J. Mol. Biol. (1990) 211, 581-594

D-Periodic

Assemblies of Type I Procollagen

A. Paul Mouldl-f-, David J. S. Hulmes2, David F. Holmes1 Christine Cummings ‘, Christopher H. J. Sear3 and John A. Chapman1 IDepartment of Medical Biophysics, University of Manchester Oxford Road, Manchester Ml3 9PT, England of Biochemistry, ‘Department George Square, Edinburgh

University of Edinburgh EH8 9XD, Scotland

River Biotechnical Services Ltd Road, Margate, Kent CT9 4LT, England

3Charles

Manston

(Received 11 July

1989; accepted 25 September 1989)

The solubility limit of purified chick type I procollagen, incubated at 37°C in phosphatebuffered saline, was found to be in the range 1 to 1.5 mg/ml. At higher concentrations large aggregates formed. These comprised: (1) D-periodic assemblies; (2) narrow filaments with no apparent periodicity; and (3) segment-long-spacing-like aggregates. The D-periodic assemblies, which predominated at high concentrations, were separated from the other types of aggregate and found to be ribbon-like. Ribbons were uniform in thickness ( N 8 nm) and up to 1 ,um wide. Staining patterns showed features similar to those in native-type collagen fibrils. Immunolabelling indicated that the carboxyl-terminal propeptide domains were close to the carboxyl-terminal gap-overlap junction, and that the amino-terminal propeptide domains were folded over into the amino-terminal side of the overlap zone. Both propeptide domains appeared to be located on the surface of the assemblies. These observations show that intact propeptide domains hinder, but do not prevent, the formation of D-periodic assemblies. The presence of the propeptide domains on the surface of a growing assembly could restrict its lateral growth and limit its final thickness.

domain the antibodies to N-propeptide (Fleischmajer et al., 1983, 1985, 1987a,6). ;Such observations have led to the suggestion that N,-propeptide domains can regulate fibril diameters (Fleischmajer et al., 1983; Hulmes, 1983; Chaplman, 1989aJ). When N-propeptide processing is defective, as in the hereditary disorder dermatosparaxis,

1. Introduction Type I collagen in the extracellular matrix is normally found as D-periodic fibrils (D=67 nm; for reviews, see Ruggeri & Motta, 1.984; Miller, 1985; Cheah, 1985). While fibrils can ble reconstituted in vitro from purified collagen (Veis & Payne, 1988), the deposition of type I collagen in viwo involves enzymic processing of type I procollagen to remove both N- and C-terminalt propeptide domains (Tanzawa et al., 1985; Hojima et aZ., 1985; Kessler et al., 1986). Several observations have indicated that procollagen N-propeptide domains can modulate the form of collagen fibril assembly (Hulmes et al., 1989a). In embryonic tissues, only narrow fibrils (approx. 20 nm in diameter) are D-periodically labelled with

$ Abbreviations used: N- and C-, amino- and carboxyl-terminal, respectively; pN-collagen and pC-collagen, intermediates in the conversion of procollagen to collagen containing, respectively, the N-propeptide domain only and the C-propeptide domain only; N-EM, N-ethylmaleimide; PMSF, phenylmethlylsulphonylfluoride; BSA, bovine serum albumin; Ig, immunoglobulin; PEG, polyethylene glycol; PBS, phosphate-buffered saline; SDS/PAGE, SDS/ polyacrylamide gel electrophoresis; proa chains, polypeptides of procollagen; pCa chains, polypeptides of pC-collagen; PBS/Tween, phosphate-buffered saline containing 0.5% (w/v) Tween 20; SLS, segment-longspacing; pNa chains; polypeptides of pN-collagen; a-chains, polypeptides of collagen; s.D., standard deviation.

7 Author to whom all correspondence should be addressed. Present address: Department of Biochemistry and Molecular Biology, University of Manchester, Stopford Building, Oxford Road, Manchester Ml3 SPT, England. 002%2836/90/030581-14 $03.00/O

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fibril morphology is severely distorted (see Olsen, 1981). pN-collagen prepared from dermatosparactic tissue forms abnormal fibrils in vitro (Bailey & Lapiere 1973; Lap&e & Nusgens, 1974; Miyahara et al., 1983): alt,hough D-periodic, the fibrils are thin and ribbon-like. Also, when pN-collagen is generated de novo, by enzymic processing of purified procollagen, it forms thin D-periodic “sheets” up to several ,um wide (Hulmes et al., 19896). The role of procollagen C-propeptide domains in collagen fibril assembly is less well understood. In embryonic skin, fibrils are not labelled in a periodic pattern with antibodies to the C-propeptide domain (Fleischmajer et al., 1983). It was suggested that the bulkiness of the C-propeptide domain prevented incorporation of procollagen or pC-collagen into D-periodic structures (Fleischmajer et aZ., 1985). This suggestion was supported by experiments in vitro: no fibril formation occurred in solutions of procollagen or pC-collagen until the C-propeptides were cleaved (Miyahara et al., 1982, 1984; Kadler et al., 1987, 1988). More recent observations suggest, however, that molecules retaining the C-propeptide domain can, under eertain conditions, participate in fibril formation. In embryonic tibiae, the C-propeptide domains have been localized at approx. 60 nm intervals on fibrils with diameters in the range 10 to 140 nm (Fleischmajer et al., 1987a). Similar results were obtained, though on smaller diameter fibrils, in human fibroblast culture medium (Phelps et al., 1985). Also, in chick embryo chondrocyte cultures, fibrils were labelled D-periodically with antibodies to the C-propeptide domain of type II procollagen (Ruggiero et al., 1988). It is generally thought that the presence of both N and C-propeptide domains prevents the assembly of procollagen molecules into D-periodic structures. Here we show, however, that purified type I procollagen can form D-periodic assemblies in vitro at sufficiently high concentrations. The assemblies are ribbon-like and have a uniform thickness of about 8 nm. The presence and position of the K and C-propeptide domains can be demonstrated by immunogold labelling and heavy-metal staining. The results suggest that both N and C-propeptide domains lie on the surface of the assemblies. The implications of in vivo are these findings for fibrillogenesis discussed. 2. Materials

