Ehlers Danlos Syndrome Type VIIB INCOMPLETE CLEAVAGE OF ABNORMAL TYPE I PROCOLLAGEN BY N-PROTEINASE IN VITRO RESULTS IN THE FORMATION OF COPOLYMERS OF COLLAGEN AND PARTIALLY CLEAVED pNCOLLAGEN THAT ARE NEAR CIRCULAR IN CROSS-SECTION*

We have shown that a child with Ehlers Danlos syn- drome (EDS) type VI1 has a G to A transition at the first nucleotide of intron 6 in one of her COLlA2 alleles. Half of the cDNA clones prepared from the proband's proa2(1) mRNA lacked exon 6. The type I procollagen secreted by the proband's dermal fibroblasts in culture was purified, and collagen fibrils were generated in vitro by cleavage of the procollagen with the procollagen N- and C-proteinases. Incubation of the procollagen with N-proteinase resulted in a 1:l mixture of pccollagen and uncleaved procollagen. Incubation of this mixture with C-proteinase generated collagen and abnormal pNcollagen (pNcollagen-""') that readily copolymerized into fibrils. By electron microscopy these fibrils resembled the hieroglyphic fibrils seen in the N-proteinase-deficient skin of dermatosparactic animals and humans and were distinct

We have shown that a child with Ehlers Danlos syndrome (EDS) type VI1 has a G to A transition at the first nucleotide of intron 6 in one of her COLlA2 alleles. Half of the cDNA clones prepared from the proband's proa2(1) mRNA lacked exon 6. The type I procollagen secreted by the proband's dermal fibroblasts in culture was purified, and collagen fibrils were generated in vitro by cleavage of the procollagen with the procollagen N-and C-proteinases. Incubation of the procollagen with N-proteinase resulted in a 1:l mixture of pccollagen and uncleaved procollagen. Incubation of this mixture with C-proteinase generated collagen and abnormal pNcollagen (pNcollagen-""') that readily copolymerized into fibrils. By electron microscopy these fibrils resembled the hieroglyphic fibrils seen in the N-proteinase-deficient skin of dermatosparactic animals and humans and were distinct from the near circular cross-section fibrils seen in the tissues of individuals with EDS type VII. Further incubation of the hieroglyphic fibrils with N-proteinase resulted in partial cleavage of the pNcollagen-""' in which the abnormal pNaS(1) chains remained intact. These fibrils were not hieroglyphic but were near circular in cross-section. Fibrils formed from collagen and pNcollagen-""' that had been partially cleaved with elevated amounts of N-proteinase prior to fibril formation were also near circular in cross-section. The results are consistent with a model of collagen fibril formation in which the intact N-propeptides are located exclusively at the surface of the hieroglyphic fibrils. Partial cleavage of the pNcollagen-""' by Nproteinase allows the N-propeptides to be incorporated within the body of the fibrils. The model provides an explanation for the morphology and molecular composition of collagen fibrils in the tissues of patients with EDS type VII. *This work was supported by The Wellcome Trust, Grant AR21557 from the National Institutes of Health, the Michael Geisman Memorial Fellowship from the Osteogenesis Imperfecta Foundation, and the South African Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adverthis fact. EDS,' a heterogeneous group of heritable disorders characterized by hypermobility of joints and abnormalities of skin, is classified into 11 types on the basis of clinical and biochemical findings (Beighton et al., 1988). EDS type VI1 is inherited in an autosomal dominant fashion and is distinct from other forms of EDS by virtue of marked joint hypermobility, multiple joint dislocations, and congenital hip dislocations that are usually bilateral (Byers, 1989). The biochemical basis of the disorder is a failure to process the N-propeptides of type I procollagen (for review see Byers, 1989).
