of biological vol. no. of july 5, pp. 9056-9064, …0 1986 by the american society of biological...

THE J O U R N A L OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc Vol. 261, No. 19, Issue of July 5, pp. 9056-9064, 1986

Printed in U.S.A.

The Molecular Defect in an Autosomal Dominant Form of Osteogenesis Irnperfecta SYNTHESIS OF TYPE I PROCOLLAGEN CONTAINING CYSTEINE IN THETRIPLE-HELICAL DOMAIN OF PRO-al(1) CHAINS*

(Received for publication, February 24, 1986)

W. Nancy de Vries and Wouter J. de Wet$ From the Department of Biochemistry, Potchefstroom University for Christian Higher Education, Potchefstroom 2520, South Africa

Synthesis of procollagen was examined in skin fibroblasts from a patient with a moderately severe autosomal dominant form of osteogenesis imperfecta. Pro- teolytic removal of the propeptide regions of newly synthesized procollagen, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, revealed the presence of type I collagen in which two al(1) chains were linked through interchain disulfide bonds. Fragmentation of the disulfide-bonded al(1) dimers with vertebrate collagenase and cyanogen bromide demonstrated the presence of a cysteine residue in al(I)CBS, a fragment containing amino acid residues 124-402 of the al(1) collagen chain. Cysteine residues are not normally found in the triple-helical domain of type I collagen chains. The heterozygous mature of the molecular defect resulted in the formation of three kinds of type I trimers: a normal type with normal pro-a(1) chains, a type I trimer with one mutant pro-al(1) chain and two normal chains, and a type I trimer containing two mutant pro- al(1) chains and one normal pro-&(I) chain. The presence of one or two mutant pro-al(1) chains in trimers of type I procollagen was found to reduce the thermal stability of the protein by 2.5 and 1 “C, respectively. In addition to post-translational overmodification, procollagen containing one mutant pro-a1 (I) chain was also cleared more slowly from cultured fibroblasts. The most likely explanation for these disruptive changes in the physical stability and secretion of the mutant procollagen is that a cysteine residue is substituted for a glycine in half of the pro-al(1) chains synthesized by the patient’s fibroblasts.

Type I collagen is a major constituent of bone, tendons, ligaments, skin, and other connective tissues. The heterotri- meric protein is first self-assembled as type I procollagen, a precursor comprised of two pro-al(1) and one pro-a2(1) chains (1, 2). After secretion of the precursor, both the amino- and carboxyl-terminal propeptides are enzymically removed to generate collagen monomers, which are then assembled and cross-linked to form extracellular fibers of exceptional tensile strength. A variety of observations, made on cultured skin

*This work was supported by a research grant (to W. J. d. W.) and a postgraduate scholarship (to W. N. d. V) from 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 ‘‘uduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

-

$ To whom correspondence should be addressed.

fibroblasts, showed that some forms of the heritable disorders of connective tissues involve changes in the metabolism of type I procollagen (for reviews, see Refs. 3-5). These observations included changes in the synthesis of pro-d(1) chains relative to pro-a2(1) chains (6, 7), increased synthesis of type I11 and/or type IV procollagen (8-11), delayed or even defective self-assembly of procollagen heterotrimers (12-14), post- translational overmodification of lysine and hydroxylysine residues (15-17), decreased thermal stability of procollagen trimers (9, 13, 14, 17-19), increased susceptibility of trimers to aspecific proteases (9, 20), decreased secretion of procollagen by skin fibroblasts (9, 10, 13, 17, 20), and defective processing of extracellular type I procollagen by procollagen N-proteinase (9, 18, 19).

Peptide mapping (9, 10, 16-19, 21) and recombinant DNA technologies (7, 11, 22-23) have recently been employed to uncover a number of molecular defects in the structure or expression of the type I procollagen genes in patients with 01,’ a heterogenous group of genetic diseases which is pri- marily characterized by increased fragility of bone (24). The majority of the known type I collagen mutations have been shown to involve in-frame deletion of one or more exons in the triple-helical domain of either the pro-al(1) or pro-aB(1) genes (11, 23). Genomic cloning of the DNA from a patient with a moderately severe form of 0 1 (12) also revealed a small frameshift deletion in the noncollagenous C-propeptide region of the pro-a2(1) collagen gene (22). In addition, a lethal variant of 01 has been demonstrated to involve the substitution of a cysteine residue for a glycine residue in fragment al(I)CB6, containing amino acid residues 817-1014 of the al(1) collagen chain (17). In another unrelated mild form of 01, half the al(1) chains contained cysteine, an amino acid not normally found in the collagenous region of type I procollagen (25).

