Matrix Vol. 12/1992, pp. 256-263 © 1992 by Gustav Fischer Verlag, Stuttgart
Self-Assembly into Fibrils of a Homotrimer of Type I Collagen DANIELJ. McBRIDE, Jr. 1 , KARL E. KADLER 2 , YOSHIO HOJIMA and DARWIN J. PROCKOP Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine,Jefferson Medical College, ThomasJefferson University, Philadelphia, PA 19107, USA.
Abstract Type I collagen, the most ahundant structural protein in vertebrates, is comprised of two a1(1) chains and one a2(1) chain. Fibroblasts from a proband with osteogenesis imperfecta, however, were shown to synthesize a type I procol!agell that was a homotrimer of proa1(1) chains. The absence of proa2(1) chains in the procollagen provided a unique opportunity to assess the role of the a2(1) chain in collagen fibrilJogenesis by examining the self-assembly de novo of the homotrimeric collagen generated in vitro. The results demonstrated that the fibrils formed by the homotrimeric collagen had an asymmetric handing pattern similar to fibrils of normal heterotrimeric type I collagen. However, the efficiency for self-assembly of the homotrimer into fibrils was markedly reduced in that the critical concentration at 37°C was 40-fold greater than for selfassembly of the heterotrimeric molecule. A van't Hoff-type plot of the data was used to determine values for ~G, ~H and ~S. The values indicated the self-assembly of the homotrimer is similar to self-assembly of the heterotrimer in that the process is entropy driven. The process is, however, less favorable in that the ~G value was 10 k]/molless negative. The results suggest that the presence of the a2(1) chain in type I collagen helps drive the self-assembly process, probably because the a2(1) chain is more hydrophobic than the aI(I) chain and, therefore, smaller amounts of structured water may be lost during self-assembly of the homotrimer than during self-assembly of the heterotrimer. Also, the absence of the additional sequence information provided by the a2(1) chain may make side chain interactions in fibrils less favorable. The data, however, did not rigorously exclude the possibility that some of the differences between the homotrimer and normal type I collagen are explained by the post-translational over-modification present in the homotrimer. Key words: fibrils, homotrimer, self-assembly, type I collagen.
Introduction Type I collagen is the most abundant member of a family of fibril-forming collagens and the most abundant struc1 Present address: Bone Metabolism Research Laboratory, Division of Geriatric Medicine and Gerontology, Johns Hopkins University, Baltimore, MD 21 224, USA. 2 Present address: Department of Biochemistry and Molecular Biology, School of Biological Sciences, University of Manchester, Manchester, M13 9PT, UK.
tural protein in vertebrates. The monomer of type I collagen is a heterotrimer of two a1(1) and one a2(1) chains, and the heterotrimeric composition is found in species as diverse as man, rodents, birds, fish, and perhaps elasmobranchs (Lewis and Piez, 1964; Kelly et aI., 1988). Therefore, the heterotrimeric structure of type I collagen has apparently been conserved through selective pressure during evolution for about the past SOO-million years. In contrast to the relative abundance of type I collagen found in animals, normal tissues contain only trace
Self-Assembly of Type I Homotrimeric Collagen amounts of homotrimeric type I collagen consisting of a 1(I) chains. However, fibroblasts from three probands with osteogenesis imperfecta were shown to synthesize a type I procollagen that was a homotrimer of proal(I) chains (Nicholls et aI., 1979; Dickson et aI., 1984; Deak et aI., 1983; Pihlajaniemi et aI., 1984; Saski et aI., 1987). The mutation in one of these probands was shown to be a frameshift deletion of four base pairs that caused synthesis of non-functional proa2(I) chains (Dickson et aI., 1984; Pihlajaniemi et aI., 1984). Here we have used the homotrimeric type I procollagen from the proband's fibroblasts to help to define the role of the a2(I) chain in self-assembly of type I collagen into fibrils.
