altered triple helical structure of type i procollagen in lethal

THE JOURNAL OF BIOLOGICAL CHEMISTRY (c, 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 3, Issue of February 10, pp. 1734-1742, 1985 Printed in U. S. A .

Altered Triple Helical Structure of Type I Procollagen in Lethal Perinatal Osteogenesis Imperfects"

(Received for publication, June 11, 1984)

Jeffrey Bonadioasb, Karen A. HolbrookCrd, Richard E. Gelinas=, Jack Jacob‘, and Peter H. Byers”.d*g*h From the Departments of “Pathology, ‘Biological Structure, and dMedicine and the gCenter for Inherited Disease, University of Washington, Seattle, Washington 98195, the %red Hutchinson Cancer Research Center, Seattle, Washington 98104, and the /Providence Hospital, Anchorage, Alaska 99510

Cultured dermal fibroblasts from an infant with the lethal perinatal form of osteogenesis imperfecta (type 11) synthesize normal and abnormal forms of type I procollagen. The abnormal type I procollagen molecules are excessively modified during their intracellular stay, have a lower than normal melting transition temperature, are secreted at a reduced rate, and form abnormally thin coliagen fibrils in the extracellular matrix in vitro. Overmodification of the abnormal type I procollagen molecules was limited to the NH2-terminal three-fourths of the triple helical domain. Two- dimensional mapping of modified and unmodified (Y

chains of type I collagen demonstrated neither charge alterations nor large insertions or deletions in the region of crl(1) and (~211) in which overmodification begins. Both the structure and function of type I procollagen synthesized by cells from the parents of this infant were normal. The simplest interpretation of the results of this study is that the osteogenesis imperfecta phenotype arose from a new dominant mutation in one of the genes encoding the chains of type I procollagen. Given the requirement for glycine in every third position of the triple helical domain, the mutation may represent a single amino acid substitution for a glycine residue. These findings demonstrate further heterogeneity in the biochemical basis of osteogenesis imperfecta type I1 and suggest that the nature and location of mutations in type I procollagen may determine phen- otypic variation.

Infants with the perinatal lethal form of osteogenesis imperfects (type 11) have bone and connective tissue fragility, bone deformity, reduced calvarial mineralization, and short stature (1). Although most affected infants die in the perinatal period from respiratory insufficiency, a small number survive for several weeks or months (2,3). Biochemical heterogeneity of type I1 osteogenesis imperfecta has been demonstrated by studies completed during the last few years, and several types of structural mutations in the pro-a chains of type I procollagen can produce the disorder. While clinical studies were

07266, AM-07171, GM-15253, and HD-17664 from the National *This work was supported in part by Grants AM-21557, GM-

Institutes of Health, by Clinical Research Grant 6-298 from the March of Dimes National Foundation, and by an award from the Poncin Scholarship Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 [J.S.C. Section 1734 solely to indicate this fact.

Recipient of National Institutes of Health Individual Fellowship Award AM-07171, To whom correspondence should be addressed.

Established Investigator of the American Heart Association.

consistent with autosomal recessive inheritance (1-3), biochemical studies have suggested that new dominant (hetero- zygous) mutations in type I collagen genes alone or on the background of a nonfunctioning allele (resulting in compound heterozygosity at one locus) may cause the phenotype.

The molecular basis of the osteogenesis imperfecta type I1 phenotype has been studied in detail in a few individuals. Cultured fibroblasts from one infant synthesized two species of the pro-d(1) chain, one of which was shorter than the normal chain because of a deletion of 50 to 100 amino acids from the triple-helical domain (4-6). Molecules that contained the short pro-d(1) chain were susceptible to proteolysis at a lower temperature than normal, were excessively modified, were secreted at a reduced rate, and were inefficiently processed to collagen molecules. Parental strains were unavailable for study. Fibroblasts from a second infant synthesized a population of pro-al(1) chains with a cysteine residue near the COOH terminus of the triple-helical domain (a region that normally contains no cysteine) (7). This structural alteration also produced an unstable procollagen molecule that was excessively modified during its intracellular stay, was secreted more slowly than normal, was degraded intracellu- larly at a greater rate than normal, and had a lower melting temperature than normal. Cells from neither parent synthesized the mutant chain. Fibroblasts from a third infant synthesized a single population of pro-a2(1) chains which were shorter than normal because of a deletion of approximately 20 amino acids from the middle of the triple helix (8). While cells from neither parent synthesized chains with this mutation, those from the father synthesized about half the normal amount of pro-a2(1). These observations were taken to imply that the disorder in this infant resulted from two mutations in the cu2(I) gene; one pro-a2(1) allele coded for the shortened chain while the other allele was “nonfunctional.”

The three infants studied in detail all had the classical and most severe form of osteogenesis imperfecta type 11, recently designated type IIA (9), with severely crumpled femurs, minimal calvarial mineralization, and broad “beaded ribs. All three also avulsed parts during birth. In an effort to under- stand the range of biochemical abnormalities in osteogenesis imperfecta type Ii, we have studied the procollagens synthesized by dermal fibroblasts from another infant with osteogenesis imperfecta type IIA whose clinical course was remarkable in that she survived for 6 months before dying of progressive respiratory insufficiency. The cells from this infant synthesized both normal and abnormal type I procollagen molecules. The abnormal molecules have a unique structural abnormality within the triple helical domain about one- quarter of the distance from the COOH terminus. The abnormal molecules are excessively modified inside the cell, have reduced thermal stability, are secreted at a reduced rate, and

1734

Collagen Triple Helix in Lethal Osteogenesis Imperfecta 1735

are organized into abnormally thin fibrils in the extracellular matrix. On the basis of these findings, we suggest that there may be a relationship between the type of mutation and its location in the type I procollagen molecule and the resultant clinical phenotype.