We report here observations on fibroblasts from a patient with a moderately severe form of 01 in which the cells synthesize type I procollagen containing a cysteine residue in fragment al(I)CB8.

The abbreviations used are: 01, osteogenesis imperfecta; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PC-al(I), partially processed pro-al(1) chains of type I procollagen containing C-propeptides but not N- propeptides; T,,,, midpoint for the helix to coil transition for procollagen; type I* and type I** procollagen, trimers containing one or two mutated pro-al(1) chains, respectively; C-propeptide, carboxyl-terminal propeptide; N-propeptide, amino-terminal propeptide.

9056

Amino Acid Substitution in Osteogenesis Imperfecta 9057

EXPERIMENTAL PROCEDURES’

RESULTS

Synthesis and Secretion of Disulfide-linked a1 ( I ) C h i n s by Fibroblasts from the Patient-Control and 01 fibroblasts were labeled with [3H]proline for 4 h, and the cell homogenates and media proteins were examined by electrophoresis in gradient polyacrylamide gels containing SDS. Pro-al(1) chains from the patient’s fibroblasts consistently migrated at a slightly slower rate than comparable chains from control fibroblasts (Fig. lA, lanes 1, 2, 7, and 8). However, when fibroblast cultures were labeled in the presence of 0.3 mM a&-dipyridyl to inhibit prolyl and lysyl hydroxylases, the pro-al(1) chains from the 01 and control fibroblasts had the same migration (Fig. lA, lanes 2 and 3). The observed difference in migration of the pro-a chains when fibroblast cultures were incubated in the absence of a,a’-dipyridyl is, therefore, indicative of more extensive post-translational modifications of the patient’s type I procollagen. Overmodification of type I procollagen is a common finding in 01, and has been shown to reflect delayed helix formation (9, 10, 13-19). Electropho- resis under nonreducing conditions, nevertheless, showed that the patient’s pro-al(1) chains were efficiently incorporated into heterotrimers (Fig. lA, lanes 5 and 6). Except for slightly slower migration rates, no other obvious differences between the 01 and control samples were observed when cellular proteins were digested with pepsin, reduced, and electrophoresed (Fig. 123, lanes 1 and 2). In contrast, separation of pepsin-digested cellular proteins from 01 fibroblasts by interrupted electrophoresis (26) resulted in the appearance of an additional species which migrated slower than normal al(1) chains, but faster than al(II1) chains (Fig. lB, lanes 3 and 4). Under nonreducing conditions, SDS-PAGE of pepsin-treated cellular and media proteins from the patient’s fibroblasts likewise showed the presence of an additional band with an apparent molecular weight of about 200,000 (Fig. lB, lanes 5- 8). The patient’s fibroblasts, therefore, synthesized and secreted material which, after pepsin digestion, co-electrophoresed with the 0 components of type I collagen and contained reducible interchain disulfide bonds.

To examine the nature of the disulfide-bonded material, labeled cellular proteins from the patient’s fibroblasts were digested with pepsin and then separated in the first dimension by SDS-PAGE in the absence of 2-mercaptoethanol. The gel strip was exposed to 2-mercaptoethanol and subjected to electrophoresis in the second dimension. As illustrated in Fig. lS, treatment of the disulfide-linked material with the reducing agent produced components which co-migrated with al(1) chains. That the patient’s type I collagen contained disulfide- bonded al(1) dimers was also substantiated by peptide mapping. Digestion of the disulfide-linked material with cyanogen bromide, followed by mercaptoethanol reduction and SDS- PAGE in the second dimension, clearly generated a distinctive peptide pattern that is characteristic of al(1) chains (Fig. 2A). Furthermore, fragment al(I)CB8 was not visualized when cyanogen bromide fragments of the al(1) dimer were separated under nonreducing conditions (Fig. 2B). Instead, an additional species corresponding to the disulfide-bonded di-

Portions of this paper (including “Experimental Procedures” and Figs 1S-6S are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Doc- ument No. 86M-599, cite the authors, and include a check or money order for $8.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

A -dip +dip -dip

+ S H * S H - S H

C P C P C P -“

i -

1 2 3 4 5 6

B + S H Z S H - S H

C P C P C P ”-

”

