form proα1(I)2proα2(I). The C-propeptides direct association within the rough endoplasmic reticulum to ensure correct chain stoichiometry, which is particularly important in cells producing more than one procollagen type. Once associated into a trimer, and after prolyl hydroxylation, triple helix formation within the collagenous region is initiated at the C terminus and proceeds in a zipper-like manner toward the N terminus (22Mclaughlin S.H. Bulleid N.J. Matrix Biol. 1998; 16: 369-377Crossref PubMed Scopus (98) Google Scholar, 25Engel J. Prockop D.J. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 137-152Crossref PubMed Scopus (269) Google Scholar). The importance of the C-propeptides in chain association has been demonstrated by both naturally occurring (26Pace J.M. Kuslich C.D. Willing M.C. Byers P.H. J. Med. Genet. 2001; 38: 443-449Crossref PubMed Google Scholar) and engineered (22Mclaughlin S.H. Bulleid N.J. Matrix Biol. 1998; 16: 369-377Crossref PubMed Scopus (98) Google Scholar, 27Lim A.L. Doyle S.A. Balian G. Smith B.D. J. Cell. Biochem. 1998; 71: 216-232Crossref PubMed Scopus (19) Google Scholar) mutations/deletions, which result in failure of or impaired procollagen molecular assembly. Very recently, it has been demonstrated that recombinant type I collagen molecules devoid of N- or C-terminal propeptides can assemble in a yeast expression system to form correctly aligned triple-helices (28Olsen D.R. Leigh S.D. Chang R. McMullin H. Ong W. Tai E. Chisholm G. Birk D.E. Berg R.A. Hitzeman R.A. Toman P.D. J. Biol. Chem. 2001; 276: 24038-24043Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), albeit with poor control of chain stoichiometry. Because expression was necessarily carried out at relatively low temperatures, however, the significance of these results for mammalian cells is unclear. Overwhelming evidence indicates that in mammalian cells the C-propeptides are essential for procollagen chain association with the correct stoichiometry. Throughout the fibrillar procollagens, the C-propeptide domain is highly conserved (1Kadler K.E. Sheterline P. Protein Profile. 2. Academic Press, Inc., London1995: 491-619Google Scholar, 29Dion A.S. Myers J.C. J. Mol. Biol. 1987; 193: 127-143Crossref PubMed Scopus (66) Google Scholar), which suggests a common overall three-dimensional structure in the C-propeptide region once the three chains have assembled to form a trimer. Within this conserved framework, a relatively variable and discontinuous sequence of 15 amino acid residues has been identified as being required for type-specific chain selection and trimer formation (30Lees J.F. Tasab M. Bulleid N.J. EMBO J. 1997; 16: 908-916Crossref PubMed Scopus (125) Google Scholar). In addition, the C-propeptide trimer (composed of three chains each of molecular mass ∼30 kDa) contains both inter- and intramolecular disulfide bonds, the former involving cysteine residues in the N-terminal region of each chain and the latter involving cysteines in the C-terminal region (31Koivu J. FEBS Lett. 1987; 212: 229-232Crossref PubMed Scopus (20) Google Scholar). Beyond this, there exists no three-dimensional structural information for any of the procollagen C-propeptide trimers that might be used to understand the mechanism of chain selection and association. Furthermore, because there are no known homologous proteins for which three-dimensional structures are available, molecular modeling by homology is precluded. Recently, a baculovirus system has been developed for the expression of recombinant human procollagen III C-propeptides in insect cells (32Zafarullah K. Brown E.M. Kuivaniemi H. Tromp G. Sieron A.L. Fertala A. Prockop D.J. Matrix Biol. 1997; 16: 201-209Crossref PubMed Scopus (8) Google Scholar). In this