CLINICAL SUMMARY

The proband, a female infant, was born a t term by vaginal delivery after an uncomplicated pregnancy and labor. The infant required brief bag and mask ventilation after birth because of poor respiratory effort and was subsequently maintained on 30% oxygen. She weighed 2660 g (10th percentile), was 47 cm long (10th percentile), and her head circumference was 33 cm (10th percentile). The remarkable features of her physical exam included widely separated cranial sutures, blue sclerae, a short neck, and short upper and lower extremities. X-rays demonstrated minimal calvarial mineralization; short, beaded ribs and a very small thorax; short crumpled femurs; and bent bones of the leg and forearm. The x-ray findings were compatible with the diagnosis of perinatal lethal osteogenesis imperfecta (type IIA) (Fig. 1).

She was weaned off supplemental oxygen by the third day of life. She was fed 12-16 ounces of formula daily but failed to gain weight appropriately and had few developmental gains. At 6 months, she developed frequent cyanotic spells, poor feeding, tachypnea, and fever of 104 OF. There were bilateral rales on auscultation of her chest. Two days after the onset of these symptoms she became apneic and bradycardiac and died. Pneumonia was not present a t autopsy, and her death was attributed to progressive respiratory insufficiency. A sib- ling born 2 years later was normal. Neither parent had any of the clinical hallmarks of osteogenesis imperfecta, and they were unrelated.

MATERIALS AND METHODS

Cell Strains Dermal fibroblast cultures were established from explants of skin

taken at post mortem from the affected baby (osteogenesis imperfecta

A

'P I FIG. 1 . X-rays of the affected infant . A , cranium. The calvaiial

cortex is extremely thin. H, thorax and upper extremities. The chest cavity is small and the rihs are broad. C, lower extremities. The right and left femur are shortened and deformed. The right and left tibia and fibula are bowed.

cells) and from explants taken from the inner aspect of the upper arm of each parent. The cells were grown to confluence in 100-mm culture dishes after the initial outgrowth and frozen in liquid nitrogen. Cells between the third and fifteenth passage were used in these studies. Cell cultures were maintained in DMEM' containing 10% fetal calf serum, penicillin a t 100 units/ml, and streptomycin a t 100 pglml. Control cells were obtained from normal newborn donors and used at similar passages. All cells were obtained with the informed consent of the patients or their parents.

Preparation and Separation of Procollagens Synthesized by Cells in Culture

Cells were plated a t a density of 2.5 X lo5 in 35-mm culture dishes and allowed to attach and spread overnight. The cells were preincu- hated for 2-4 h in 0.7 ml of serum-free DMEM containing 50 pg/ml of ascorbic acid. The medium with ascorbic acid was replaced with fresh medium and ascorbate and either [2,3-'H]proline (40 Ci/mmol, New England Nuclear) or [2,4,4,5-3H]proline (101 Ci/mmol, Amer- sham Corp.) was added for the indicated labeling times. The medium was harvested into an inhibitor solution which yielded a final concentration of 35 mM EDTA plus 25 mM phenylmethanesulfonyl fluoride. The cell layer was rinsed twice with phosphate-buffered saline containing 25 mM EDTA, scraped into 1.3 ml of the same inhibitor solution, and homogenized with 20 strokes in a Teflon/glass homog- enizer. Medium and cell homogenate samples were dialyzed into 1 mM ammonium bicarbonate containing the same inhibitors and then lyophilized (unless otherwise indicated). The dried samples were dissolved in electrophoresis sample buffer, and the proteins were denatured by boiling for 5 min. The proteins were analyzed by SDS- PAGE with 2 M urea added to the buffers to enhance the separation of pro-cu chains (10). Gels were processed for fluorography with dimethyl sulfoxide and 2,5-diphenyloxazole (11) or with EN3HANCE (New England Nuclear).

Peptide Mapping Proteins were separated in the first dimension on 5% sodium

dodecyl sulfate-polyacrylamide gels under reducing conditions. The lanes were cut out and the proteins were digested in the gel with CNBr (50 mg/ml in 70% formic acid, Sigma) for 2 h a t room temperature. The longer digestion period of 10-18 h that was previously recommended (12) results in reduced resolution of many large CNBr peptides and loss of low molecular weight peptides. Each gel strip was washed with water for three 20-min periods, equilibrated for 20 min with sample buffer lacking SDS and urea, and then placed horizontally over a second dimension 12.5% gel which contained no urea. The strips were overlaid with sample buffer that contained dithiothreitol and 5% SDS, and the cyanogen bromide peptides were separated by electrophoresis.

Alternatively, CNBr peptides were mapped in a two-dimensional system which employed isoelectric focusing in the first dimension. Hydroxylated and unhydroxylated (see below) collagens were incubated for 5 h at 30 "C in 70% formic acid that contained CNBr, 20 mg/ml, under a N2 atmosphere. To terminate the reaction, samples were diluted with water and lyophilized ( ~ 2 ) . The peptides were dissolved in 4 M urea, 2.5% Triton X-100, and 4% ampholines (Pharmacia) and then separated by isoelectric focusing on 2 X 115- mm cylindrical polyacrylamide gels as described by Benya (13). The gels were equilibrated after electrophoresis in 0.5% ammonium per- sulfate and then sample buffer lacking SDS and urea and then overlaid on 12.5% gels that contained no urea. Second-dimension electrophoresis was performed as described above.