-dip

+SH

C P

- p r o x ( 1 1

7 8

- S H

il ** I ! 1 2 3 4 5 6

FIG. 1. SDS-polyacrylamide gel electrophoresis of cellular and media proteins before (A) and after (B) digestion with pepsin. 01 and control fibroblasts were labeled with [3H]proline for 4 h. The gels were 4-8% gradients of polyacrylamide. Lanes 1-6, cellular proteins; lane 7 and 8, media proteins. -dip, cells labeled in absence of cY,cY’-dipyridyl; +dip, cells labeled in presence of a,&‘- dipyridyl; +SH, electrophoresis in presence of 2-mercaptoethanol; -SH, electrophoresis in absence of 2-mercaptoethanol; G’H, electrophoresis first in absence and then in presence of 2-mercaptoethanol (26); C, control fibroblasts; P, fibroblasts from the patient; FN, fibronectin.

mer of fragment al(I)CB8 was observed. Digestion with vertebrate collagenase (27) prior to cyanogen bromide peptide mapping was employed to confirm that the al(1) chains of the al(1) dimer were joined by a disulfide bond contained in fragment al(I)CB8. Treatment of the patient’s labeled media proteins with the enzyme generated an ~ ~ l ( 1 ) ~ dimer (Fig. 2C). The cyanogen bromide peptide pattern of the al(I)A dimer was characterized by the presence of fragment (~1(1)CB7~ and the absence of fragments al(I)CB6 and al(I)CB7. The disulfide-bonded peptide dimer did not contain the vertebrate collagenase recognition site.

On the basis of the results represented in Fig. 2, we concluded that the mutation resulted in the incorporation of a cysteine residue in fragment al(I)CB8.

Thermal Stability of the Patient’s Type I Procollagen-To assess the effect of the mutation on the helix stability of the patient’s type I procollagen, a technique pioneered by Bruck- ner and Prockop (28) was employed to examine the thermal denaturation of cellular procollagen. In essence, control and

9058 Amino Acid Substitution in Osteogenesis Imperfecta

A B C

FIG. 2. Two-dimensional gel electrophoresis of cyanogen bromide fragments of disulfide-linked media collagens. 01 fibroblasts were labeled by incubation with 100 pCi/ml of [3H]proline for 4 h. Media proteins were digested with pepsin ( A and B ) and then with vertebrate collagenase (C) prior to SDS-PAGE under nonreducing conditions. The electrophoretogram in C represents partial digestion of media proteins with vertebrate collagenase. Following electrophoresis in the first dimension, the proteins were digested with cyanogen bromide. The gel strips were equilibrated with sample buffer, and the cyanogen bromide fragments were electrophoresed in the second dimension in the presence ( A ) or absence (B and C) of 2-mercaptoethanol. -SH, nonreducing conditions; +SH, reducing conditions; +CNBr, digestion with cyanogen bromide.

0 1 fibroblasts were labeled with [3H]proline for 3 h and then homogenized. Aliquots of the cell homogenates were incubated for 10 min at temperatures ranging from 34 to 43 "C, and a mixture of trypsin and chymotrypsin was added as a proteo- lytic probe for triple-helical conformation. After brief digestion at 20 "C, the samples were subjected to SDS-PAGE under nonreducing conditions (Fig. 2s). In addition to the proteo- lytic digestion of the collagen domain of denatured or partially unfolded procollagen, both the N- and C-propeptides of native triple-helical procollagen were efficiently removed by the combined action of the two enzymes. The procedure, therefore, allowed examination of the thermal stability of not only type I procollagen containing disulfide-bonded al(1) chains (fluo- rographed in the form of al(1) dimers), but also of type I procollagen which did not contain al(1) dimers (represented by the al(1) or a2(I) bands). Densitometry of fluorograms such as those presented in Fig. 2S, showed that type I procollagen which contained disulfide-bonded al(1) dimers had a melting temperature (T,) of about 39 "C (Fig. 3B). Densitom- etry of the patient's al(1) band produced a biphasic melting curve (Fig. 3C). About two-thirds of the material denatured with a T, value of 38 "C. The remainder of the patient's type I procollagen exhibited a T,,, of about 40.5 "C, a value also observed for type I procollagen from control fibroblasts (Fig. 3A).

The results, therefore, indicated that the pro-al(1) chains synthesized by the patient's cells were incorporated into three kinds of heterotrimers: normal type I with normal pro-al(1) chains; a type I trimer comprised of one mutant pro-al(1) chain, one normal pro-al(1) chain, and one pro-a2(1) chain (defined here as type I*); and a type I trimer containing two mutant pro-al(1) chains and one pro-a2(1) chain (defined here as type I**). The three entities defined here as type I, type I*, and type I** procollagen were present in a stoichiometry of about 1:2:1. A second conclusion was that procollagen which contained only one mutant pro-al(1) chain had