Early studies of EDS type VI1 suggested that the impaired conversion of procollagen to collagen was the result of a deficiency of N-proteinase (Lichtenstein et al., 1973). EDS type VI1 was therefore thought to be the human counterpart of dermatosparaxis, a recessively inherited disorder of cattle (Lenaers et al., 1971), cats (Counts et al., 1980;Holbrook et al., 1980), sheep (Fjdstad andHelle, 1974), and humans' that is characterized by skin fragility and is caused by the absence of N-proteinase activity. However, in the five individuals with EDS type VI1 in whom the molecular defects are known, all were heterozygous for mutations in either the COLlAl or COLlA2 collagen genes. The mutations resulted in the synthesis of proa chains that lacked the amino acid sequences encoded by exon 6 in either COLlAl or COLlA2 as a result of exon skipping (Weil et al., 1988(Weil et al., , 1989(Weil et al., , 1990Vasan et al., 1991;Nicholls et al., 1991). In both genes exon 6 encodes the N-proteinase cleavage site and surrounding residues. Biochemical studies of tissues from one proband with EDS type VIIB showed that the abnormal and normal ot2(I) chains occurred in almost equal amounts in the extracellular matrix of the skin and bone (Eyre et al., 1985). Also, whereas in 'The abbreviations used are: EDS, Ehlers Danlos syndrome; pNcollagen, intermediate in the normal processing of type I procollagen to type I collagen containing the N-propeptides hut not the Cpropeptides; pNcollagen-""6, pNcollagen that lacks the 18-amino acid residues encoded by exon 6 of the COLlA2 gene; pCcollagen, intermediate in the normal processing of type I procollagen to type I collagen containing the C-propeptides but not the N-propeptides; Nproteinase,, the enzyme that removes the N-propeptides of type I procollagen in viuo; C-proteinase, procollagen C-proteinase, the enzyme that removes the C-propeptides of type I procollagen in uivo; STEM, scanning transmission electron microscopy; a-chains, the chains of collagen; proa-chains; the chains of procollagen; pr0cu2(1)-""~ and pN~r2(1)-~~' chains, proal(1) and pNaB(1) chains, respectively, that lack the amino acid residues encoded by exon 6 of the COLlA2 gene; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.

Ehlers Danlos Syndrome
Type VIIB normal tissues the collagen fibrils are circular in cross-section, the fibrils in the skin and bone of that individual had rough borders and were near circular in cross-section (Eyre et ul.,

1985).
We show here that an individual with EDS type VI1 has a G to A transition at the 5' donor splice site of exon 6 in one of her alleles for the COLlA2 gene. Type I procollagen purified from the medium of the proband's dermal fibroblasts in culture and incubated with N-proteinase generated a mixture of pCcollagen and N-proteinase-resistant procollagen. Fibrils generated in vitro by cleavage of the mixture with C-proteinase (Kadler et al., 1987) initially formed hieroglyphic fibrils that could be resolved to fibrils with near circular crosssections with additional N-proteinase. On the basis of our findings, we propose that the collagen fibrils in the tissues of individuals with EDS type VIIB result from copolymerization of collagen and pNcollagen-ex6 and partial cleavage of the abnormal pNcollagen by N-proteinase.

Clinical History of the Patient and Her Family
The clinical details of the proband and members of her family have been previously described (Viljoen et al., 1987). The proband was the third child of a family where the mother and her four children had an inherited connective tissue disorder characterized by generalized articular laxity, joint dislocations and subluxations, and wormian bones in the skull. The latter feature may be common in EDS type VI1 but is not generally evaluated. A skin biopsy was taken from the proband for biochemical and molecular analyses. Dermal fibroblasts that grew from the biopsy in culture were used in the studies described here. Dermal fibroblasts from an unrelated healthy individual were used in control experiments.

Source of Materials
Radiochemicals were from ICN Radiochemicals; sodium ascorbate was from Sigma; Dulbecco's modified Eagle's medium was from Northern Biologicals Ltd.; DEAE-cellulose was from Whatman; YM-100 ultrafiltration membranes were from Amicon; Sephacryl S-300 resin was from Pharmacia-LKB Ltd.; fertile hen eggs were from Northern Biological Supplies; spectroscopically pure carbon (rods) were from Agar Aids, copper grids were from Gilder Grids; Sequenase' was from United States Biochemicals; water used in the preparation and analyses of the proteins was from a commercial water purification system that comprised tap water feeding into a Millipore R06 Plus cartridge pack (Millipore) connected in-line to a Millipore Milli-Q Plus Ultrapure water purification for final delivery.

DNA Sequence Determination
Total RNA was prepared from cultured dermal fibroblasts (Chromcgynski and Sacchi, 1987;Greenberg, 1987). Ten pg of RNA were precipitated with 1 pg of a SalI-tailed oligonucleotide primer, A, complementary to coding sequence in exons 8 and 9 of COLlA2 (sequence 5': TCACGTCGACGTCCGGGTTTCCAGGGTG). The cDNA was prepared as described elsewhere (Maniatis et al., 1982;Willing et al., 1990). An EcoRI-tailed primer, B, identical to coding sequence in exons 2 and 3 (sequence 5': GCGAATTCTTTACAA-GAGGAAACTGTAAG), and primer A were used to amplify cDNA synthesized from the COLlA2 gene spanning exon 6 using the polymerase chain reaction (Saiki et al., 1988). The amplified cDNA fragment was cloned into M13 mp19. Single-stranded DNA was prepared (Messing et al., 1984) and sequenced by the dideoxy chain termination method (Sanger et al., 1979) with T7 polymerase Sequenasb. Genomic DNA was prepared from the patient's dermal fibroblasts in culture using standard procedures. The genomic DNA sequence spanning exon 6 and including the intron 5 acceptor and intron 6 donor splice sites was amplified using an EcoRI-tailed primer, C, within intron 5 (sequence 5': AATAGAATTCGAACTACATGACATGTA-AC) and a SalI-tailed primer, D, within intron 6 (sequence 5': CACG CCATTTATTTAGCTACCTAAGTTAAC) using the polymerase chain reaction. The amplified fragment was cloned and sequenced as above.