and Methods

(a) Materials Trypsin solution (2.5 %, w/v) and penicillin/streptomycin (10,000 units penicillin/ml, 10,000 ng streptomycin/ ml) were supplied by Gibco Ltd, Paisley, Scotland, U.K. L-[5-3H]proline (25 Ci/mmol) was obtained from the Radiochemical Centre, Amersham, Bucks, U.K. Highly purified bacterial collagenase was obtained from Boehringer-Mannheim, Lewes, East Sussex, U.K. N-Ethylmaleimide (N-EM), phenylmethylsulphonylfluoride (PMSF), p-aminobenzamidine dihydrochloride, Brij 35, Tween 20, Nonidet P40, bovine serum albumin (BSA) and sodium ascorbate were purchased from Sigma Chemical

Co., Poole, Dorset, U.K. All other chemicals were obtained from BDH Chemicals, Poole, Dorset. U.K. DEAE-Sephacel, Sephacryl S500 and protein ASepharose CL-4B were supplied by Pharmacia Ltd, Milton Keynes, Bucks, U.K. Electron microscope grids were purchased from Agar Aids, Stansted, Essex: U.K. The 10 nm-gold conjugated to goat anti-mouse TgG (IO nm goat a,nti-mouse gold) and 5 nm-gold conjugated to goat anti-rabbit IgG (5 nm goat anti-rabbit gold) were obtained from Bio Cell Research Laboratories, Cardiff; Wales, U.K.; the 2 probe sizes were non-overlapping. A monoclonal antibody to the C-propeptide domain of human type I procollagen (McDonald et al.; 1986) was obtained from the Development Studies Hybridoma Bank, Baltimore, MD, U.S.A. (supplied as partly purified IgG). Polyclonal antiserum to the N-propeptide of bovine proal(1) (Fisher et al., 1987) was generously donated by Dr L. Fisher, National Institutes of Health, Bet,hesda, MD, U.S.A. (Both antibody preparations cross-react with chick type I procollagen.) Lathyritic chick embryo tendon collagen (Ward et aZ., 1986) was a gift from Dr N. Ward, University of Manchester. Manchester, U.K. (b) Procollagen purification

and characteksatio?z

Metatarsal tendons were removed from 10 to 20 dozen 17 day-old chick embryos, incubated with trypsin/bacterial collagenase (Dehm & Proekop, 1971) and the released tendon cells cultured as described (Mould 85 Hulmes, 1987) in the presence of 5 to 10 PCi of L-[5-3H]proline/ml, 50 units penicillin/ml, 50 ,ng streptomycin/ml and 50 fig ascorbate/ml. After 4 to 5 h in culture, the cells were removed by centrifugation and oneninth volume of 250 ma+EDTA in 1 rvr-Tris . HCl, (pH 7.4). containing 50 mg BSA/ml was added to the medium. (The pH of all buffers was adjusted at 20°C.) Procollagen was precipitated by adding polyethylene glycol (PEG) 4009 to a final concentration of 5 o/0 (w/v) and stirring the medium gently at 4°C overnight (Ramshaw et al., 1984). All subsequent operations were at this temperature. The precipitate was pelleted by centrifugation at 3000g for 30 min and the supernatant discarded. To remove excess PEG and BSA the pellets were washed by resuspension in distilled water and repelleted by centrifugation at 3000 g for 10 min. Pellets were then dissolved over several hours in approximately 40 ml of 94 M-NaCl, 0.1 MM-Tris HCI (pH 7.4), 2.5 mM-EDTA, and the resulting solution was clarified by centrifugation at 40,OOOg for 1 h. The supernatant was dialysed against 2 1 of 2 x-urea, 50 mM-Tris. HCl (pH 78), 25 mM-EDTA, clarified by centrifugation at 3000g for 20 min and purified by ionexchange chromatography on DEAE-Sephacel as described (Mould & Hulmes, 1987). Procollagen peak fractions were dialysed against PBS (150 rnx-NaCl, 7 mM-Na,HPO,, 3 mM-KH,PO,, 15 mM-NaN,, (pH 72), with or without protea,se inhibitors: 25 rnivr-EDTA, I mx-N-EM, 82 mM-PMSF, 61 mr\l-p-aminobenzamidine. Finally, procollagen was concentrated by PEG precipitation as described above, the pellets washed severai t.imes in distilled water to remove any remaining PEG and the pellets redissolved in small volumes of PBS. Concentrated procollagen solutions were clarified by eentrifugation at 3000 g for 10 min and stored at - 70°C. Labelled polypeptides were examined by SDS/PAGE (Laemmli, 1970) and fluorography (Skinner & Griswold; 1983). Fluorograms were scanned using an LKB 2202 Ultroscan laser densitometer. Electrophoresis under reducing conditions in SO,/, polyacrylamide gels showed that purified procollagen samples contained 90 t.o 98%