Materials and Methods Procollagen purification Fibroblasts from the proband were grown in DMEM to confluency in 175-cm 2 flasks in the presence of 10% fetal calf serum and without antibiotics (see Deak et aI., 1983; Kadler et aI., 1987; 1988; 1990). To obtain [14C]-labeled procollagen, fresh DMEM containing 1 [tCi/ml of [14C]labeled amino acids (lCN) and 25 [tg/ml of ascorbic acid was added. The medium was recovered at 24 h and the cells were then incubated for one additional 24-h period with the [14C]-labeling medium followed by one 24-h period without [14C]-labeled amino acids. The three samples of medium were combined with VIO volume of buffer consisting of 250mM EDTA/0.2% NaN]11 M Tris-HCI buffer (pH 7.4) and cooled at 4 °C for 30 min. Protein was precipitated by the addition of ammonium sulfate (176 mg/ml) and stirred overnight slowly. The pellets from the three collection periods were combined and resuspended in storage buffer consisting of O.4M NaCI, 0.04% NaN], and 0.1 M Tris-HCI, pH 7.4. The combined suspensions were stirred at 4 °C for a minimum of 4 h. After a second centrifugation, the pellet was discarded and the supernatant was dialyzed twice against 2 M urea/0.2 M NaCII0.04% NaN]/ 0.1 M Tris-HCI, adjusted to pH 7.5. The sample was loaded onto a 1.6 x 5 em column of DEAE cellulose (DE52, Whatman) that was equilibrated and eluted with the dialysis buffer (Fiedler-Nagy et aI., 1982). The pooled flow-through fractions were dialyzed twice against 21 of buffer consisting of 2 M urea/0.04% NaN]/ 0.075 M Tris-HCI (pH7.8). The sample was chromatographed on a second column (1.6 x 5 cm) of DEAE cellulose that was eluted with a linear gradient of 0 to 0.20 M NaC!. Fractions containing the type I procollagen were pooled and twice dialyzed against 2 L of storage buffer. Pooled fractions were concentrated by pressure ultrafiltration using an Amicon YM30 or YMI00 membrane. For reasons that were not apparent, the yields of the homotrimer through the purification and concentration steps were consistently lower than the yields of normal type I procollagen.
257
Formation and purification ofpCcollagen Partially purified chick procollagen N-proteinase (EC 3.4.24.14) was used to form pCcollagen enzymically (Hojima et aI., 1989; Kadler et aI., 1987; 1988; 1990). Procollagen (50-100 [tg/ml) was digested with 50-70 units/ml of the N-proteinase in the presence of 5 mM Ca + + and 0.15 M NaC! at 30°C. The reaction was stopped by the addition of 1/10 volume of 0.25 M EDTA and 1 M Tris-HCI buffer (pH 7.4). After dialysis against 2 M urea and 75 mM Tris-HCI buffer (pH 7.8), the sample was loaded onto a third column of DEAE cellulose, and the pCcoliagen was collected in 5-ml fractions in the flow through. The pCcollagen was then concentrated by ultrafiltration as described above. Specific activity was determined using a colormetric hydroxyproline assay and assuming a 10% hydroxyproline content for type I collagen (Fiedler-Nagy et aI., 1982).
Cleavage ofpCcollagen by C-proteinase to generate collagen fibrils Concentrated pCcoliagen and a purified chick C-proteinase (Hojima et aI., 1985) were dialyzed twice against 500 ml of a fibril-forming buffer at 4°C and under an atmosphere of 10% CO 2 and 90% air. The fibril formation buffer was 20mM NaHCO], 117mM NaC!, 5.4mM KCI, 1.8mM CaCh, 0.81 mM MgS0 4 , 1.03 mM NaH 2 P0 4 , and 0.04% NaN], pH 7.3. Fibril formation was initiated by mixing pCcoliagen with C-proteinase. The concentration of Cproteinase was varied with temperature in order to adjust for 3.6-fold change per 10°Cin enzyme activity between 20 and 41 °c (Hojima et aI., 1985). C-proteinase activity was determined using a rapid assay procedure of Hojima et al. (1985). One unit of enzyme activity was defined as the amount of enzyme that cleaved l[tg of type I procollagen under standard conditions in 1 h at 35°C.