system, disulfide-linked trimers are formed and secreted into the culture medium that appear to have the same secondary structure as native C-propeptide trimers from chick procollagen I. Here we have used a variety of biophysical approaches to study the overall shape of the procollagen III C-propeptide trimer (CPIII)1in solution. We show that the trimer has an elongated shape, which by small angle x-ray scattering appears to be a cruciform structure with three large lobes and one small lobe, consistent with each large lobe corresponding to one of the three constituent chains. Trichoplusia ni (BTI-TN-5B1–4, High Five) insect cells (Invitrogen) maintained in suspension culture at 1.0 × 106 cells/ml were infected with rBac-TyIII-3.1 (a kind gift from Dr. D. Prockop (32Zafarullah K. Brown E.M. Kuivaniemi H. Tromp G. Sieron A.L. Fertala A. Prockop D.J. Matrix Biol. 1997; 16: 201-209Crossref PubMed Scopus (8) Google Scholar)) and incubated at 27 °C in Express Five serum-free medium (Invitrogen) complemented with 16 mm l-glutamine (Invitrogen), 100 units/ml penicillin, 0.1 mg/ml streptomycin (Sigma), and 0.1% Pluronic® F-68 (Invitrogen). Conditioned medium was then centrifuged at 900 ×g for 15 min followed by the addition of enzyme inhibitors to the supernatant to final concentrations 10 mm EDTA, 10 mmN-ethylmaleimide, 10 mm4-aminobenzamidine dihydrochloride, and 0.5 mmphenylmethylsulfonyl fluoride (all from Sigma). After a second centrifugation for 15 min at 20,000 × g and 4 °C, the supernatant was stored at −80 °C for up to 10 months without significant loss of protein integrity. Three chromatographic steps were used to purify recombinant CPIII. Typically 800 ml of conditioned medium was processed. Unless otherwise stated, all procedures were carried out at 4 °C. After the addition of 4-aminobenzamidine dihydrochloride (final concentration, 1 mm), phenylmethylsulfonyl fluoride (final concentration, 1 mm) and adjustment of the pH to 7.4 using a stock solution of 1 m Tris-HCl, pH 8.5, medium was centrifuged for 15 min at 10,000 × g to pellet any suspended material. Clarified medium was loaded at 75 ml/h onto a 5 × 2.5-cm column of concanavalin A-Sepharose (Amersham Biosciences, Inc.) pre-equilibrated in buffer A (50 mm Tris-HCl, 300 mm NaCl, 1 mmCaCl2, 1 mm MnCl2, pH 7.4). After extensive washing to remove non-bound material, CPIII was eluted at 10 ml/h using buffer A containing 1 m methyl α-d-mannopyranoside (Sigma). The eluate was then diluted with 5 volumes of 50 mm Tris-HCl, pH 8.5, and loaded onto a 10 × 2.6-cm column of DEAE-Sephacel (Amersham Biosciences, Inc.) preequilibrated in Buffer B (50 mm Tris-HCl, 50 mm NaCl, pH 8.5). After extensive washing, bound proteins were eluted with a 500 ml of linear gradient of Buffer B containing 50–300 mm NaCl. Fractions of interest were then pooled, 3.4 m (NH4)2SO4 was added to a final concentration of 1 m, and the sample was loaded at 10 ml/h and at room temperature on to a 5 × 1.6-cm column of butyl-Sepharose (Amersham Biosciences, Inc.) pre-equilibrated in buffer C (50 mm Tris-HCl, 1 m(NH4)2SO4, pH 8.0). After washing, bound proteins were eluted at 10 ml/h and at room temperature using a linear gradient of buffer C containing 1 to 0 m(NH4)2SO4. After analysis by SDS-PAGE, fractions of interest were pooled, concentrated up to 10 mg/ml using an UltraFree 15 device (Millipore, 30-kDa cut off), and stored at −80 °C. CPIII concentrations were measured by absorbance at 280 nm using a calculated extinction coefficient of 1.23 ml mg−1 cm−1, based on the presence of 8 cysteine, 5 tryptophan, and 7 tyrosine residues in the sequence of each polypeptide chain (33Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3472) Google Scholar). This value was confirmed using a commercial