Preparation of Unhydroxylated Procollagen Cells in culture were preincubated for 2 h in 0.7 ml of serum-free

DMEM and 0.5 mM cu,tu'-dipyridyl (Sigma). The cells were then pulse labeled with ['Hlproline in fresh medium plus n,tr'-dipyridyl for 4 h. The medium and cell layer proteins were harvested separately as described, and the constituent proteins were separated by SDS-PAGE under reducing conditions.

' T h e abbreviations used are: DMEM, Dulbecco-Vogt modified Eagle's medium; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 01, osteogenesis imperfecta.

1736

A

Collagen Triple Helix in Lethal Osteogenesis Imperfecta

B """"

L-.

C F O I M C F O I M C F O I M Medium Cells Medium

FIG. 2. Autoradiofluorogram of the electrophoretic 'separation of A pro-a chains and B (Y chains synthesized by osteogenesis imperfecta (OZ) cells, parental (father (F), mother ( M ) ) cells, and control (C) cells in culture. (A similar notation is used in all subsequent figures.) Procollagens from the medium and cell layer were reduced with dithiothreitol prior to electrophoresis on 5% SDS-PAGE. All of the radiolabeled proteins produced by 250,000 cells were applied to each lane. Two forms of the pro-nl(1) and pro- &(I) chains were secreted into the medium by the osteogenesis imperfecta cells. One chain from each doublet migrated more slowly than normal (arrows), and the other chain co-migrated with the control. Under these conditions, approximately 90% of the procolla- pen produced by normal fibroblasts appeared in the medium. The osteogenesis imperfecta cells retained more procollagen than the control or parental cells. The majority of the retained osteogenesis imperfecta procollagen consisted of pro-nl(1) and pro-n2(I) chains with delayed mobility (arrows). To prepare a chains, procollagens from the medium were digested with pepsin for 12-16 h a t 4 "C prior to electrophoresis on 5% SDS-PAGE. The osteogenesis imperfecta pro-tu chains were converted into two forms of nl(1) and cu2(I) with one chain from each doublet migrating more slowly than normal (arrow) and the other chain co-migrating with the control and parental (r chains.

-. . .

ap'-dipyridyl - + - + - + - + + C + - F 4 + 0 1 d ~ M +

FIG. 3. Autoradiofluorogram of unmodified pro-al(1) and pro-a2(1) chains synthesized by osteogenesis imperfecta, parental, and control cells. Cells in culture were pulse labeled with ["Hlproline in the presence of 0.5 mM n,n'-dipyridyl for 4 h, and cell layer samples were prepared for electrophoresis under reducing conditions on 5% SDS-PAGE. Pro-cu chains from the medium of replicate cultures labeled for 4 h in the absence of n,n'-dipyridyl have been separated in adjoining lanes for comparison. In the presence of n,a'- dipyridyl unmodified pro-tul(I) and pro-cu2(I) chains migrated faster than their modified counterparts, and the osteogenesis imperfecta pro-(u chain doublets appeared as single bands that co-migrated with the control and the parental chains.

Enzymatic Digestion of Procollagen Pepsin-Medium and cell layer proteins were harvested as de-

scribed, dialyzed into 0.11 N acetic acid, pH 2.6, and digested with pepsin (50 pglml, Worthington) for 12-16 h at 4 "C. The reaction

mm

ad- " a 2 ( I ) C ,; - -0

I" - collagenase - + - - & -

C O I F M FIG. 4. Autoradiofluorogram of medium collagens synthe-

sized by osteogenesis imperfecta. parental, and control cells after cleavage with fibroblast collagenase. Cells in culture were pulse labeled with ['Hlproline for 16 h, medium procollagens were digested with pepsin and then with fibroblast collagenase, and the samples were prepared for electrophoresis on 8% SDS-PAGE under reducing conditions. Pro-n chains from the medium of replicate cultures labeled for 16 h and digested with pepsin have been separated in adjoining lanes for comparison. The osteogenesis imperfecta collagens yielded normal and slow migrating forms of nl(UA and ( ~ 2 ( 1 ) ~ which include the NHt-terminal three-fourths of the triple helix. However, the ~ u l ( I ) ~ and ~ 2 ( 1 ) ~ chain fragments of the osteogenesis imperfecta collagens migrated as single bands with the same mobility as the control.

was stopped with pepstatin (0.5 pg/ml, Sigma), and the samples were prepared for electrophoresis after lyophilization as described above. Unhydroxylated procollagens were harvested from within cells and then digested in the same manner to produce unmodified collagen- sized molecules.

Fibroblast Collagenase-Collagen-sized molecules from the medium (after pepsin digestion) were dialyzed into 50 mM Tris-HCI buffer, pH 7.5, that contained 10 mM CaCI2 and then digested for 24 h a t room temperature with fibroblast collagenase (final concentration, approximately 1.0 pg collagenase/lO pg collagen). The mammalian collagenase was a gift from Dr. Eugene Bauer, Division of Dermatol- ogy, Department of Medicine, Washington University, St. Louis.