D-Periodic

Assemblies of Procollagen

proa chains and 2 to 10% pCa chains. Coomassie blue staining showed that the purified procollagen was essentially free of BSA and other proteins. Concentration measurements were made by hydroxyproline assay (Woessner, 1961) assuming 85 residues/1000 hydroxyproline (Fiedler-Nagy et al., 1981). The specific activity (30,000 to 50,000 cts/min per pg) was used to estimate all subsequent concentrations. For most of the experiments described here two procollagen preparations were used. Preparation A contained - 98 y. proct chains and had a concentration of 1.2 mg/ml. Preparation B contained -92 y. procl chains and had a concentration of 35 mg/ml. (c) Immunoprecipitation

Precipitation of procollagen molecules from solution using antibodies to the N and C-propeptides followed the procedure of Cooper et al. (1981). Briefly, purified procollagen in PBS was diluted to approx. 10 pg/ml in buffer A (0.4 M-NaCl, 50 m&r-Tris. HCl, 5 mM-EDTA 1 y. (v/v) Nonidet P40, pH 8.0), a portion of antiserum or partly purified antibody added, and the solution incubated at room temperature for 1.5 h. Normal mouse serum or normal rabbit serum were used as controls. Then 30 ~1 of a 50% suspension of protein A-Sepharose in buffer A was added to each sample and the samples were incubated with shaking at 4°C for 30 min. Samples were then centrifuged for 30 s at 3000g and the pellets washed twice in buffer A and then twice in 10 mivr-Tris’ HCl (pH 68). The pellets were resuspended in electrophoresis sample buffer, boiled for 3 min, centrifuged for 30 s at 3000g and the supernatants were electrophoresed as described above for purified procollagen samples. Labelled polypeptides were visualized by fluorography as described above. (d) Incubation

of purified

procollagen

Procollagen solutions (0.5 to 3.5 mg/ml) in PBS were clarified by centrifugation at 12,000 g for 5 min at 4°C. Portions (50 to 200 ~1) of the supernatant were incubated at 37°C in a waterbath under a water-saturated atmosphere for 24 h. For turbidity measurements a 220 ,a1 portion was preheated for 5 min in a waterbath at 37 “C, transferred to a preheated quartz microcuvette (path length 10 mm), and layered with an atmosphere of water-saturated air. The 5 min incubation time prior to turbidity measurements was used to prevent bubble formation in the cuvette, and thereby avoided the necessity of degassing solutions (Kadler et aZ., 1987). Changes in turbidity were assayed by absorbance at 313 nm in a LKB Ultrospec spectrophotometer fitted with a temperature-controlled cuvette holder. For assays of procollagen solubility, 50 ~1 portions were incubated as described above and then centrifuged at 12,000g for 5 min at room temperature. The concentration of procollagen in the supernata,nt was measured by scintillation counting of 3H cts/min. Supernatants and pellets were also analysed by SDS/PAGE on 6% gels, as described above. (e) Gel-jiltration

chromatography

Supernatants from incubated procollagen samples centrifuged at 12,000 g for 5 min were analysed using procedures similar to those described by Mould & Hulmes (1987). Supernatants were fixed by adding one-ninth volume of 10% formaldehyde in PBS and incubating at

37°C for 15 min. Samples were then diluted to ap:prox 1 ml with running buffer (PBS containing 0.5% (w/v) Brij 35) and loaded onto a 1.5 cm x 95 cm column of Sephacryl S500. The column was eluted with running buffer at 10 ml/h and fractions of - 2 ml were collected. Portions (100 ~1) were used for scintillation counting. (f) Electron

microscopy

(i)

Staining procedures Incubated procollagen solutions were prepared for electron microscopy by diluting the solutions, where necessary, with PBS at 37°C. A 10 ~1 drop was applied to a carbon-coated copper grid. After allowing 30 E: for adsorption of aggregates to the carbon film, the grid was blotted, washed with several drops of distilled water and either negatively stained with 1 y0 (w/v) sodium phosphotungstate, (pH 7.4), or positively stained with 1 %(w/v) phosphotungstic acid, (pH 2.2), followed by 1 y. (lw/v) uranyl acetate, (pH 44). The same procedures were used to examine resuspended procollagen aggregates (see Results).