protein assay (Pierce) based on the Bradford method (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using bovine serum albumin as the protein standard. SDS-PAGE (10% acrylamide for samples reduced with dithiothreitol, 6% acrylamide in non-reducing conditions) was carried out according to Laemmli (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) using reagents from Bio-Rad. Western blotting and immuno-labeling on polyvinylidene difluoride membranes were done according to Towbinet al. (36Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). The three monoclonal antibodies used for the immuno-labeling (kind gifts from Dr. E. Burchardt (37Burchardt E.R. Heke M. Kauschke S.G. Harjes P. Kohlmeyer J. Kroll W. Schauer M. Schroeder W. Voelker M. Matrix Biol. 1998; 17: 673-677Crossref PubMed Scopus (6) Google Scholar)) were produced against single chains of CPIII expressed in Escherichia coli. Their epitopes are as follows: 48D34, amino acids 1–30; 48B14, amino acids 80–207; 48D19, amino acids 207–245 (numbering follows the TrEMBL CPIII sequence, GenBankTM accession number Q15112). Detection was with a commercial anti-mouse secondary antibody (Dako) coupled with alkaline phosphatase followed by color development using an alkaline phosphatase conjugate substrate kit (Bio-Rad). Deglycosylation in native conditions was carried out in 50 mm Hepes, 100 mm NaCl, pH 7.4, using CPIII at a concentration of 1 mg/ml andN-glycosidase F (Roche Molecular Biochemicals) at 0.1 unit/μg substrate and incubating for 4 h at 37 °C. Deglycosylation of denatured CPIII was done on protein previously heated for 10 min at 100 °C in the presence of 1% SDS then diluted to 0.1% SDS with buffer containing 0.15% Nonidet P-40 and applying the same procedure as used for native conditions. Samples were analyzed in Buffer D (20 mm Hepes, 150 mm NaCl, pH 7.4) by both static and dynamic light scattering (38Cantor C.R. Schimmel P.R. Biophysical Chemistry Part II: Techniques for the Study of Biological Structure and Function. W. H. Freeman and Co., San Francisco1980Google Scholar) using a Malvern 4700 spectrometer and 7132 256-channel correlator with a 40-mW He-Ne laser (Siemens). Before analysis, solutions were centrifuged at 4 °C for 15 min at 15,000 × g, then supernatants were transferred to 10-mm-diameter sample cells and examined at 25 °C. Samples were analyzed in the concentration range of 1.8–4.2 mg/ml. For static light scattering, samples were analyzed in the angular range 30–130°, and the molecular mass of CPIII was calculated using a Zimm plot (38Cantor C.R. Schimmel P.R. Biophysical Chemistry Part II: Techniques for the Study of Biological Structure and Function. W. H. Freeman and Co., San Francisco1980Google Scholar). Rayleigh ratios were determined with reference to a toluene standard, and a value of 0.182 ml/g was assumed for the refractive index increment. For dynamic light scattering, samples were analyzed at 90°, and correlation curves were analyzed using the Contin program provided by the manufacturers. Diffusion coefficients (experimentally observed D or correctedD20,w) were related to the frictional factor f and hydrodynamic diameter Dh by the relation D = RT/Nf =RT/3πηDh, where R is the gas constant, T the absolute temperature, N is Avogradro's number, and η the solvent viscosity. Sedimentation velocity experiments were performed using a Beckman XL-I analytical ultracentrifuge and an AN-60 TI rotor (Beckman Instruments). The experiments were carried out at 25 °C in Buffer D. Three samples of 400 μl at protein concentrations of 0.24, 0.44, and 0.81 mg/ml were loaded into 1.2-cm path cells and centrifuged at 42,000 rpm. Scans were recorded at 278 nm every 5 min using a 0.003-cm radial spacing. Sedimentation profiles were