Cytoplasmic mRNA Dot Hybridization

Cells in culture were preincubated for 2 h in serum-free DMEM and ascorbic acid (50 pg/ml). The cells were then incubated for 1 h in fresh DMEM containing 10% fetal calf serum plus ascorbic acid, and cytoplasmic mRNA was prepared according to the procedure described by White and Bancroft (14). Serial 2-fold dilutions of the cytoplasmic preparations were made in 2.25 M NaC1, 0.225 M Na citrate (1.5) and applied with suction to nitrocellulose paper (Schleicher & Schuell) by employing a 96-well Minifold apparatus (Bethesda Research Laboratories). Prehybridization of the nitrocellulose, preparation of the 32P-labeled DNA probes of the a l ( I ) and cuZ(1) collagen genes by nick translation, autoradiography, and quan- titation by scanning gel densitometry were performed as described previously (16, 17). Hybridization was performed under conditions of "P-labeled DNA excess and saturation of hybridization. The probes Hf677 (cul(1)) and Hf32 (cu2(I)) were provided by Dr. Francesco Ramirez, Rutgers University, Piscataway, NJ.

Kinetics of Procollagen Secretion

Pulse-chase experiments were performed essentially as described by Kao et al. (18). Replicate cultures were preincubated for 2 h in DMEM that contained 0.5% fetal calf serum and ascorbic acid (50 pglml). The cells were then pulse labeled with ['Hlproline for 4 h in fresh medium that contained ascorbic acid and serum. At the end of the pulse, the cells were washed twice with chase medium (DMEM, ascorbic acid, and 2.8 mM L-proline) and then incubated for up to 4

Collagen Triple Helix in Lethal Osteogenesis Irnperfecta 1737

C F 01 M

al(l) : : : ~ 0 : : * O . . O

1 2 4 5 8 3 7 6 FIG. 5. Autoradiofluorogram of medium collagens synthe-

sized by osteogenesis imperfecta, parental, and control cells after cleavage with cyanogen bromide. For CNBr peptide mapping, (r chains from the medium (after pepsin) were separated in the first dimension on 5% SDS-PAGE, the lanes were cut out and proteins digested in the gel with CNBr, and the CNBr peptides were separated by SDS-PAGE in the second dimension. CNBr peptides from the normal nl(1) and n2(I) chains synthesized by the osteogenesis imperfecta cells co-migrated with those of the parental and control cells. By contrast, the overmodified nl(1) chain of the osteogenesis imperfecta cells yielded three CNBr peptide spots: a single spot in the region of nl(I)CB7 and nl(I)CB8 that migrated just slower than trl(I)CB7, a single spot that co-migrated with al(I)CB6, and a single spot that migrated slower than nl(I)CB3. A schematic diagram of the trl(1) chain with its CNBr peptides indicated by numbers is presented at the bottom of the figure. The relative electrophoretic mobilities of n(I) CNBr peptides from normal nl(1) chains (0) and overmodified cul(1) chains (0) is shown.

h. Medium and cell layer proteins were harvested separately, and the proteins were analyzed by SDS-PAGE under reducing conditions.

Enzymatic Assay of Triple-helix Conformation Melting t.emperatures of the triple helix of normal and abnormal

collagens were measured as described by Bruckner and Prockop (19). Pepsin-digested procollagens from the medium were dissolved in 700 pl of 0.4 M NaCI, 0.01 M Tris-HCI, pH 7.4. The samples were placed in a water bath a t 30 "C and then warmed at a rate of 5 "C/h to 43 "C. Aliquots of 36 p1 were removed at 0.5 "C increments. Each aliquot was rapidly cooled to 20 "C and then digested with trypsin (100 pg/ ml, final concentration, Worthington) for 2 min at 22 "C. The reaction was stopped by adding phenylmethanesulfonyl fluoride and SDS to achieve a final concentration of 2 mM and 2%, respectively. The whole mixture was placed into boiling water immediately and heated for 3 min. The proteins were separated by electrophoresis on 5% polyacrylamide gels that contained 0.5 M urea.

Morphology Cells were plated a t a density of 2.5 x 10' in 35-mm dishes and

allowed to attach and spread overnight. They were then incubated with medium that contained 50 pg/ml of ascorbic acid in addition to 10% fetal calf serum for 7 days with daily changes of the medium. The medium was then decanted, and the cells were fixed in half- strength Karnovsky's fixative (20) and postfixed in osmium tetroxide. The cells were embedded in Epon, and sections were cut parallel and perpendicular to the culture dish surface, stained with phosphotung- stic acid, uranyl acetate, and lead acetate, and observed in a Philips 420 transmission electron microscope as previously described (21).

RESULTS

Localization and Nature of the Mutation-The osteogenesis imperfecta cells secreted two forms of pro-al(1) and pro-a2(I)

' . *

Control 01

FIG. 6. Autoradiofluorogram of CNBr peptides of the A fragments of crl(1) and &(I) of osteogenesis imperfecta and control cells. Partial collagenase cleavage products were separated in the first dimension on 8% SDS-PAGE, the lanes were cut out and proteins digested in the gel with CNBr, and CNBr peptides were separated by SDS-PAGE in the second dimension. The overmodified a l ( I )A chain fragment from osteogenesis imperfecta cells (arrow) yielded c~1(1)CB7~, al(I)CB8, and nl(I)CB3 peptides which migrated more slowly than their normal counterparts as indicated by their slanted appearance. CNBr peptides from al(1) and a2(I) chains uncleaved by fibroblast collagenase serve as convenient markers of al(I)CB7 and al(I)CB8 in the gel. The results, presented schemati- cally at the bottom, provide evidence that the large CNBr peptide from the overmodified al(1) chain migrating just slower than al(I)CB7 (Fig. 5) contains overmodified al(I)CB7 and nl(I)CB8 peptides. The arrow indicates the fibroblast collagenase cleavage site.