(ii) Embedding and sectioning Pelleted procollagen aggregates were fixed with 4% formaldehyde in 200 mM-sodium cacodylate buffer, (pH 7.2), stained sequentially with 1 o/0 phosphotungstic acid (pH 2.2), and 1% uranyl acetate (pH 44), dehydrated in ethanol and embedded in Araldite using standard procedures. Thin (-40 nm) sections were poststained with phosphotungstic acid and uranyl acetabte as described above. Image analysis of transverse sections of pelleted aggregates was carried out using a modified Microsemper software package (Synoptics, Cambridge, U.K.) on an Olivetti M28/Matrox PIP1024 frame store system with TV input. (iii) Immunolabelling Resuspended aggregates (see Results) were adsorbled to carbon-coated nickel grids which were then washed with distilled water. To block non-specific reactions, grids were placed face downwards on 20 ~1 drops of PBS contabining 65% Tween 20 (PBS/Tween) for 15 min. They were then incubated for 16 h with (1) antibodies against the C-propeptide domain (2.4 pg partially purified IgG/ml in PBS/ Tween), (2) antibodies against N-propeptide domain (100 pg lyophilized antiserum/ml in PBS/Tween) or (3) a mixture of the two antibodies (2.4pg/ml and 50 ;lg/ml, respectively). After washing with PBS/Tween, grids! were incubated for 1 to 1.5 h with either 10 nm-goat anti-mouse or 5 nm-goat anti-rabbit gold probes 1 : 20 dilution in PBT/Tween) or a mixture of the probes (same final dilutions). Finally, grids were washed with distilled water (adjusted to pH %O with sodium hydroxide) and positively stained as described above. Normal mouse serum and normal rabbit serum (1 : 200 dilution in PBS/Tween) were used as controls. (iv) Rotary shadowing Rotary shadowing of fractions from the gel-filtration of supernatants (see above) was carried out at described (Mould et al., 1985; Mould & Hulmes, 1987). Briefly, fractions were diluted to approx. 025 pg/ml in running buffer and a 5 ~1 drop sandwiched between 2 freshly cleaved mica sheets. After washing in 62 M-ammonium acetate solution (pH 7.2) and freeze-drying, specimens were rotary shadowed with platinum and coated, with

584

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1

2.7

4

i 1

0 Incubation

time

24

(h)

Figure 1. Turbidimetric behaviour of procollagen solutions at 37°C. Procollagen concentrations (mg/ml) are as indicated in the Figure. No turbidity increase was observed for procollagen solutions of 1 mg/ml or below (not shown).

carbon. Replicas were floated off onto distilled water and picked up on 400 mesh copper grids. (g) Collagen j&i1

formation

Collagen fibrils were formed by neutralizing a one-ninth volume of chick embryo tendon collagen in 10 mM-&C&C a.cid with 1.1 x strength PBS and incubating the solution at 37°C for 4 h. The final collagen concentration was approx. 30 pg/ml. Collagen fibrils were stained for electron microscopy as described above for procollagen aggregates.

3. Results (a) Assembly

of procollagen

aggregates

Type 1 procollagen concentrated by PEG precipitation was soluble in PBS up to a maximum concentration of approximately 4 mg/ml at 4°C.

Incubation of procollagen solutions at 37°C for 24 hours resulted in a concentration-dependent increase in turbidity (Fig. 1). Incubated solutions were examined by electron microscopy after negative staining. Several t’ypes of aggregate were observed in solutions with concentrations above 1 mg/ml (see Fig. 2(a)). Most striking were the periodic assemblies, which possessed large, globular protrusions every period (arrowheads in Fig. 2(a)). The periodicity (62( + 3) nm, mean (&- s.D.), n = 10) was close to the D-periodicity of collagen fibrils prepared under the same buffer conditions (68 (+2nm), mean (+s.D.), n=7). These and other observations (see below) confirmed that the assem-

et, al. blies possessed a native D-periodicity. Two further types of aggregate were present: thin, filamentous material with no apparent periodicity and bundles of molecules packed as -300 nm-long segments (SLS-like aggregates; between the arrows in Fig. 2(a)). This latter type of aggregate was mere readily visible in sections of pelleted material (see below). At 1.2 mg/ml D-periodic assemblies constituted only a small percentage of the total aggregated material (as assessed by negative staining). concentrations at higher However, (up to 3.5 mg/ml) D-periodic assemblies predominated. No D-periodic structures were observed in solutions before incubation at 37°C or in incubated solutions of concentrations below 1 mg/ml (results not shown). The D-periodic assemblies could be largely separated from the non-periodic aggregates by repeated cycles of centrifugation at room temperature (5 min at 12,OOOg) and resuspension of the pelleted material in the original volume of PBS at 37°C. (Resuspension was essential to obtain “clean” preparations of the D-periodic assemblies for analysis by staining and other electron-optical techniques.) esuspended aggregates formed at an initial procollagen concentration of 3.5 mg/ml, are shown in Figure 2(b) (negatively stained) and (c) (positively stained). The assemblies appear ribbon-like and frequently twisted. Procollagen aggregates were also studied by pelleting using lowspeed centrifugat,ion (5 min, 112,000g at room temperature). Figure 3 shows thin sections of pellets obtained from samples equivalent to those in Figure 2. All three types of aggregate described above were present in t,he initial pellets from incubated procollagen solution (Fig. 3(a)). At the highest concentrations of procollagen the initial pellets contained mainly D-periodic assemblies (not shown). Pellets of resuspended aggregates (Fig. 3(b)) consisted almost entirely of D-periodic assemblies. The arrows in Figure 3(b) show D-periodic assemblies in transverse section. The result’s confirm that the assemblies are ribbon-like, with widths up to 1 pm and have a near uniform thickness of 7.8 (+l.l)nm (mean (fS.D.), n=92). Supernatants from incubated procollagen solutions (following an initial 5 min, 12,000 g centrifugation at room temperature) were analysed by gelfiltration on Sephacryl S500 after formaldehyde fixation. Figure 4 shows the elution profile of the supernatant from a solution incubated at 1.2 mg/ml. Rotary shadowing demonstrated that the void-volume peak (peak A) contained mainly large aggregat,es, which frequently consisted of bundles of inregister molecules (SLS-like aggregates) (Fig. 3). About 25% of the total recovered cts/min was present in the void volume peak. However, total recoveries were only about 70%, compared to - 9076 for monomeric samples (not shown). This suggests that some of the largest aggregates in the sample could not penetrate the gel-filtration column. Rotary shadowing also showed that peak B consisted mainly of dimers and peak C of monomers