Assessment ofcollagen fibril formation Fibril formation was assayed by absorbance-time measurements and direct visualization of fibrils by light and electron microscopy. A 120-[t1 reaction mixture of pCcollagen and C-proteinase in fibril formation buffer was preheated in a water bath at the indicated temperature for 5 to 10 min prior to transfer to a preheated quartz microcuvette that was subsequently sealed with a greased stopper. The reaction mixture in the cuvette was equilibrated with water-saturated 10% CO 2 and 90% air. Changes in the absorbance of the reaction mixture were measured at 313 nm in a Gilford Response spectrophotometer fitted with a temperature-controlled cuvette holder (Kadler et aI., 1987). Four measurements of absorbance were taken per minute by the spectrophotometer for a period of 999 min. For light microscopy, a 50-[t1 sample was placed on a concave glass slide. After charging the sample with watersaturated 10% CO 2 and 90% air, the sample was sealed
258
D.]. McBride et al.
with a greased coverslip and incubated at 35°C for 24 h. Fibrils formed on the slide were examined at room temperature with dark-field light microscopy. For electron microscopy, 50-,d samples were incubated for 24 h and centrifuged for 4 min at 13 000 rpm to form a pellet of fibrils. The pellet was fixed in 1% formaldehyde and 0.2 M sodium cacodylate buffer (pH 7.4) for 12 to 24 h. The pellet was then stabilized with 0.5% agarose with 0.01 % NaN). Prior to embedding, the pellet was stained en bloc with 1% uranyl acetate in 50% ethanol. Thin sections were cut and stained with 0.4% phosphotungstic acid and 1% uranyl acetate and examined in a Philips CM 10 transmission electron microscope. Critical concentration measurements
Results
The critical concentration was determined for reaction mixtures incubated at 22 to 40°C. The 50-Ill samples contained 65 Ilg/ml of pCcollagen and 50 Vlml of procollagen C-proteinase and were incubated for 24 or 48 h in 1.5-ml microcentrifuge tubes. At the end of the incubation period, the samples were centrifuged at room temperature for 4 min at 13 000 rpm in a Model 5414 Eppendorf centrifuge. The supernatants were carefully drawn off and placed in a second microcentrifuge tube. Pellets were then resuspended in 40 III of fibril formation buffer. Supernatants and pellets were prepared for SDS-polyacrylamide gel electrophoresis by adding 0.25 vol of 5 x sample buffer and heating at 100°C for 3 min. The 5 x sample buffer was 50% glycerol, 10% SDS, 0.005% bromphenol blue, and 0.625 M TrisHCI buffer, pH 6.8. The sample was reduced by adding ~HEAT TEST
mercaptoethanol and heated at 100°C for 3 min. The samples were separated by electrophoresis on a 3.5% polyacrylamide stacking gel and a 7 or 7.5% polyacrylamide resolving gel (Laemmli, 1970) and processed for fluorography using 20% 2,5-diphenyloxazole in glacial acetic acid. Dried gels were exposed to pre-flashed Kodak XAR-5 film at - 70°C. Multiple exposures of the gels were scanned with an LKB Vltrascan XL laser densitometer. The concentration of collagen in the supernatant was determined from the band intensity measurements of the a1(1) chains in the pellets and supernatants corrected by the total [14C]-labeled collagen in the sample and its specific activity (see Kadler et aI., 1987).
pC
+
Isolation ofhomotrimeric type I pCcoliagen
Protein in the medium from the proband's cultured skin fibroblasts was precipitated with ammonium sulfate and then chromatographed on two columns of DEAE cellulose. The homotrimer of type I procollagen co-eluted in the flow through volume with other procollagens in the first DEAEcellulose column. In the second DEAE-cellulose column, the homotrimer of type I procollagen eluted at approximately 0.055 M NaCl and just before type III procollagen that eluted at 0.065 M NaCl. To separate the homotrimer of type I procollagen from the type III procollagen, the mixture of the two proteins was digested with procollagen Nproteinase that cleaves the N-propeptide from type I procollagen but not from type III procollagen (Hojima et
C- PROTEI ASE
OEm LOAD
pC a 1(1) nl
Self-Assembly into Fibrils of a Homotrimer of Type I Collagen DANIELJ. McBRIDE, Jr. 1 , KARL E. KADLER 2 , YOSHIO HOJIMA and DARWIN J. PROCKOP Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine,Jefferson Medical College, ThomasJefferson University, Philadelphia, PA 19107, USA.