Preparation of Procollagen "C-Labeled type I procollagen was purified from the culture medium of normal and proband dermal fibroblasts (passage 7-10) using the methods described previously (Kadler et al., 1987). In brief, fibroblasts were grown to confluence and incubated in Dulbecco's modified Eagle's medium supplemented with 1 pCi/ml of a mixture of uniformly labeled "C-~-amino-acids, 25 pg/ml ascorbic acid, and no serum. Proteins in the culture medium were precipitated by ammonium sulfate, and the type I procollagen was chromatographed on two consecutive columns of DEAE-cellulose (Fiedler-Nagy et al., 1981;Peltonen et al., 1980). The procollagen was concentrated by ultrafiltration and stored at -20 "C in storage buffer consisting of 0.1 M Tris buffer (pH 7.4 at 20 "C) containing 0.4 M NaCl and 0.01% NaN3. Procollagen concentration was determined by a colorimetric hydroxyproline procedure (Woessner, 1961), assuming 10.1% hydroxyproline by weight procollagen (Fiedler-Nagy et al., 1981). The procollagens had a specific radioactivity of 1500 cpmlpg.
Procollagen Nand C-proteinases The C-proteinase was purified from the culture medium of the leg tendons of 250 dozen 17-day chick embryos as described previously (Hojima et al., 1985). The preparation had 400 units/ml activity where 1 unit is the amount required to cleave 1 pg of type I procollagen/h at 34 "C in a reaction system containing procollagen at a concentration of 10 pg/ml. The N-proteinase was purified from extracts of the used tendons as described previously (Hojima et al., 1989). The preparation had 800 units/ml activity. Preparations of highly purified N-and C-proteinases used in some of the experiments were a generous gift from Dr. Yoshio Hojima (Jefferson Institute of Molecular Medicine, Jefferson Medical College, Philadelphia).

Preparation of Substrates for Fibril Formation
For control experiments, pccollagen was generated by incubating type I procollagen (2500 pg) with partially purified N-proteinase (140 units) in a volume of 6 ml for 8 h at 34 "C in 0.05 M Tris-HC1 buffer (pH 7.4 at 20 "C) containing 0.15 M NaCl, 5 mM CaCI2, 0.01% NaN3, and 0.01% Brij (35). The reaction was stopped by the addition of 0.1 volume of 1 M Tris buffer (pH 7.4 at 20 "C) containing 0.25 M EDTA and 0.1% NaN3. 0.2 volume of 50% sucrose was added and the pccollagen was isolated by Sephacryl S-300 gel filtration as described previously (Kadler et al., 1987). The pccollagen was concentrated by ultrafiltration and stored in storage buffer at -20 "C. Type I procollagen (2166 pg) from the EDS type VI1 cells was incubated with Nproteinase (140 units) under the same conditions as for control samples (6 ml, 8 h, 34 "C), the reaction was stopped by the addition of Tris-EDTA, and the resultant mixture pCcollagen/uncleaved procollagen was purified by Sephacryl s-300 gel filtration. The pccollagen and uncleaved procollagen were found in the void fractions of the column. The mixture was concentrated by ultrafiltration and stored in the storage buffer at -20 "C. In subsequent experiments that examined the effects of partial cleavage of the abnormal procollagen, the mixture of pccollagen and procollagen (6 pg) was incubated with N-proteinase (12 units) in a final volume of 83 pl, and the tated by the addition of 28 pl of 81% ethanol (-20 "C). The proteins resultant pccollagen and partially cleaved procollagen were precipiwere collected by centrifugation at 15,000 X g for 15 min (at 4 'C) and dried in a flow of dry nitrogen. The mixture pCcollagen/partially cleaved procollagen was resuspended in fibril formation buffer (see below) and used directly in experiments.

Analysis of N-proteinase Cleavage
Products of N-proteinase cleavage were separated by SDS-PAGE using 7% separating and 3.5% stacking gels according to Laemmli (1970). The collagens were visualized by fluorography. Fluorograms were prepared by equilibrating gels in 20% diphenyloxazole in glacial acetic acid and exposing dried gels to preflashed Kodak X-OMAT-AR film at -70 'C. Fluorograms of the pellet fractions of fibrils were analyzed by laser densitometry and the amounts of collagen and pNcollagen"x6 molecules in fibrils were calculated, after correction for molecular mass, from the relative intensities of the a2(I) and pN~t2(1)-"'~ bands, respectively.