(Fig. 2A). One pro-a chain in each doublet migrated more slowly than normal on SDS-PAGE. The other chain could not be distinguished from pro-al(1) and pro-a2(1) chains synthesized by control and parental cell strains. The altered electrophoretic mobility of some pro-a chains of type I procollagen made by the osteogenesis imperfecta cells indicated that there was a structural abnormality which changed the behavior of molecules into which the abnormal chains were incorporated. To determine whether the structure of the pre- cursor-specific extensions or the triple helical domain of the molecule was affected, medium and cell layer procollagens were treated with pepsin to remove the globular NH2- and COOH-terminal peptide extensions and examined by SDS- PAGE (Fig. 2B). The osteogenesis imperfecta pro-a chains were converted into two forms of al(1) and a2(I). One form of each chain migrated more slowly than normal and the other co-migrated with the control and parental a chains. These results indicate that the delay in electrophoretic mobility was due, at least in part, to structural alteration of the triple- helical domain. Resistance to proteolytic digestion with pepsin also indicates that the abnormal pro-a chains were incorporated into molecules which have a triple-helical conformation a t 4 "C.

The delay in mobility could result from exaggeration of a process that normally occurs along the full length of the molecule, i.e. post-translational modification of prolyl and lysyl residues, or from a more localized event such as the insertion of peptidyl material. To determine if excessive post- translational modification could account for differences in electrophoretic mobility of the two populations of osteogenesis imperfecta procollagen, cells were pulse labeled for 4 h in the presence of @,a'-dipyridyl to block prolyl and lysyl hydroxylation and, secondarily, hydroxylysyl glycosylation. Under these conditions, a,d-dipyridyl markedly inhibited procolla-

1738 Collagen

acidic

-a.a'dipyridyl

+ 0.a' dipyridyl

Triple Helix in Lethal Osteogenesis Imperfecta

basic acidic baric

4 a2fOCB3-5 .e d U I C 8 3 - 5 " .. . . . ,

d U I C B 4

FIG. 7. Autoradiofluorogram of the two-dimensional electrophoretic separation of CNBr peptides of al(1) and a2(I) chains from osteogenesis imperfecta and control cells. Hydroxylated collagens were prepared by pulse labeling 250,000 cells for 16 h with [3H]proline and digesting the medium procollagens with pepsin as described above. Unhydroxylated collagens were prepared by pulse labeling a similar number of cells with ['HI proline for 4 h in the presence of a,a'-dipyridyl and digesting the cell layer samples with pepsin. Hydroxylated and unhydroxylated collagens were then cleaved with CNBr, and the peptides were separated on the basis of charge in the first dimension and molecular weight in the second dimension. With the exception of nl(I)CB6, all of the hydroxylated CNBr peptides migrated as doublets with one spot from each doublet showing delayed mobility and the other peptide co-migrating with the control (arrows). By contrast, each of the unhydroxylated osteogenesis imperfecta CNBr peptides migrated as single peptides with the same apparent molecular weights and charges as the control. The charge heterogeneity observed here is consistent with the experience of others (22.23).

gen secretion, and the osteogenesis imperfecta cells synthesized pro-al(1) and pro-a2(1) chains that co-migrated with those produced by the control and parental cell strains (Fig. 3). The unhydroxylated pro-a chains were identified by their conversion to a-sized chains after pepsin treatment and by CNBr peptide mapping (not shown). These results exclude the possibility that a large insertion of peptidyl material caused the delayed mobility of the pro-a chains of type I procollagen and indicate that molecules which contain the abnormal chains undergo excessive intracellular modification.

To determine if the increased modification affected the entire length of the triple-helical domain, medium collagens (after pepsin) were cleaved asymmetrically with fibroblast collagenase (Fig. 4). The osteogenesis imperfecta collagens yielded normal and slow migrating forms of ~ l ( 1 ) ~ and a2(1)* which include the NH2-terminal three-fourths of the triple helix. By contrast, the ~ y l ( I ) ~ and ~ 2 ( 1 ) ~ chain fragments of the osteogenesis imperfecta collagens migrated as single bands with the same mobility as the control. These results indicate that post-translational modification is increased along all or part of the NH2-terminal three-fourths of the helix.

Additional evidence that the excessive modification occurs NH2-terminal to the fibroblast collagenase cleavage site was provided by cyanogen bromide peptide mapping of a chains from the medium of the osteogenesis imperfecta cells (Fig. 5 ) . This technique allows resolution of the large CNBr peptides of al(1) (al(I)CB7, al(I)CB8, al(I)CB6, and al(I)CB3) and of a2(I) (a2(I)CB4 and the uncleaved a2(I)CB3-5). CNBr peptides from the normal al(1) and a2(I) chains synthesized by the osteogenesis imperfecta cells co-migrated with those

of the parent and control cells. By contrast, the overmodified al(1) chain of the osteogenesis imperfecta cells yielded three CNBr peptide spots: a single spot in the region of al(I)CB7 and al(I)CB8 that migrated just slower than al(I)CB7; a single spot that co-migrated with al(I)CB6; and a single spot that migrated slower than al(I)CB3. Co-migration of the al(1)CBG peptides from the normal and abnormal al(1) chains indicates that the COOH-terminal region of the abnormal chain is not excessively modified.

Although we assumed that the mobility of al(1)CBS from the overmodified al(1) chain was altered, its position in the CNBr mapping gel was difficult to determine. To determine the fate of the abnormal al(I)CB8 peptide, medium collagens were digested with fibroblast collagenase, the cleavage products were separated in the first dimension on SDS-PAGE and then cleaved in the gel with CNBr and mapped in the second dimension (Fig. 6). Fibroblast collagenase cleaves the al(1) chain within al(I)CB7, and the resultant (~1(1)CB7~ fragment migrates further into the gel than al(1)CBS. The overmodified ~ l ( 1 ) ~ chain fragment gave rise to (~1(1)CB7~ and al(I)CB8 peptides which migrated more slowly than their normal counterparts. The results of this experiment indicate that the large CNBr peptide from the overmodified osteogenesis imperfecta al(1) chain that migrated just slower than normal al(I)CB7 (Fig. 5 ) contains overmodified al(I)CB7 and al(I)CB8 peptides.