Figure 2. Procollagen aggregates formed at 37°C. (a) Procollagen concentration 1.2 mg/ml. Note the D-periodic assemblies (centre). When viewed edge-on these structures are seen to be thin and to possess globular projections on both sides (arrowheads). Much unbanded filamentous material is present in the background. Narrow SLS-like aggregates, approx. 300 nm long (between the arrows) can also be detected amongst the background material. Negatively stained. (see the text) to purify the D-periodic assemblies, (b) Procollagen concentration 3.5 mg/ml. Sample was “resuspended” These structures appear twisted and ribbon-like, and have a prominent stain-excluding band every D-period (arrows), again seen edge-on as globular projections on both sides (arrowheads). Negatively stained. (c) As (b) but pos.itively stained. The broad dark band every D-period (arrows) appears to correspond to the prominent white band observed after negative staining. Scale bars represent 300 nm.

Figure 3. Thin sections of pelleted procollagen aggregates (positively stained). (a) Equivalent sample to that jn Fig. 2(a). Note the D-periodic structure (left of centre) surrounded by fine, filamentous material and SLS-like aggregates (arrows). (b) Equivalent sample to that in Fig. Z(b) and (c). Only D-periodic assemblies are observed (at various angles of sectioning) together with small amounts of filamentous material. Arrows show D-periodic assemblies in transverse section. Image analysis of transversely sectioned material showed that the assemblies had an approximately uniform thickness of 7.8 (f 1.1) nm (mean (*SD.), n=92) and widths ranging from -20 nm to N 1 pm with a mean value of 250 ( f 190) nm (s.D., n= 140). Measurements were made from electron micrographs taken at a nominal 50,000 x (t,hickness measurements) or 10,000 x magnification (width measurements). The scale bar (located in (b)) represents 300 nm.

D-Periodic

pNa2 chains could be detected. All the samples shown in Figure 6 had similar amounts of these components. Immunoprecipitation of procollagen with antibodies to either the N or the C-propeptides precipitated all the processed procollagen chains (p&l, pCa2, al and pNa2), indicating that they were part of “nicked” procollagen molecules which co-purify with intact procollagen (see Hulmes et al., 19893; results not shown).

A

C

6C

587

Assemblies of Procollagen

(b) Xolubility

measurements

Supernatants from 1.2 mg procollagen/ml solutions incubated for 24 hours at 37°C and then centrifuged (12,000g for 5 min at room temperature) had a concentration of 1.01 ( kO.06) mg/ml (mean (*s.D.), n=4). Supernatants from procollagen solutions at 3.5 mg/ml gave less reproducible results, with minimum values of approximately may have 1.5 mg/ml. This lack of reproducibility been due to the high viscosity of these solutions and the large gelatinous pellet that formed upon centrifugation, making it difficult to separate supern,atant from pelleted material.

50

40

L

Fraction

Figure 4. Gel-filtration chromatography of procollagen supernatant on Sephacryl 5500. The procollagen sample (1.2 mg/ml) was incubated at 37°C for 24 h then centrifuged 5 min at 12,000g. The supernatant was fixed with formaldehyde and applied to the column. Vc indicates the void volume (~74 ml). Three peaks of activity are observed: peak A eluting at the void volume, and peaks B and C, which have Vcz:84 ml and 100 ml, respectively.

(about 15% and SO%, respectively, of total recovered cts/min, results not shown). Similar results were obtained with supernatants from procollagen solutions incubated at 3.5 mg/ml. Solutions were also examined by SDS/PAGE before and after incubation at 37°C. Figure 6 shows the fluorogram from an experiment in which a procollagen sample of 3.5 mg/ml was incubated at 37 “C for 24 hours and then centrifuged at 12,000 g for five minutes. Supernatant and pellets had the same proportion of proa and pCa chains. Similar results were obtained at 1.2 mg/ml. Resuspended aggregates

(almost

entirely

D-periodic

structures)

showed an identical electrophoretic pattern (Fig. 6, lane 4). Fluorograms also showed that there was essentially no degradation or processing of the procollagen during the incubation. On prolonged exposure of the fluorograms, traces of polypeptides (< 1 y0 of total radio-labelled protein) migrating in the positions of the al and