Fibril Formation
The substrates (pccollagen in control samples and the mixture pCcollagen/procollagen in the EDS type VI1 samples) and C-proteinase were dialyzed separately against 2 X 600 volumes of fibril forma-tion buffer consisting of 20 mM NaHC03, 117 mM NaCl, 3.4 mM KCl, 1.8 mM CaC12, 0.81 mM MgS04, 1.03 mM NaH2P04, and 0.01% NaN3 (pH 7.4 at 20°C). Fibril formation was initiated by mixing the substrate (100 pg/ml) and C-proteinase (50 units/ml) in a 1.5-ml microcentrifuge tube, and the tube was incubated at 37 "C for 24 h. To prevent changes in pH and volume of the solutions, the tube was gassed with water-saturated 5% co2/95% air and a truncated plunger from a 1-ml syringe was inserted and held in position by the closed cap. To examine the composition of the fibrils formed, the reaction mixture was centrifuged at 15,000 X g for 5 min, and the pellet and supernatant fractions were analyzed separately by SDS-PAGE and fluorography as described above.
Electron Microscopy of Thin Cross-sections of the Fibrils Fibrils were collected by centrifugation and secured in a drop of warm 0.1% agar to facilitate handling. The fibrils were fixed in 4% formaldehyde, stained with 1% phosphotungstic acid and 1% uranyl acetate (pH 4.4), and embedded in araldite epoxy resin. Thin sections were obtained using a diamond knife and a LKB-ultracut ultramicrotome. The sections were post-stained in 1% phosphotungstic acid and 1% uranyl acetate (pH 4.4) and examined in the JEOL 1200EX transmission electron microscope operated in the conventional mode. Images were recorded on Ilford E.M. film.

Scanning Transmission Electron Microscopy (STEM)
(i) Sample Preparation-Carbon films were prepared by evaporation onto freshly cleaved mica, using carbon rods as a source. Carbon film thickness was typically 2-2.5 nm, as measured by electron scattering in STEM. The films were floated onto a clean water surface and collected on 400-mesh copper grids that had previously been ultrasonically cleaned for 15 min in acetone. A drop of the sample was placed on a filmed grid, and the sample was allowed to adsorb for 15 s. The grid was flushed with six drops of water and allowed to air dry.
(ii) Use of a Conventional Instrument in the STEM Mode-The basic instrument was a JEOL 1200EX transmission electron microscope equipped with a JEOL ASIDlO scanning unit and the standard bright field and annular dark-field detectors. The instrument was interfaced with a microcomputer system (Holmes et al., 1991) that permitted digital control of the scan and digitization of the detector signals. The STEM was operated at 120 kV with the standard lens settings. The collection angle of the annular dark-field detector ranged from 25 X to 75 X radians; the effective camera length in the STEM mode was determined from the diffraction pattern of an evaporated aluminum film. The signal from the darkfield detector was linearly dependent on carbon film mass thickness up to approximately 50 kDa nm-'.
(iii) Measurement of Transverse Mass Distributions-Mass mapping procedures were similar to those developed for a dedicated STEM instrument with a field emission gun (Engel, 1978(Engel, , 1982Engel et all, 1981;Engel and Reichelt, 1984;Freeman and Leonard, 1981). Micrographs were acquired using an -3-nm spot size and exposing the specimen to a low electron dose (510' electrons nm-*). A diffraction grating replica (2160 lines/mm) was used for magnification calibration and was estimated to be accurate to better than 2%. At the instrumental magnification setting of X25,OOO used here, the pixel size was 8.5 nm. Specimens were at room temperature during electron distributions of fibrils were written in the SEMPER 5 image analysis microscopy. Image analysis routines for obtaining transverse mass program (Synoptics Ltd., Cambridge, United Kingdom).