Abstract Type I collagen, the most ahundant structural protein in vertebrates, is comprised of two a1(1) chains and one a2(1) chain. Fibroblasts from a proband with osteogenesis imperfecta, however, were shown to synthesize a type I procol!agell that was a homotrimer of proa1(1) chains. The absence of proa2(1) chains in the procollagen provided a unique opportunity to assess the role of the a2(1) chain in collagen fibrilJogenesis by examining the self-assembly de novo of the homotrimeric collagen generated in vitro. The results demonstrated that the fibrils formed by the homotrimeric collagen had an asymmetric handing pattern similar to fibrils of normal heterotrimeric type I collagen. However, the efficiency for self-assembly of the homotrimer into fibrils was markedly reduced in that the critical concentration at 37°C was 40-fold greater than for selfassembly of the heterotrimeric molecule. A van't Hoff-type plot of the data was used to determine values for ~G, ~H and ~S. The values indicated the self-assembly of the homotrimer is similar to self-assembly of the heterotrimer in that the process is entropy driven. The process is, however, less favorable in that the ~G value was 10 k]/molless negative. The results suggest that the presence of the a2(1) chain in type I collagen helps drive the self-assembly process, probably because the a2(1) chain is more hydrophobic than the aI(I) chain and, therefore, smaller amounts of structured water may be lost during self-assembly of the homotrimer than during self-assembly of the heterotrimer. Also, the absence of the additional sequence information provided by the a2(1) chain may make side chain interactions in fibrils less favorable. The data, however, did not rigorously exclude the possibility that some of the differences between the homotrimer and normal type I collagen are explained by the post-translational over-modification present in the homotrimer. Key words: fibrils, homotrimer, self-assembly, type I collagen.
Introduction Type I collagen is the most abundant member of a family of fibril-forming collagens and the most abundant struc1 Present address: Bone Metabolism Research Laboratory, Division of Geriatric Medicine and Gerontology, Johns Hopkins University, Baltimore, MD 21 224, USA. 2 Present address: Department of Biochemistry and Molecular Biology, School of Biological Sciences, University of Manchester, Manchester, M13 9PT, UK.
tural protein in vertebrates. The monomer of type I collagen is a heterotrimer of two a1(1) and one a2(1) chains, and the heterotrimeric composition is found in species as diverse as man, rodents, birds, fish, and perhaps elasmobranchs (Lewis and Piez, 1964; Kelly et aI., 1988). Therefore, the heterotrimeric structure of type I collagen has apparently been conserved through selective pressure during evolution for about the past SOO-million years. In contrast to the relative abundance of type I collagen found in animals, normal tissues contain only trace
Self-Assembly of Type I Homotrimeric Collagen amounts of homotrimeric type I collagen consisting of a 1(I) chains. However, fibroblasts from three probands with osteogenesis imperfecta were shown to synthesize a type I procollagen that was a homotrimer of proal(I) chains (Nicholls et aI., 1979; Dickson et aI., 1984; Deak et aI., 1983; Pihlajaniemi et aI., 1984; Saski et aI., 1987). The mutation in one of these probands was shown to be a frameshift deletion of four base pairs that caused synthesis of non-functional proa2(I) chains (Dickson et aI., 1984; Pihlajaniemi et aI., 1984). Here we have used the homotrimeric type I procollagen from the proband's fibroblasts to help to define the role of the a2(I) chain in self-assembly of type I collagen into fibrils.