(c) Analysis of staining patterns Staining patterns of the D-periodic assemblies were compared with those of fibrils formed from extracted and purified chick embryo tendon collagen. In Figure 7, these patterns are suitably aligned with the corresponding D-staggered array of collagen molecules. The procollagen assemblies showed native-type D-periodic staining patterns (see Chapman & Hulmes, 1984) and a gap-overlap structure. However, both negatively st’ained and positively stained assemblies Pig. 7(a)) (Fig. 7(c)) showed features not present in colllagen fibril staining patterns (Fig. 7(b) and (d)). ‘These included a prominent stain-excluding (white) band at the C-terminal ends of the procollagen molecules (arrows in Fig. 7(a)) and pronounced additional positive staining in the same region (between the a, and b, lines) centred about the C-terminal gap overlap junction. Differences between the staining patterns at or near the N-terminal ends of the apparent. molecules were much less readily However, the c2 line in the positive-staining pattern of the D-periodic assemblies (close to the N-terminal gapoverlap junction) was frequently reduced in intensity, relative to the other lines in a D period. (d) lmmunogold

labelling

Indirect immunolabelling with secondary antibodies conjugated to colloidal gold was used to gain more information about the location of the propeptide domains within the D-periodic procollagen assemblies. Labelling with a monoclonal antibody to the C-propeptide domain and a 10 nm goat antimouse gold probe (Fig. S(a) and (b)) showed a skewed distribution with a peak in the gap region near to the C-terminal gapoverlap junction. IWith a

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Figure 5. Rotary shadowing of the void volume peak. A sample from peak A of Fig. 4 was diluted to approx. 025 ,ag/ml, adsorbed to mica, washed in 0.2 M-ammonium acetate solution, freeze-dried and rotary-shadowed. The large aggregates shown contain units of in-register molecules (arrows). The scale bar represents 300 nm. polyelonal antibody to the N-propeptide domain and a, 5 nm goat anti-rabbit gold probe, higher concentrations of label were required to visualise

the D-periodic binding of the antibody (Fig. S(c)). The majority of the labelling was in the overlap region, close to the N-terminal gap-overlap junction (Fig. B(d)). Labelling with bot’h antibodies and a mixture of the two gold probes (Fig. B(e)) demonstrated the presence of both N and C-propeptide domains in the same structure. All the D-periodic assemblies labelled with both antibodies. Neither amibody showed any periodic labelling of collagen fib& (results not shown). Control samples incubated with mixtures of normal mouse serum and normal rabbit serum followed by a mixture of the two gold probes showed only sparse, non-specific labelling (Fig. S(fj).

pro a I

PC a I pro a 2 PC a 2

Figure 6. SDS/PAGE of procollagen solutions. Lane 1, Lanes 2 to 4, procollagen sample before incubation. samples (35 mg/ml) incubated at 37°C for 24 h. Lane 2, supernatant from sample centrifuged for 5 min at 12,000 g. Lane 3, pellet from sample centrifuged for 5 min at 12,000g. Lane 4, pellet (5 min at 12,000g) of resuspended aggregates (see the text). Equal numbers of counts were loaded in each lane. All the samples have essentially the same electrophoretic pattern. (Fluorogram of the gel is shown.)

4. Discussion (a) Preparation

of puri$ed

procollagen

Since chick type I procollagen has been extensively studied (Miyahara et al., 1982, 1984; Berg et al., 1986; Mould & Hulmes, 1987); medium from suspension cultures of chick embryo tendon fibroblasts was chosen as the source of procollagen for the present investigation. However, like other workers (Kadler et al., 1987), we found that this medium is heavily contaminated by proteases, which slowly degrade native procollagen and rapidly degrade denatured procollagen. It was found that addition of a large excess of BSA to the medium at the end of the culture period (see Materials and Methods) largely eliminated the contaminating proteases, and the purified procollagen was essentially free of protease activity. Addition of protease inhibitors to the purified procollagen before concentration gave a further slight improvement in stability. In the experiments reported here there was no degradation of purified procollagen

D-Periodic

Assemblies

qf Procollagen

OTp

589 Gap

l--r--Y

a40 e2’

I

Figure 7. Comparison of staining patterns of D-periodic procollagen assemblies with those of collagen fibrils. (a) and (c) Procollagen assemblies; (b) and (d) collagen fibrils; (a) and (b) negatively stained; (c) and (d) positively stained. The corresponding D-staggered array of collagen molecules is shown at the top of the Figure, with telopeptides shown in condensed conformations (see Chapman & Hulmes, 1984). At the bottom of the Figure the lines in the collagen positivestaining pattern are indicated. The staining patterns’of the procollagen assemblies are similar to those of the collagen fibrils. However, the contrast is relatively weak, presumably because the assemblies are much thinner than the collagen fibrils. Arrows in (a) show the prominent stain-excluding band of the negatively stained assemblies. This band shares the same position in the D period as the broad dark band present in (c), which occupies the region between the a, and b, lines. These bands are centred about the C-terminal gap-overlap junction (C-terminal end of the molecules). The bar (located in (d)) indicates the D period.

590

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;b) Gap

N

c B-4

Overlap

-4

Gap I

701

0.4’

Oi

Distance

Gap -B-s

N

’ f:om

overiop

0.8

0,6 ‘$’

I.0

line

C

GOP

5.6

0.8

100,

0’

ok

0.4 Distance

from

.”