Mutation
Analysis-Using the methods described by Cohn and Byers (1990) for the routine screening of the type I collagen synthesized by dermal fibroblasts, 50% of the patient's proa2(1) chains was found to be resistant to cleavage by pepsin at the N-terminal end and 50% was cleaved to a2(I). In control samples all the proa2(1) chains were converted to a2(I) chains (results not shown). The pattern of cleavage in the proband's samples was like that observed for patients with EDS type VIIB . cDNA clones spanning exon 6 of COLlA2 were prepared from the proband's RNA and sequenced. Half of the clones lacked precisely the sequences encoded by exon 6 (Fig. IA). On sequencing the genomic DNA clones spanning exon 6, a G to A transition was found at the first position in the consensus donor splice site of intron 6 in some of the clones (Fig. 1, B  and C ) . Cleavage of Control and Proband Type I Procollagen with N-proteinase-To examine the consequences of the mutation on processing of the procollagen by N-proteinase, type I procollagen was purified from the medium of control and EDS type VI1 dermal fibroblasts. The procollagens were incubated with N-proteinase and the reaction products were examined by SDS-PAGE and fluorography (Fig. 2). No differences in migration were observed between control and EDS type VI1 procollagens (compare lanes 1 and 4, Fig. 2). After treatment with N-proteinase all the proal(1) and proa2(1) chains were converted to pCal(1) and pCa2(I) chains in control samples (Fig. 2, lanes 2 and 3). In the EDS type VI1 samples, 50% of the proal(1) chains and 50% of the proaS(1) chains were converted to pCal(1) and pCa2(I) chains, respectively (Fig.  2, lanes 5 and 6). Thus, the absence of the N-proteinase cleavage site in the pr0a2(1)""~ chains conferred resistance to cleavage by N-proteinase to the proal(1) chains in the same molecules.
Copolymerization of Collagen and the N-proteinase-resistant pNcollagen-ex6-Fibrils were formed by incubating the mixture of pCcollagen/N-proteinase-resistant procollagen with C-proteinase. Similarly, fibrils were generated in control samples by incubating pccollagen with C-proteinase (Kadler et al., 1987(Kadler et al., , 1988(Kadler et al., , 1990a. The fibrils formed from control samples were circular in cross-section, cross-striated, and were like those generated previously in the fibril-forming system (Kadler et al., 1990b) (Figs. 3A and 4A). Fibrils formed from the EDS type VI1 samples were highly irregular in crosssection, cross-striated, and resembled the hieroglyphic fibrils seen in the skin of dermatosparactic animals and man (Figs. 3B and 4B). We noted that the circularity of fibrils generated in control samples was often difficult to assess because the fibrils tended to clump and coalesce. This was attributed to the forces imparted on the fibrils by the centrifugation step used to collect the fibrils. A gentler method of preparing fibrils for electron microscopy, and consequently a more reproducible way of accessing their circularity, was to deposit the fibrils on carbon-coated electron microscope grids and to examine the distribution of mass across the fibril (the transverse mass distribution) using STEM. An additional feature of STEM was that the transverse mass distribution along the entire length of the 20-100 fibrils collected/grid could be examined. The analyses showed that the transverse mass distributions of all the fibrils generated in control samples were symmetrical and that the fibrils were uniform diameter cylinders (Fig.  4A). No fibrils were observed that did not have symmetrical, uniform transverse mass distributions. In contrast, the transverse mass distributions of all the fibrils generated in the EDS type VI1 samples were asymmetric (Fig. 4B), and the fibrils exhibited a marked deviation from circularity. The STEM results were in good agreement with the hieroglyphic patterns of the fibrils observed in cross-section (Fig. 3B). The STEM analyses also showed that the fibrils were as long and apparently as flexible as those generated in control samples.
To examine the molecular composition of the hieroglyphic Perspective displays are shown from two different angles. A , collagen fibril formed by incubating pccollagen with C-proteinase at 37 "C for 24 h. The fibril is highly uniform and exhibits the cross-striated banding pattern characteristic of collagen fibrils. The perspective displays showed a symmetrical transverse mass distribution consistent with the fibril being a symmetrical and uniform cylinder. B, hieroglyphic fibril formed by copolymerization of collagen and pNcollagen"x6. The fibril exhibits the cross-striated banding pattern seen in control samples and has a "ribbon-like" appearance. The perspective displays shows an asymmetric distribution of mass in transverse cross-section. C, fibril formed by copolymerization of collagen and partially cleaved pNcollagen"x6 in which some of the pNal(1) chains are cleaved. The transverse mass distribution of the fibril is symmetrical along the long axis of the fibril and demonstrates that the copolymer is a uniform cylinder with near circular crosssection. D, hieroglyphic fibril treated with N-proteinase. The symmetry of the transverse mass distribution is consistent with the fibril having a near circular cross-section. Typically 20-50 fibrils were examined in each sample. There were no indications that the distribution of diameters (mass/unit cross-section) of the fibrils were different in any of the samples examined. Bar = 1 pm. fibrils, fibrils were collected by brief centrifugation and the pellet (fibril) and supernatant (soluble) fractions were analyzed separately by SDS-PAGE and fluorography (Fig. 5 ) . In control samples, the pellet fractions contained al(1) and a2(I) chains, and the supernatant fractions contained the cleaved C-propeptides and small amounts of al(1) and a2(I) chains that were only visible after long exposures of the gels to film (data not shown). The al(1) and a2(I) chains in the supernatant were from the critical concentration of collagen in sectioned. A , control; B, EDS type VII. The bar = 500 hm. equilibrium with fibrils (Kadler et al., 1987;Na et al.,-1989). T h e pellet fraction of the hieroglyphic fibrils contained pNtrl(I), pNn2(1)"'"'', n l ( I ) , and tr2(I) chains. Quantitation of t,he amounts of tr2(1) and pNnS(I)""" in fibrils by laser densitometry of fluorograms and correction for molecular mass, showed t,hat pNcollagen""" accounted for 52 ? 6% S.D. ( n = 3 ) of the protein in the fibrils. Collagen accounted for 48% of the protein. The supernatant fract.ion of the fibrils contained t,he cleaved C-propeptides and pNnl(I), pNn2(1)"'"'', with small amounts of c r l ( I ) and trZ(1) chains.