analyzed by different methods using time derivative analysis or direct modeling of boundary profiles in terms of one non-interacting component (dcdt+ and Svedberg from J. Philo, Sedfit from Ref. 39Schuck P. Biophys. J. 1998; 75: 1503-1512Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Sedfit takes advantage of a radial and time-independent noise subtraction procedure (40Schuck P. Demeler B. Biophys. J. 1999; 76: 2288-2296Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). These procedures allow the evaluation of both sedimentation (s) and diffusion (D) coefficients, from which the molar mass is derived using the Svedberg equation: M = sRT/D(1 − ρV̄). We estimated the partial specific volume V̄ of the protein to be 0.721 ml/g (assuming one high mannose glycan Man9GlcNAc2(41Jarvis D.L. Kawar Z.S. Hollister J.R. Curr. Opin. Biotechnol. 1998; 9: 528-533Crossref PubMed Scopus (85) Google Scholar) per polypeptide chain), the solvent density ρ to be 1.004 g/ml, and the solvent viscosity η to be 0.908 mPa·s, at 25 °C using Sednterp software (V1.01; developed by D. B. Haynes, T. Laue, and J. Philo) for the calculation of the corrected S20w and D20,wvalues. For SAXS, CPIII samples in buffer D were analyzed at 20 °C on beamline ID2 (42Narayanan T. Diat O. Bösecke P. Nucl. Instrum. Methods Phys. Res. A. 2001; 467–468: 1005-1009Crossref Scopus (283) Google Scholar) at the European Synchrotron Radiation Facility, Grenoble. Samples (25 μl) in the concentration range 4–10 mg/ml were placed in a quartz capillary (GLAS, 2-mm diameter, 10-μm thickness) mounted in a thermostatted flow-through cell. Scattering was measured using a two-dimensional detector, either an x-ray Image Intensifier FReLoN CCD camera (at 2.5 m from the sample) or a multiwire proportional gas-filled detector (at 1 m) using x-rays of wavelength λ = 0.995 Å. Data were averaged from individual exposures of 500 ms (CCD detector, 1024 × 1024 pixels) or 600 s (gas-filled detector, 512 × 512 pixels). Two-dimensional data reduction consisted of normalization for detector response, exposure time and sample transmission, absolute intensity calibration, azimuthal integration, and background subtraction from buffer alone to obtain the normalized scattered intensity I as a function of Q ors, where Q = 2πs = 4πsin(θ)/λ, and 2θ is the scattering angle. Data from the CCD detector in the Q range 0.01–0.15 Å−1 were merged with data from the gas detector in the Q range 0.15–0.3 Å−1. No concentration dependence in the scattering curves was observed at the concentrations used. SAXS data were analyzed using Guinier plots (43Guinier A. Fournet G. Small-angle Scattering of X-rays. John Wiley & Sons, Inc., New York1955Google Scholar) to determine the radius of gyration Rg as well as the apparent molecular mass of CPIII, where the latter was obtained by extrapolation to zero angle with reference to a bovine serum albumin standard. The program GNOM (44Semenyuk A.V. Svergun D.I. J. Appl. Crystallogr. 1991; 24: 537-540Crossref Scopus (575) Google Scholar) was used to determine the distance distribution functionp(r) after eliminating data for Q< 0.0272 Å−1 to suppress subsidiary maxima at large distances (45Chacon P. Diaz J.F. Moran F. Andreu J.M. J. Mol. Biol. 2000; 299: 1289-1302Crossref PubMed Scopus (112) Google Scholar). For modeling of the structure, three programs were used: SASHA (46Svergun D.I. Volkov V.V. Kozin M.B. Stuhrmann H.B. Acta Crystallogr. Sect. A. 1996; 52: 419-426Crossref PubMed Scopus (124) Google Scholar), DAMMIN (47Svergun D.I. Biophys. J. 1999; 76: 2879-2886Abstract Full Text Full Text PDF PubMed Scopus (1756) Google Scholar), and DALAI_GA (45Chacon P. Diaz J.F. Moran F. Andreu J.M. J. Mol. Biol. 2000; 299: 1289-1302Crossref PubMed Scopus (112) Google