Because the unhydroxylated pro-a chains (synthesized in the presence of a,a'-dipyridyl) of type I procollagen from the osteogenesis imperfecta cells co-migrated with those from control cells, the mutation likely is either a single amino acid

Collagen Triple Helix in Lethal Osteogenesis Irnperfecta 1739

Pulse-lobel t lme (hr)

FIG. 8. Production of type I collagen by osteogenesis imperfecta, parental, and control cells. Replicate cultures were pulse labeled with ['Hlproline for 1, 2, 4, and 8 h, procollagens from the medium cell layer were separated on 5% SDS-PAGE under reducing conditions, and the amount of radioactivity incorporated into type I collagen was measured by scanning densitometry of x-ray films. The osteogenesis imperfecta cells produced about one-third the amount of type I collagen produced by the parental and control cells.

substitution or an insertion or deletion that is not detected in the system of electrophoresis employed here (<lo-20 amino acids). To determine whether the mutation resulted in a charge change in the domain of al(I)CB7 or a2(I)CB3-5, medium collagens and unhydroxylated cell layer collagens were cleaved with CNBr, and the peptides were separated on the basis of charge in the first dimension and molecular weight in the second dimension (Fig. 7). Consistent with the experience of others (22, 23), some charge heterogeneity was observed for most of the large CNBr peptides. With the exception of al(I)CB6, all of the hydroxylated CNBr peptides of tu1 (I) and tu2(1) demonstrated size heterogeneity consistent with that shown previously (Figs. 5 and 6). By contrast, the unhydroxylated osteogenesis imperfecta a1 (I) and (u2( I) chains yielded CNBr peptides which migrated with the same apparent molecular weights and charges as the control. These results indicate that the osteogenesis imperfecta mutation does not result in a detectable change in either the molecular weight or the charge of CNBr peptides from pro-al(1) or pro- tY2(I).

Effect of the Mutation on Procollagen Production and Sta- bility-After a 16-h pulse with ['HH]proline (Fig. a), the osteogenesis imperfecta cells appeared to produce less type I procollagen than the control or parental cells. To confirm this observation, replicate cultures were preincubated with ascorbic acid and 0.5% fetal calf serum and then labeled with ['HI proline for up to 8 h. Medium and cell layer proteins were then separated on SDS-PAGE under reducing conditions, and type I collagen production was quantitated by scanning gel densitometry (Fig. 8). Over an 8-h period the osteogenesis

_. -

A al(l) mRNA B a2(1) mRNA .... 0 . 0 0 .... e o * * . e 0 0 . . e . 0 . e 0 . . 0

C F O I M C FOI M

C I hour pulse label

proaI(I)- . " proaZ(1).

C F 0 1 M C F O I M Medium Cells

FIG. 9. Autoradiofluorogram of the steady state amount of pro-al(1) and pro-a2(1) cytoplasmic mRNA produced by osteogenesis imperfecta, parental, and control cells. Serial 2-fold dilutions of total cytoplasmic mRNA from replicate cultures were applied to nitrocellulose paper, hybridization was performed with cDNA probes specific for rul(1) and n2(I) collagen genes, and the amount of radioactivity was measured by scanning gel densitometry of x-ray films. The ratio of cytoplasmic pro-cu(I) and pro-n2(I) mRNA was approximately 2:l for the osteogenesis imperfecta, parental, and control cells. Further, there were no significant differences in the hybridization intensities of mRNAs from the 4 cell strains. As a parallel experiment, cells in culture were pulse labeled with ['HI proline under similar conditions, and procollagens from the medium and cell layer were separated on 5% SDS-PAGE under reducing conditions. Significantly less procollagen was produced by the osteogenesis imperfecta cells.

imperfecta cells secreted about one-third the amount of type I procollagen as the control or parental cells.

Decreased procollagen production could result from decreased synthesis or from increased degradation of pro-a chains or the assembled molecules. As one estimate of the rate of type I procollagen synthesis (24,25), the relative steady state amounts of pro-al(1) and pro-a2(1) cytoplasmic mRNA were determined under experimental conditions analogous to those used to assess procollagen production (Fig. 9). The ratio of cytoplasmic pro-cul(1) to pro-a2(I) mRNA was the same for the osteogenesis imperfecta, control, and parental cell strains. Further, the hybridization intensities of mRNAs from the 4 cell strains were similar. Although the amount of pro- al(1) and pro-a2(1) mRNA produced by the osteogenesis imperfecta cells closely approximated those of the control and parental cells, significantly less type I procollagen was harvested from the osteogenesis imperfecta cells after pulse labeling with ['Hlproline for 1 h. These findings suggest that some of the osteogenesis imperfecta procollagen molecules are unstable and rapidly degraded (or, alternatively, that the translational efficiency of pro-a chains was decreased). Deg- radation might increase if procollagen were unstable at normal culture temperatures. Indeed, when the melting temperature (T,) was measured (Fig. lo), that of type I collagen from the medium of two control cell strains and the parental cells was 41-42 "C. By contrast, the melting temperature curve of the collagen was complex; molecules which contained al(1) and n2(I) chains with normal mobility had a T,,, of 40.5 "C while molecules which contained al(1) and a2(I) chains with delayed mobility had a T, of 39.0 "C.