Cleavage of the pronI(I) Chains in Abnormal Procollagen Molecules b+v Elevated Amounts of N-proteinase-Extracts of skin and bone from individuals with EDS type VIIR contain t r l ( 1 ) chains and about equal amounts of normal n2(I) chains and abnormal tr2(I) chains that migrate at the position expected of pNn2(I). pNtrl(1) chains were not detected (Eyre et al., 1985). To test the hypothesis that absence of pNnl (1) chains in extracts of E D S t-ype VIIR tissues was the result of N-proteinase activity, the abnormal type I procollagen was incubated with the highest concentration of N-proteinase experimentally practical. The pronl(1) chains in the abnormal procollagen molecules were cleaved by 145 units/ml N-proteinase at 34 "C for 8 h (Fig. 6 ) , a concent,ration that was 7fold higher (24-fold the amount of enzyme protein) than was normally used in t.he "system" to generate pCcollagen (Kadler et al., 1987). The reaction was completely inhibited by 25 mM EDTA (lane 3 , Fig. 6) and was dependent on the presence of active enzyme (lane 4 , Fig. 6). Laser densitometry of fluorograms, such as the one shown in Fig. 6, showed that 78% of t h e pronl(1) chains were cleaved to pCnl(1) chains and 40% of the pron2 chains were cleaved in the reaction. Complete cleavage of the pronl(1) chains by prolonged incubation with enzyme was never OhSeNed.
Copolymerization of Collagen and Partially Cleaved pNcol1agen"""-Fibrils formed from collagen and part.ially cleaved pNcollagen""" contained tul(I), &(I), pNn2(I)"'"", and small amounts of pNnl(1) chains (Fig. 7, lane I ) . Quantitation of t r l ( I ) , n2(I), pNnl(I), and pNn2(I)"'"" chains in fibrils (by laser densitometry of fluorograms and correction for molecular mass) showed t.hat the ratio n2(1)/pNruZ(I)-""' chains in fibrils was approximately 4:l. Only small amounts and resnspentled in fil~ril formation hrffrr antl inrll1)nte.d with rproteinase at 37 "C for 24 h. The fihrils were rollrrtctl t)y brief centrifrlKation. and the pellet nnd supernntnnt frnrtions a r v qhotvn in lnnrs 1 and 2, respect ivrly. of pNtrl(1) chains were detected. Consequently. the fibrils comprised approximately 80T collagen, approximately 2 0 5 pNcollagen""'; in which both the pN(rl(1) chains were cleaved to n l ( I ) chains, and trace amounts of pNcollngen""" in which one or both of the pNtrl(1) chains remained intact. The analyses also showed that the amounts of trl(1) chains were equal to twice the sum of the amounts of the tr2cI) and pNn2(I)"'"' chains. These data indicated that the pNtr2(1)""' chains were part of molecules in which some pNnl(1) chains had been cleaved to c r l ( 1 ) chains. Fibrils formed from these molecules differed from the hieroglyphic patterns seen previously in that they had symmetrical transverse mass distributions (Fig. 4C) and were therefore uniform cylinders.