Scholar, 48Chacon P. Moran F. Diaz J.F. Pantos E. Andreu J.M. Biophys. J. 1998; 74: 2760-2775Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Model building using spherical harmonics (SASHA) was carried out up to a maximum harmonic order of 4, corresponding to 19 independent parameters for 11.9 Shannon channels. The dummy atom-simulated annealing program DAMMIN automatically subtracted a small constant from each data point to force the Q−4 decay of the intensity at higher angles (49Porod G. Glatter O. Kratky O. Small Angle X-ray Scattering. Academic Press, Inc., London1982Google Scholar). Subtraction of the same constant before modeling using SASHA had no effect on the final structure. Modeling using the genetic algorithm DALAI_GA was carried out using an initial conformational space in the form of a sphere or a prolate ellipsoid with maximum dimensions at least 30 Å greater than that given by GNOM using cycles with progressively smaller dummy atoms starting at a radius of 10 Å decreasing to 5 Å in steps of 1 Å. A small constant determined by DAMMIN was also subtracted from the data before modeling with DALAI_GA to suppress internal cavities in the structure (45Chacon P. Diaz J.F. Moran F. Andreu J.M. J. Mol. Biol. 2000; 299: 1289-1302Crossref PubMed Scopus (112) Google Scholar). Model structures were visualized using the program ASSA (50Kozin M.B. Volkov V.V. Svergun D.I. J. Appl. Crystallogr. 1997; 30: 811-815Crossref Google Scholar). Optimal production of recombinant CPIII was found when insect cells were infected with baculovirus for 45 h with a multiplicity of infection (expressed as number of viral particles per cell) = 3. As previously reported (32Zafarullah K. Brown E.M. Kuivaniemi H. Tromp G. Sieron A.L. Fertala A. Prockop D.J. Matrix Biol. 1997; 16: 201-209Crossref PubMed Scopus (8) Google Scholar), the presence in the medium of the ∼90-kDa disulfide-linked C-propeptide trimer was confirmed by SDS-PAGE in reducing and non-reducing conditions (not shown). In reducing conditions, two bands (I and II) were systematically observed with apparent molecular masses of 34 and 32 kDa, respectively. Both bands were identified by immunoblotting using monoclonal antibody 48B14, specific for the central region of the human procollagen III C-propeptide (not shown). The relative intensities of these bands varied as a function of multiplicity of infection with the upper band (band I), predominating at a multiplicity of infection = 3. In these conditions CPIII production, representing 30% of total protein in the medium, was about 20 mg/liter. Recombinant CPIII was purified using a three-step procedure (Fig.1). As previously reported (32Zafarullah K. Brown E.M. Kuivaniemi H. Tromp G. Sieron A.L. Fertala A. Prockop D.J. Matrix Biol. 1997; 16: 201-209Crossref PubMed Scopus (8) Google Scholar), initial purification was by concanavalin-A affinity chromatography. Subsequent cation exchange chromatography at low pH led to considerable losses due to proteolysis; hence, this step was replaced by DEAE anion-exchange at pH 8.5. A final purification step by hydrophobic interaction on butyl-Sepharose resulted in essentially pure CPIII that was enriched in band I in the early part of the elution gradient (see Fig. 1). The total yield of purified CPIII from 1 liter of conditioned medium was 6.4 mg. To determine the nature of bands I and II seen by SDS-PAGE in reducing conditions, purified CPIII was probed by immunoblotting using monoclonal antibodies specific for the N-terminal 30 residues (antibody 48D34) and C-terminal 39 residues (antibody 48D19) of human CPIII (37Burchardt E.R. Heke M. Kauschke S.G. Harjes P. Kohlmeyer J. Kroll W. Schauer M. Schroeder W. Voelker M. Matrix Biol. 1998; 17: 673-677Crossref PubMed Scopus (6) Google Scholar). Both bands I and II were recognized by both antibodies (not shown), indicating that the difference in apparent molecular mass was not due to partial proteolytic degradation. A further possibility was differences in post-translational modifications, in particular,N-linked glycosylation. The observed molecular mass difference (∼2 kDa) corresponds to the presence of a single high mannose glycan Man9GlcNAc2, as commonly found in glycoproteins produced in insect cells (41Jarvis D.L. Kawar Z.S. Hollister J.R. Curr. Opin. Biotechnol. 