Materials and Methods Procollagen purification Fibroblasts from the proband were grown in DMEM to confluency in 175-cm 2 flasks in the presence of 10% fetal calf serum and without antibiotics (see Deak et aI., 1983; Kadler et aI., 1987; 1988; 1990). To obtain [14C]-labeled procollagen, fresh DMEM containing 1 [tCi/ml of [14C]labeled amino acids (lCN) and 25 [tg/ml of ascorbic acid was added. The medium was recovered at 24 h and the cells were then incubated for one additional 24-h period with the [14C]-labeling medium followed by one 24-h period without [14C]-labeled amino acids. The three samples of medium were combined with VIO volume of buffer consisting of 250mM EDTA/0.2% NaN]11 M Tris-HCI buffer (pH 7.4) and cooled at 4 °C for 30 min. Protein was precipitated by the addition of ammonium sulfate (176 mg/ml) and stirred overnight slowly. The pellets from the three collection periods were combined and resuspended in storage buffer consisting of O.4M NaCI, 0.04% NaN], and 0.1 M Tris-HCI, pH 7.4. The combined suspensions were stirred at 4 °C for a minimum of 4 h. After a second centrifugation, the pellet was discarded and the supernatant was dialyzed twice against 2 M urea/0.2 M NaCII0.04% NaN]/ 0.1 M Tris-HCI, adjusted to pH 7.5. The sample was loaded onto a 1.6 x 5 em column of DEAE cellulose (DE52, Whatman) that was equilibrated and eluted with the dialysis buffer (Fiedler-Nagy et aI., 1982). The pooled flow-through fractions were dialyzed twice against 21 of buffer consisting of 2 M urea/0.04% NaN]/ 0.075 M Tris-HCI (pH7.8). The sample was chromatographed on a second column (1.6 x 5 cm) of DEAE cellulose that was eluted with a linear gradient of 0 to 0.20 M NaC!. Fractions containing the type I procollagen were pooled and twice dialyzed against 2 L of storage buffer. Pooled fractions were concentrated by pressure ultrafiltration using an Amicon YM30 or YMI00 membrane. For reasons that were not apparent, the yields of the homotrimer through the purification and concentration steps were consistently lower than the yields of normal type I procollagen.
257
Formation and purification ofpCcollagen Partially purified chick procollagen N-proteinase (EC 3.4.24.14) was used to form pCcollagen enzymically (Hojima et aI., 1989; Kadler et aI., 1987; 1988; 1990). Procollagen (50-100 [tg/ml) was digested with 50-70 units/ml of the N-proteinase in the presence of 5 mM Ca + + and 0.15 M NaC! at 30°C. The reaction was stopped by the addition of 1/10 volume of 0.25 M EDTA and 1 M Tris-HCI buffer (pH 7.4). After dialysis against 2 M urea and 75 mM Tris-HCI buffer (pH 7.8), the sample was loaded onto a third column of DEAE cellulose, and the pCcoliagen was collected in 5-ml fractions in the flow through. The pCcollagen was then concentrated by ultrafiltration as described above. Specific activity was determined using a colormetric hydroxyproline assay and assuming a 10% hydroxyproline content for type I collagen (Fiedler-Nagy et aI., 1982).
Cleavage ofpCcollagen by C-proteinase to generate collagen fibrils Concentrated pCcoliagen and a purified chick C-proteinase (Hojima et aI., 1985) were dialyzed twice against 500 ml of a fibril-forming buffer at 4°C and under an atmosphere of 10% CO 2 and 90% air. The fibril formation buffer was 20mM NaHCO], 117mM NaC!, 5.4mM KCI, 1.8mM CaCh, 0.81 mM MgS0 4 , 1.03 mM NaH 2 P0 4 , and 0.04% NaN], pH 7.3. Fibril formation was initiated by mixing pCcoliagen with C-proteinase. The concentration of Cproteinase was varied with temperature in order to adjust for 3.6-fold change per 10°Cin enzyme activity between 20 and 41 °c (Hojima et aI., 1985). C-proteinase activity was determined using a rapid assay procedure of Hojima et al. (1985). One unit of enzyme activity was defined as the amount of enzyme that cleaved l[tg of type I procollagen under standard conditions in 1 h at 35°C.