The supernatant formed after centrifugation of the fibrils contained predominantly nl(1) chains antl also pNtrl(1) and pNnZ(I)""" chains. Only small amounts of tr2(1) chains were detected (Fig. 7, lane 2). T h e relative proportions of the chains in the supernatant were consistent with the presence of pNcollagen""'; molecules in which one of the pNtrl(1) chnins Ehlers Danlos Syndrome Type VIIB were converted to al(1) chains. The virtual absence of a2(I) chains indicated that collagen represented a very small fraction of the soluble molecules. Molecules containing ~Na2(1)-~"' and cleavedpNal(1) chains were not incorporated into fibrils as readily as molecules in which no cleavage had occurred (compare Fig. 7, lune 1, and Fig. 5, lane 3 ) . N-proteinase Treatment of Hieroglyphic Fibrils-Although the fibrils formed by copolymerization of collagen and pNcollagen""' had near circular cross-sections, they differed from those found in tissues in that they contained only small amounts of abnormal molecules (approximately 20%). To determine if continued exposure of fibrils to N-proteinase could modify fibril structure, hieroglyphic fibrils were formed by incubating the mixture of pccollagen and abnormal procollagen with C-proteinase and then treated with N-proteinase (12 units in 85 p1 at 34 "C for 12 h). The fibrils were collected by brief centrifugation, and the pellet and supernatant fractions were examined separately. The fibrils comprised al(I), a2(I), and ~Na2(1)-~"' chains and no pNal(1) chains. Laser densitometry of fluorograms demonstrated that the fibrils contained 39 & 5% S.D. (n = 4) pNcollagen""' molecules in which the pNal(1) chains were cleaved to al(1) chains. The supernatant of the fibrils contained al(1) (the major component), pNal(I), and ~Na2(1)-~~' chains. Only small amounts of a2(I) chains were detected. These results indicated that the soluble phase was a mixture of molecules containing ~N a 2 ( 1 ) -~~' chains and pNal(1) chains that had been cleaved to al(1) chains, and, molecules containing ~Na2(1)-~"' chains and intact pNal(1) chains. Noteworthy, all of the pNal(1) chains in fibrils were cleaved to al(1) chains by N-proteinase (Fig. 8, lune 1 ), and therefore, all were most likely to be located at the surface of the fibrils. Intact pNal(1) chains were found in the supernatant of the fibrils (Fig. 8,  lane 2). This suggested that N-proteinase may cleave the pNal(1) chains more rapidly when the pNcollagen""' molecules are part of fibrils than when they are in free solution.
Treatment of hieroglyphic fibrils with N-proteinase (12 units in 85 pl at 34 "C for 12 h) had a dramatic effect on fibril morphology. Examination of 18 fibrils from three preparations in 10 fields of STEM view showed that the fibrils had a symmetrical transverse mass distribution (Fig. 4 0 ) consistent with a near circular cross-section. Hieroglyphic were not seen. In control experiments in which glyphic fibrils were diluted with buffer not containing Nproteinase, the fibrils had a highly irregular transverse mass distribution (data not shown).

DISCUSSION
In uiuo, type I procollagen is converted to type I collagen by specific enzymic removal of N-and C-propeptides by the Nand C-proteinases (Hojima et al., 1985(Hojima et al., ,1989). The collagen then spontaneously self-assembles into the cylindrical, crossstriated fibrils characteristic of the extracellular matrix of connective tissues. In EDS type VIIB, individuals have mutations in their type I collagen genes that result in incomplete processing of procollagen to collagen and the accumulation in tissues of pNaP(1) chains. Also, the tissues of these people contain collagen fibrils that are near circular in cross-section. We wanted to learn more about the pathway of events occurring between the formation of the abnormal type I procollagen, its proteolytic processing, and the way in which these abnormal molecules participate in collagen fibril formation.
We showed that a proband with EDS type VIIB had a G to A transition in the obligate -GT-dinucleotide at the 5' donor splice site of exon 6 in one allele of the COL1A2 gene.
Consequently, half of the proband's type I procollagen molecules contained a proa2(1) chain that lacked the sequences containing the N-proteinase cleavage site. N-proteinase is a conformational-dependent proteinase that will not cleave heat-denatured procollagen . It cleaves the proa chains in a sequential manner such that, during the initial stages of the reaction, proal(1) and procu2(1) chains are cleaved at about the same rate. An intermediate is formed that is slowly converted to pccollagen by cleavage of the remaining proal(1) chain . Also, the enzyme readily cleaves a procollagen molecule constructed from three proal(1) chains in which the procu2(1) chain is substituted by a proal(1) chain (Dombrowski and Prockop, 1988). Experiments here showed that the absence of the 18 amino acids that surround and include the N-proteinase cleavage site in the proa2(1) chain drastically slowed the rate of cleavage of the proal(1) chains. The results demonstrate that cleavage of the proal(1) chains of procollagen by N-proteinase occurs at a maximal rate only when the procollagen molecule contains three proa chains and when the cleavage sites are in their native conformation and spatial organization.