Cs’ line

Figure 8. Immunogold labelling of D-periodic procollagen assemblies (positively stained). (a) Monoclonal antibody to C-propeptide domain and 10 nm goat anti-mouse gold probe. Labelling is D-periodic (arrows) and occurs at, or close to. the broad dark band in the positive staining pattern. (c) Polyclonal antibody to the N-propeptide domain and 5 nm goat anti-rabbit gold probe. Labelling is D-periodic (arrows). (e) Mixture of C and N-propeptide antibodies followed by 10 nm and 5 nm gold probes. The assembly is labelled with probes to both a.ntibodies. (f) Control incubated with a mixture of

D-Periodic

Assemblies of Procollagen

when it was incubated at 37°C for 24 hours at concentrations up to 3.5 mg/ml (Fig. 6). (b) Formation of procollagen aggregates Two previous approaches have been used to study the aggregation of procollagen at high concentrations. In the first (Hulmes et al., 1983; Gross & Bruns, 1984), culture medium was concentrated to a pellet by prolonged ultracentrifugation and thin sections of the pellet were analysed by electron microscopy. In the second (Mould & Hulmes, 1987), purified procollagen was concentrated by ultrafiltration and studied using gel-filtration chromotagraphy and density-gradient ultracentrifugation. However, both approaches are limited by the concentration range that can be investigated. In the first, the concentration of procollagen could be estimated only approximately (from the dimensions of the pellet) as > 16 “g/ml, and in the second, procollagen concentrations in excess of 1 mg/ml could not be obtained by ultrafiltration (A. P. Mould, unpublished results). The approach used here (concentration by PEG precipitation) allows procollagen concentrations to be obtained up to 4 mg/ml in a carefully controlled manner. Furthermore, previous studies were carried out at room temperature; with procollagen free of contaminating proteases it has now been possible to use the more physiological temperature of 37 “C. Incubation of concentrated solutions at 37°C led to the formation of several types of aggregate (Figs 2 and 3). SLS-like aggregates of procollagen have been described by many authors. They were first observed within the secretory vacuoles of epithelial cells (Trelstad, 1971) and have been seen subsequently in a number of other cell types (Weinstock & Leblond, 1974; Weinstock, 1975; Bruns et al., 1979; Cho & Garant, 1981; Wright & Leblond, 1981). Similar structures have been observed in fibroblast culture medium after concentration to a pellet by prolonged ultracentrifugation (Hulmes et al., 1983; Gross & Bruns, 1984). This type of aggregate was also seen in samples of culture media or purified procollagen observed after negative staining or rotary shadowing (Bruns et al., 1979; Mould & Hulmes, 1987). However, when analysed by density-gradient ultracentrifugation or gelfiltration chromatography, the same samples were shown to contain essentially only procollagen

monomers. We proposed that aggregation of procollagen is strongly favoured when samples are adsorbed to a mica or carbon surface during preparation for electron microscopy, and termed this phenomenon surface-induced aggregation (Mould & Hulmes, 1987). It is now apparent that SLS-like aggregates can form in solution at concentrations of procollagen higher than those previously studied. These aggregates could be separated from procollagen monomers by gel filtration and then examined by freeze-drying and rotary shadowing (Figs 4 and 5). It now seems likely that surface-induced aggregation takes place as a result of high, local concentrations of procollagen molecules at, or close to, the mica or carbon surface during preparation of samples for electron microscopy. Our observation of D-periodic assemblies in incubated procollagen solutions was unexpected; previous studies (performed at lower concentrations) had suggested that unprocessed procoll.agen molecules cannot assemble into fibrillar structures (Miyahara et al., 1982, 1984). D-periodic aggregates were observed in culture medium concentrated to a pellet by prolonged ultracentrifugation (Gross & Bruns, 1984). However, these results were based on heterogeneous preparations which contained mostly processed forms of procollagen, as well as other components of crude culture medium. The formation of D-periodic assemblies in the present experiments raised the question of possible contamination of purified procollagen with small amounts of pC-collagen, pN-collagen or collagen. These contaminants could have co-polymerized with procollagen to give rise to the D-periodic assemblies. In support of the view that the assemblies do consist entirely, or almost entirely, of procollagen, we would emphasize the following. (1) Resuspended aggregates (which comprised very largely D-periodic structures) had essentially the same eleetrophoretic pattern as the starting material (Fig. 6). Hence the assemblies must have contained mainly procollagen molecules. (2) The small amounts of pCcl, pNa and a-chains observed by SDS/PAGE and fluorography were probably part of “nicked” procollagen molecules (rather than pC-collagen, pN collagen or collagen molecules) because they co-purified with intact procollagen and because they co-precipitated with procl chains using antibodies to the N or C-propeptides (see Results). Furthermore, the cr2 chain

normal mouse serum and normal rabbit serum followed by 10 nm and 5 nm gold probes. (b) and (d) Histograms

of the distribution of C-propeptide and N-propeptide antibody labelling, respectively. In (b) labelling is close to the C-terminal gap-overlap junction. In (d) the majority of labelling occurs on the N-terminal side of the overlap zone. To measure the distribution of labelling, micrographs of single-labelled procollagen assemblies were enlarged to a final magnification of -500,000 x . The polarity was established from the banding pattern and the position of the d lines marked. (The d line was the strongest and most readily identifiable band within a D period.) For each gold probe, the axial distance from the d line on its N-terminal side was measured and converted to a fraction of the periodicity, D (the distance between successive d lines). Data in (b) were taken from several micrographs, as in (a) (n=304). Data in (d) were taken from micrographs as in (c) (n= 511). The resolution of the immunolabelling data is determined mainly by the size alf the antibody-anti-antibody-gold probe complex (see Beesley, 1985) and is expected to be only -20 nm. The spread of the histograms probably reflects this resolution limit, rather than indicating a variable position for the propeptide domains. The scale bar (located in (f)) represents 300 nm.