Assessment ofcollagen fibril formation Fibril formation was assayed by absorbance-time measurements and direct visualization of fibrils by light and electron microscopy. A 120-[t1 reaction mixture of pCcollagen and C-proteinase in fibril formation buffer was preheated in a water bath at the indicated temperature for 5 to 10 min prior to transfer to a preheated quartz microcuvette that was subsequently sealed with a greased stopper. The reaction mixture in the cuvette was equilibrated with water-saturated 10% CO 2 and 90% air. Changes in the absorbance of the reaction mixture were measured at 313 nm in a Gilford Response spectrophotometer fitted with a temperature-controlled cuvette holder (Kadler et aI., 1987). Four measurements of absorbance were taken per minute by the spectrophotometer for a period of 999 min. For light microscopy, a 50-[t1 sample was placed on a concave glass slide. After charging the sample with watersaturated 10% CO 2 and 90% air, the sample was sealed
258
D.]. McBride et al.
with a greased coverslip and incubated at 35°C for 24 h. Fibrils formed on the slide were examined at room temperature with dark-field light microscopy. For electron microscopy, 50-,d samples were incubated for 24 h and centrifuged for 4 min at 13 000 rpm to form a pellet of fibrils. The pellet was fixed in 1% formaldehyde and 0.2 M sodium cacodylate buffer (pH 7.4) for 12 to 24 h. The pellet was then stabilized with 0.5% agarose with 0.01 % NaN). Prior to embedding, the pellet was stained en bloc with 1% uranyl acetate in 50% ethanol. Thin sections were cut and stained with 0.4% phosphotungstic acid and 1% uranyl acetate and examined in a Philips CM 10 transmission electron microscope. Critical concentration measurements
Results
The critical concentration was determined for reaction mixtures incubated at 22 to 40°C. The 50-Ill samples contained 65 Ilg/ml of pCcollagen and 50 Vlml of procollagen C-proteinase and were incubated for 24 or 48 h in 1.5-ml microcentrifuge tubes. At the end of the incubation period, the samples were centrifuged at room temperature for 4 min at 13 000 rpm in a Model 5414 Eppendorf centrifuge. The supernatants were carefully drawn off and placed in a second microcentrifuge tube. Pellets were then resuspended in 40 III of fibril formation buffer. Supernatants and pellets were prepared for SDS-polyacrylamide gel electrophoresis by adding 0.25 vol of 5 x sample buffer and heating at 100°C for 3 min. The 5 x sample buffer was 50% glycerol, 10% SDS, 0.005% bromphenol blue, and 0.625 M TrisHCI buffer, pH 6.8. The sample was reduced by adding ~HEAT TEST
mercaptoethanol and heated at 100°C for 3 min. The samples were separated by electrophoresis on a 3.5% polyacrylamide stacking gel and a 7 or 7.5% polyacrylamide resolving gel (Laemmli, 1970) and processed for fluorography using 20% 2,5-diphenyloxazole in glacial acetic acid. Dried gels were exposed to pre-flashed Kodak XAR-5 film at - 70°C. Multiple exposures of the gels were scanned with an LKB Vltrascan XL laser densitometer. The concentration of collagen in the supernatant was determined from the band intensity measurements of the a1(1) chains in the pellets and supernatants corrected by the total [14C]-labeled collagen in the sample and its specific activity (see Kadler et aI., 1987).
pC
+
Isolation ofhomotrimeric type I pCcoliagen
Protein in the medium from the proband's cultured skin fibroblasts was precipitated with ammonium sulfate and then chromatographed on two columns of DEAE cellulose. The homotrimer of type I procollagen co-eluted in the flow through volume with other procollagens in the first DEAEcellulose column. In the second DEAE-cellulose column, the homotrimer of type I procollagen eluted at approximately 0.055 M NaCl and just before type III procollagen that eluted at 0.065 M NaCl. To separate the homotrimer of type I procollagen from the type III procollagen, the mixture of the two proteins was digested with procollagen Nproteinase that cleaves the N-propeptide from type I procollagen but not from type III procollagen (Hojima et
C- PROTEI ASE
OEm LOAD
pC a 1(1) nl