Effect of the Mutation on Procollagen Secretion-Over an

1740 Collagen Triple Helix in Lethal Osteogenesis Irnperfecta

.- 6 0.6 c 0 0

h 0.2 b -

I I I

37" 38O 39O 40° 41' 42 Temperature ("C)

FIG. 10. Melting curves of collagens from the osteogenesis imperfecta. parental, and control cells. Cells in culture were incubated with ['HH]proline for 16 h, collagens from the medium (after pepsin) were placed in a water bath and warmed at a rate of 5 "C/h, and equal aliquots were removed at 0.5 "C intervals and digested with trypsin. The proteins were separated on 5% SDS-PAGE, and the amount of radioactivity was quantitated by scanning densitometry of x-ray films exposed in the linear range. The T,,, of the control collagen (0, bottom), the parental collagen (not shown), and the osteogenesis imperfecta collagen with normal mobility (0, top) was 40.5 "C. The T,,, of osteogenesis imperfecta collagen consisting of cul(1) and n2(I) chains with delayed mobility (0, top) was 39.0 "C. The fluorograms have been arranged to reflect the temperature of digestion of samples.

Chase FIG. 11. Secretion of type I procollagen by osteogenesis im-

perfects, parental, and control cells. Replicate cultures were pulse labeled with ['Hlproline for 4 h, the cells were washed twice with chase medium and then incubated with a large excess of L- proline for up to 2 h. The amount of radioactivity was quantitated by scanning densitometry of x-ray films exposed in the linear range. Relative amounts of medium (0) and cell layer (0) procollagen are depicted.

8-h pulse labeling period the osteogenesis imperfecta cells secreted approximately the same proportion of type I procollagen (by scanning gel densitometry) as the control and the parent cells (Fig. 8). However, the fluorograms from the continuous labeling experiment indicate that the normal and abnormal procollagens synthesized by the osteogenesis imperfects cells were cleared a t different rates; after pulse labeling for 1 h, only pro-al(1) and pro-a2(1) chains with normal

electrophoretic mobility were identified in the medium of osteogenesis imperfecta cell cultures, while at the end of 8 h both normal and overmodified species of pro-al(1) and pro- a2(I) chains had been secreted while most of the procollagen retained by the osteogenesis imperfecta cells consisted of overmodified pro-a chains. The delay in secretion of the overmodified type I procollagen molecules can be seen by comparing the mobilities of the chains secreted at 1, 4, and 16 h (Figs. 9, 3, and 2, respectively). To study procollagen secretion further, cells were incubated in the presence of ["HI proline for 8 h, a large excess of unlabeled L-proline was added, and the incubation was continued for up to 2 h (Fig. 11). During the subsequent chase more procollagen was retained by the osteogenesis imperfecta cells than the control. The majority of the retained procollagen consisted of pro-a chains with delayed mobility (not shown).

Effect of the Mutation on the Structure of the Extracellular Matrix-The amount and organization of the matrix, and the size of the collagen fibrils differed in the cultures from the 01 patient and an age-matched control. In the 01 cultures, there was less total matrix and fewer banded fibrils. The organization of the matrix was more random than in control cells (Fig. 12, A and 23). The fibrils in the 01 culture stained faintly and had indistinct banding patterns. Their borders were poorly defined because of the large amount of diffuse matrix that was associated with them. The fibrils in the control cultures were well stained, had a clear banding pattern, and sharply defined edges (Fig. 12, C and D). Fibrils from multiple areas of each culture were photographed in the electron microscope at a magnification of 30,000 X and printed at 90,000 x for measurement. The mean diameter of the 01 fibrils was 227 f 19 nm (n = 130) compared with 490 & 37 nm in the control cultures (Fig. 12, C and D).

DISCUSSION

The mutation within pro-a chains synthesized by this osteogenesis imperfecta cell strain affected the post-translational modification of molecules into which the abnormal chains were incorporated. Co- and post-translational hydroxylation of prolyl and lysyl residues and the glycosylation of hydroxylysyl residues begin on nascent chains soon after they enter the cisternal lumen of the rough endoplasmic reticulum (26, 27). Virtually all prolyl residues that occur in position Y of the Gly-X-Y triplet are hydroxylated by the enzyme prolyl 4-hydroxylase (28). A smaller proportion of lysyl residues in the Y-position is also hydroxylated (by a different enzyme), and the hydroxylation of lysyl residues creates potential sites for 0-linked glycosylation. Modification of prolyl and lysyl residues is regulated, in part, by the substrate requirements of the hydroxylase enzymes; the hydroxylation of prolyl residues promotes and stabilizes the formation of the triple helix, but a triple helical conformation inhibits further modification of prolyl and lysyl residues (28). I t is clear from our study that the mutation leads to increased post-translational modification of the NH2-terminal three-fourths of type I procollagen molecules which contain the mutant chain. Because triple-helix formation is propagated from the COOH terminus toward the NH2 terminus (29, 30) it appears that the rate at which some of the osteogenesis imperfecta pro-a chains fold into a stable triple-helical conformation is normal at first but then is delayed. Because both the pro-al(1) and pro-a2(I) chains are excessively modified, the delay in triple helix formation appears to allow all of the chains in abnormal molecules to remain in equilibrium with the post-translational modifying enzymes longer than normal.