A. P. Mould was barely detectable even after prolonged exposure of fluorograms and had an intensity -tenfold less than that of al chain. This supports the suggestion that the a-chains were not derived from collagen molecules, where the ratio of the al/a2 chains is 2 : 1. (3) The D-periodic assemblies labelled with antibodies to both the N and C-propeptides, indicating that molecules possessing both propeptide domains were present. In summary, the results suggest that the formation of D-periodic assemblies is due to high concentrations of procollagen rather than to the presence of small amounts of processed forms. The observed solubility of procollagen in these experiments (1 to 1.5 mg/ml) is more than three orders of magnitude greater than the solubility of collagen at 37°C under similar buffer conditions (Kadler et al.: 1987). In this respect the collagen/ procollagen system appea,rs analogous to that of fibrin and its precursor, fibrinogen. At very high concentrations fibrinogen precipitates to form periodic polymers similar to those formed from fibrin at much lower concentrations (Voter et al., 1986a,b). The high solubility of procollagen appears to be conferred mainly by the C-propeptide domain since pN-collagen produced by processing of C-propeptides in vitro has a solubility of approximately 0.15 mg/ml at 37°C (Hulmes et al.: 1989a). It is not clear whether SLS-like aggregates or the unbanded filaments are precursors of the D-periodic assemblies. The continued presence of SLS-like aggregates and unbanded filaments when the formation of D-periodic assemblies is complete (as assayed by turbidity), suggests either that these structures are in slow equilibrium with the D-periodic assemblies or that they may not be intermediates but rather separate products. (c) Structure

of the D-periodic

assemblies

Heavy-metal staining and immunolabelling (Figs 7 and 8) were used to elucidate the structure of the D-periodic assemblies. The globular projections visualized after negative-staining (Fig. 2(a) and (lo)), and seen as broad dark bands after positive staining (Fig. 2(c)), were tentatively identified from their size and shape (see e.g. Mould & Hulmes, 1987) and position in the D-period (Fig. 7(a) and (c)) as the C-propeptide domains. Their identity was confirmed by immunolabelling (Fig. 8(a) and (b)). Hence the C-propeptide domains lie on the surface of the assemblies at the C-terminal gap-overlap junction. In contrast, the size, and shape and location of the Pi-propeptide domains in the D-periodic assemblies could not be determined as accurately because there was little change in the staining patterns at or near the N-terminal ends of the molecules. However, the immunolabelling studies (Fig. 8(c)and (d)) suggested that the N-propeptide domains were present in the “overlap” zone. We have recently confirmed that additional mass is present in the Nterminal side of the overlap zone using dark-field electron microscopy (D. F. Holmes & A. P. Mould, unpublished results). The observation that the

et al.

N-propeptide domains were readily detectable by immunolabelling also suggests that, like the Cpropeptide domains, they were present on the surface of the assemblies rather than buried in the interior. A location in the overlap zone of the Dperiodic assemblies would suggest that the N-propeptide domains were folded back along the centra,l triple-helical region of the procollagen molecules. Folding back of the N-propeptide domain was observed after rotary shadowing of procollagen monomers (Mould & Hulmes, 1987). This folding back may be due to the “hair-pin bend” eonformation of the N-telopeptides (Helseth et al., L979: Dombrowski 8: Prockop, 1988). Observations on dermatosparactic tissues, both by negative staining (Fjelstad & Helle, 1974) and by X-ray diffraction (Mosler et al., 1986), suggested that the N-propept,ide domains partly filled t’he “gap” zone of dermatosparactie fibrils. However, these fibrils are hybrids containing fully processed collagen as well as pN-collagen. In these hybrid structures the preferred position of the N-propeptide domains within the D-period may not be the same as in structures in which all molecules possess N-propeptide domains. The D-periodic assemblies described here appear similar to the D-periodic pN-collagen “sheets” reported by Hulmes et al. (19896), although lateral widths are smaller. The thickness of the D-periodic asssemblies (7.8 ( f 1.1) nm) was identical with t,hat of the pN-collagen sheets (7.9 (+ 1.3) nm): suggesting that they share a common growth-limiting mechanism in this dimension. It was proposed that the pN-collagen sheets consisted of two “zones” of close-packed molecules (each of thickness -4 nm) arranged back to back in such as way as to keep all the N-propeptide domains on the surface of the sheets. It seems likely that the procollagen assemblies described here also consist of two “zones” of molecules, but with both N and C-propeptide domains on the surface of the assemblies. The surface location of these domains appears to block further accretion of molecules once a certain critical thickness is reached.

(d) Implications

for Jibrillogenesis

in vivo

The exist’ence of D-periodic procollagen assemblies poses the question: why, within cells that are actively synthesizing procollagen, does fibril formation not commence b