The self-assembly of collagen into fibrils is similar to other protein self-assembly systems, such as tobacco mosaic virus (Lauffer, 1975), actin filaments (Frieden and Goddette, 1983;Pollard and Cooper, 1986;Frieden, 1989), and microtubules (Timasheff and Grisham, 1980;Mitchison and Kirschner, 1984;Olmsted, 1986) in that it is a spontaneous, entropydriven process in which the driving force is the displacement of solvent molecules bound to the monomers of protein (Lauffer, 1975). Type I collagen fibrils are cylindrical, needle-like (Kadler et al., 1990a;Birk et al., 1989) and are near circular in cross-section (Kadler et al., 199Ob;Birk et al., 1989). Type I pNcollagen self-assembles in vitro into extended sheet-like structures of thickness 8 nm in which the N-propeptides are found at the surface of the sheets in a "folded-back" conformation (Holmes et al., 1991). The hieroglyphic structures formed here by copolymerization of collagen and pNcollagen""' had a morphology that was intermediate between that of cylinders and sheets. As reviewed by Oosawa and Asakura (1975), molecules that polymerize to form similar structures can copolymerize if they have similar polymerforming tendencies. The fact that a single population of fibrils was seen, as opposed to distinct populations of sheets and fibrils, demonstrates that the hieroglyphs were copolymers of collagen and pNcollagen-""'. Treatment of the hieroglyphic fibrils with N-proteinase resulted in cleavage of all the pNal(1) chains in pNcollagen-ex6 molecules demonstrating that the N-propeptides were located exclusively at the fibril surface. We conclude that the hieroglyphic nature of the fibrils was the result of incorporation of large amounts of pNcollagen-""' and close packing of the N-propeptides at the fibril surface.
Treatment of the hieroglyphic fibrils with N-proteinase resulted in cleavage of the pNal(1) chains in pNcollagen-'"' molecules and had drastic consequences on fibril morphology. The remarkable observation that hieroglyphic fibrils were resolved to cylinders lends support to the suggestion that there is considerable fluidity between collagen molecules within a collagen fibril (Chapman, 1989). That the relative proportions of collagen and pNcollagen-'"' changed little after cleavage of the pNal(1) chains by N-proteinase, and that the partially cleaved pNcollagen-e"6 molecules were present in the large diameter (small surface area/volume ratio) fibrils, suggested that some of the abnormal molecules must have been located within the body of the cylindrical fibril. The ability of the partially cleaved N-propeptides to be incorporated into the fibril suggests that they undergo a conformational change following cleavage of the pNal(1) chains. The nature of this conformational change is unknown but one possibility is that the N-propeptides, instead of being folded back, straighten following cleavage of the pNal(1) chains by N-proteinase.
Collagen molecules are divided into 4.4 D units and associate in parallel array so that, in the fibril, they are staggered by one or more D units relative to their nearest neighbor. This arrangement of collagen molecules generates regions of gap and overlap in the fibril. The "folded" conformation of the Npropeptides of pNcollagen molecules presumably prevents this side-by-side association whereas an "extended" conformation would not. Wirtz et al. (1990) showed that the pNal(1) chains in the abnormal pNcollagen from an individual with EDS type VIIB were cleaved in vivo in the region of the al(1) N-proteinase cleavage site and that the cleaved al(1) N-propeptides remained noncovalently associated with the pNaB(1) chain. It was not determined whether the pNal(1) chains were cleaved by N-proteinase or by another proteinase. We showed here that N-proteinase in elevated concentrations can cleave the pNal(1) chains in the abnormal molecules and that these partially processed molecules copolymerize with collagen to form roughly cylindrical fibrils. However, even at the highest concentrations of N-proteinase that were experimentally practical, cleavage of all the pNal(1) chains to al(1) chains in pNcollagen-'"' molecules in solution could not be attained. Yet, treatment of hieroglyphic fibrils with N-proteinase did result in cleavage of all the pNal(1) chains that were in fibrils.
Although not conclusive, these observations suggest that the rate of cleavage of the pNal(1) chains by N-proteinase is greater when the N-propeptides are at the surface of a collagen fibril. Two scenarios are possible for the assembly of collagen fibrils with near circular cross-sections in tissues of people with EDS type VIIB. In the first, cleavage of the abnormal molecules by N-proteinase occurs prior to fibril formation. The partially cleaved pNcollagen-""' molecules have a low affinity for the fibril, but once bound they are buried in the body of the fibril. Fibrils formed in this way have a small fraction of pNcollagen-ex6. In the second, collagen and pNcollagen""' copolymerize, and the N-propeptides are restricted to the surface of the fibril. The intact pNcollagen-""' has a high affinity for the fibril so that the fibrils formed are approximately 1:1 copolymers of collagen and pNcollagen. The exposed al(1) N-propeptides are then readily cleaved by N-proteinase and the partially cleaved pNcollagen-""6 molecules are incorporated into the body of the fibril. Whereas both sequences of events lead to the formation of fibrils with near circular cross-sections, the copolymerization of collagen and pNcollagen-'x6 and subsequent cleavage by N-proteinase provides an explanation for the large amounts of pNa2 (1) chains in the tissues of individuals with EDS type VIIB.