We propose that the delay in triple-helix formation occurs

Collagen Triple Helix in Lethal Osteogenesis Imperfecta 1741

FIG. 12. Fibroblasts and ma t r ix f rom 01 ( A and C) and control ( B a n d D) cultures. The collagen in the 0 1 culture is sparse and less well oriented compared with the control cultures. The fibrils synthesized by the 01 cells have a smaller diameter (227 nm), poorly defined borders, and an indistinct banding pattern ( E ) . Fibrils from control cells are 490 nm in diameter and have distinct margins and banding patterns (D). A and E , X 3,600; C and D, X 90,000.

as the region of the molecule that harbors the mutation is reached. In support of this proposal, Steinmann et al. (7) have demonstrated in cells from another infant with type I1 osteogenesis imperfecta that a Cys substitution in al(I)CB6 results in excessive modification along the entire length of the triple- helical domain of molecules that contain the mutant chain. Further, we and others (4-6)* have shown in cells from a third infant that a deletion of 84 amino acids from the domains of al(I)CB8 and al(I)CB3 (the deletion encompasses the Met residue at the boundary of these domains) results in excessive modification beginning NH2-terminal to the al(I)CB7 domain. Together, the results of the two previous studies and

Barsh, G. S., Roush, C. L., Bonadio, J., Byers, P. H., and Gelinas, R. E., Proc. Natl. Acad. Sci. U. S. A., in press.

those reported here suggest that localization of the region within type I procollagen a t which increased post-translational modification begins may be a valuable probe for the identification of the site of the mutation which can direct a molecular approach to these genes."

A mutation that results in substitution of another amino acid for a Gly residue in the triple-helical domain or in a small deletion or insertion which disrupts the Gly-X-Y sequence could produce the effects on the molecule we have observed. Single base substitutions in the glycine codons can result in eight different possibilities: Ala, Val, Cys, Trp, Arg, Asp, Ser, and a terminator codon. Examination of the cyanogen bromide peptides of modified and unmodified molecules revealed

J. Bonadio and P. H. Byers, submitted for publication.

1742 Collagen Triple Helix in Lethal Osteogenesis Irnperfecta

no charge changes within either al(I)CB7 or aZ(I)CB3-5. Furthermore, treatment of pro-a chains with N-chlorosucci- nimide (which cleaves at Trp residues) failed to fracture either chain within the triple helical domain (not shown). Finally, neither chain incorporated cysteine within the triple-helical domain, and no disulfide-bonded dimers of d (1) chains could be identified (not shown). These results reduce the number of possible substitutions for a glycine residue to those that do not change the charge but nevertheless interfere with triple- helix formation (Ala, Val, Ser). We are still uncertain whether the mutation which alters molecular behavior resides in the al(1) or a2(I) allele although previous studies suggest that it is most likely to be d ( 1 ) (7, 30). The precise nature of the mutation in this infant, however, will have to be determined by gene or message sequence studies.

This cell strain provides additional evidence that minimal lesions can produce major alterations in the behavior of procollagen molecules. The effects on the behavior of molecules which contain mutant chains are several: there is increased post-translational modification of molecules between the apparent site of the mutation and the NH2 terminus of the triple helix; there is delayed secretion and, probably, increased intracellular degradation of abnormal molecules; the melting temperature of the abnormal molecules is sub- stantially lower than normal; and the manner in which molecules pack into fibrils in the extracellular matrix is altered. Although we are still uncertain which chain contains the mutation, the likely sequence of events in assembly and modification appears to be as follows. The rate of synthesis of pro-al(1) and pro-&(I) and the ability of chains to incor- porate into molecules is normal. Molecules which contain the mutant chain begin triple-helix formation normally but experience a delay in the region of the collagenase cleavage site leaving the NH2-terminal three-fourths of the triple helix accessible to the modifying enzymes. The abnormal molecules are less stable to thermal denaturation, and a portion is degraded prior to secretion. Once outside cells, the abnormal molecules interfere with normal fibril formation, in part because of the nontriple-helical conformation and in part because of the increase in post-translational modification which has created a bulky molecule. The effects of the abnormal molecules on bone mineralization and tissue integrity are unknown.

It seems reasonable to conclude that the type I procollagen synthesized by cells from the parents of the proband was normal because these molecules were structurally indistin- guishable from the control, the rates of procollagen production and secretion were appropriate under the experimental conditions employed, and the morphology of collagen fibrils in the extracellular matrix of these cells in uitro was normal. Although these studies do not formally exclude the possibility of a silent mutation in one (or more) of the parental type I procollagen genes, the data are consistent with the concept that the osteogenesis imperfecta phenotype resulted from a new dominant mutation in one allele of one of the proband's genes for type I procollagen.

Our studies suggest that lethal mutations in type I procollagen may be used to study the requirements for molecular survival within cells, the mechanisms which determine the extent of prolyl and lysyl modifications, the pathway of secretion and the mechanisms by which cells transport molecules within the secretory pathway, and the means by which certain collagen mutations are lethal. These studies lend further

support to the concept that a significant proportion of infants with the lethal perinatal form of osteogenesis imperfecta (type 11) have mutations in only one allele of the genes for type I collagen and that the clinical phenotype can result from new dominant mutations. Finally, our findings suggest that the effect on procollagen molecules (and on the clinical phenotype) may depend on the site of the mutation as well as the nature of the substitution.

Acknowledgments-We are indebted to Dr. Francesco Ramirez for providing the al(1) and a2(I) cDNA probes, to Dr. Eugene Bauer for providing the fibroblast collagenase, and to Dr. Marian Witt for additional clinical information. The technical assistance of Tina Roush and Susan Anderson is gratefully acknowledged. We thank Nancy Higgins for preparing the manuscript.

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