1998; 9: 528-533Crossref PubMed Scopus (85) Google Scholar). In addition, there is a single asparagine linked N-glycosylation site in each of the three identical chains of CPIII. Recombinant CPIII, both native and denatured, was thus subjected to treatment by N-glycosidase F, then analyzed by SDS-PAGE in reducing and non-reducing conditions. As shown in Fig. 2,N-glycosidase F treatment of native CPIII led to the appearance of four bands migrating in the region of 100 kDa by SDS-PAGE in non-reducing conditions as well as a relative increase in the intensity of band II in reducing conditions. When exposed toN-glycosidase F after denaturation of CPIII, deglycosylation was complete, resulting in a single fast migrating band in non-reducing conditions and the total disappearance of band I in reducing conditions. We conclude that the four bands observed in non-reducing conditions correspond to CPIII trimers glycosylated at a single site on 0, 1, 2, or 3 chains. Thus, the recombinant CPIII used in these experiments consists of a mixture of mostly fully glycosylated (i.e. on all three chains) and partially glycosylated (i.e. on two of three chains) trimers. Analysis of CPIII by static light scattering (not shown) revealed no significant angular dependence in scattering intensity, indicating the absence of large aggregates in the concentration range studied (2–4 mg/ml). Extrapolation to zero concentration and zero angle using a Zimm plot gave a molecular mass of 103 ± 8 kDa, consistent with the mass calculated from the amino acid sequence of 88 kDa (TableI). We conclude that CPIII behaves as a freely soluble trimer (composed of three polypeptide chains) in solution.Table IHydrodynamic and scattering data for CPIIIMethodMolecular massStructural parametersD20,w (×107, cm2s−1) s20,w(×1013, s−1) Rg (Å)Diameter of equivalent spherekDaÅCalculated1-aAssuming one high mannose glycan Man9GlcNAc2 per chain (41).88.158.61-bUnhydrated.65.81-cAssuming hydration of 0.3 g of water/g of protein.Static light scattering103 ± 8Dynamic light scattering901-dMass calculated usings20,w from analytical ultracentrifugation. ± 6D20,w 4.8 ± 0.389 ± 6Analytical ultracentrifugation81 ± 6s20,w 5.0 ± 0.0580 ± 6D20,w 5.4 ± 0.4Small angle x-ray scattering100 ± 10Rg 33.4 ± 0.386.2 ± 0.41-a Assuming one high mannose glycan Man9GlcNAc2 per chain (41Jarvis D.L. Kawar Z.S. Hollister J.R. Curr. Opin. Biotechnol. 1998; 9: 528-533Crossref PubMed Scopus (85) Google Scholar).1-b Unhydrated.1-c Assuming hydration of 0.3 g of water/g of protein.1-d Mass calculated usings20,w from analytical ultracentrifugation. Open table in a new tab Dynamic light scattering of CPIII at 4 mg/ml gave an apparent diffusion coefficient (Table I) corresponding to a hydrodynamic radius 1.35 times greater than for a normally hydrated spherical protein of mass 88 kDa. This indicated that CPIII has a highly elongated shape equivalent to a prolate (cigar-like) or oblate (disc-like) ellipsoid with axial ratios 6.6 or 7.5, respectively. Sedimentation velocity profiles were nicely modeled by considering one single non-interacting species, giving similar results whatever the data treatment
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