Temperoture I "C I

FIG. 3. Melting curves of type I procollagen from control and 01 fibroblasts. Values for the relative survival of al(1) chains or d ( 1 ) dimers in triple-helical trimers after incubation of cellular proteins at the indicated temperatures and digestion with a mixture of trypsin and chymotrypsin at 20 "C were obtained by densitometry of fluorograms such as those shown in Fig. 2s. A, thermal stability of type I procollagen from control fibroblasts (T, about 40.5 "C); B, thermal stability of the patient's type I procollagen containing disulfide-linked d ( 1 ) dimers (T, about 39 "C); C, biphasic melting curve of the patient's type I procollagen which did not contain disulfide- linked al(1) dimers (T, values 38 "C and about 40.5 "C, respectively).

a thermal stability that was 2.5 "C lower than that of normal type I procollagen. Paradoxically, the type I** species was thermally more stable than procollagen containing only one mutant pro-al(1) chain.

Extracellular Processing of the Procollagen-To examine the metabolic processing of the patient's procollagen, 01 and control fibroblasts were labeled for 2-16 h. Visual examination of the fluorograms in Fig. 3 s suggested that procollager,

Amino Acid Substitution in Osteogenesis Irnperfecta 9059

comprised of two altered pro-d(I) chains did not accumulate in the patient’s fibroblasts, but was efficiently cleared from these cells. In contrast to the electrophoretograms shown in Fig. 3S, media proteins from the same clearance experiment were not pepsin-treated prior to SDS-PAGE, but were directly prepared for electrophoresis in the presence of 2-mercaptoethanol. This approach resulted in the identification of pC- collagen, as well as smaller amounts of pN-collagen and collagen in the media of both control and 01 fibroblasts (Fig. 4S), suggesting that the secreted procollagen was partially processed to pC-collagen by procollagen N-proteinase present in the culture medium.

For a more detailed analysis of the conversion of procollagen to PC-collagen by the endogenous procollagen N-proteinase, the methods described under “Experimental Procedures” were employed. Prolonged exposure of the patient’s labeled media procollagen to unlabeled culture medium, obtained from control fibroblast cultures, clearly resulted in an increased conversion of procollagen to PC-collagen (Fig. 5% panel A ) . A similar result was obtained when pulse-labeled 01 fibroblasts were incubated with chase medium from unlabeled control fibroblast cultures (Fig. 5S, panel B). Because of the much lower activity of the endogenous procollagen C-proteinase (29), it was not possible to distinguish between the processing of type I* and type I** procollagen by procollagen N- proteinase (Fig. 5S, panel C). Nevertheless, quantitation of the fraction of procollagen converted to PC-collagen, as re- flected by the ratio of PC-al(1) to the sum of PC-al(1) and pro-d(1) in the electrophoretograms depicted in Fig. 5S, demonstrated that the patient’s type I procollagen was processed at the same rate and to the same extent as procollagen from control fibroblasts (Fig. 4). In conclusion, no evidence for an adverse effect of the mutation on the processing of the patient’s procollagen by procollagen N-proteinase was found.

Kinetics of Secretion of Procollagen by 01 Fibroblasts-As noted above, mutant procollagen containing two disulfide- linked al(1) chains was efficiently cleared from the patient’s fibroblasts. In fact, an increase in the relative presence of type I** procollagen in the extracellular space was seen after longer incubation periods (Fig. 3s). The experiment, therefore, suggested that type I** procollagen was more efficiently cleared from the fibroblasts than type I* procollagen.

To explore the observation in greater detail, the kinetics of secretion of type I procollagen by 01 and control fibroblasts were examined in pulse-chase experiments. To facilitate dis- crimination between the secretion of the disulfide-linked and

0 e 16 24 0 Time ( h

8

I 16 24

FIG. 4. Time course for conversion of type I procollagen to PC-collagen by endogenous procollagen N-proteinase. Values for the ratio of PC-d(1) to the sum of pC-cyl(I) and pro-d(1) were determined by densitometry of fluorograms shown in Fig. 5s. A, processing of media procollagen in the absence of fibroblasts; B, processing of procollagen secreted by pulse-labeled fibroblasts. 0, control; 0 , O I patient.

. . \, I I 1 I

0 2 4 6 Chase Time ( h )

FIG. 5. Kinetics of secretion of type I procollagen from pulse-labeled control and the patient’s fibroblasts. The per cent labeled type I procollagen remaining in the cells at various time points after the chase was determined by densitometry of fluorograms such as those in Fig. 6s. Data for the secretion of type I* procollagen by 0 1 fibroblasts were simulated as described under “Experimental Procedures.” 0, control type I procollagen; M, patient’s type I procollagen containing disulfide-linked al(1) dimers; A, patient’s type I procollagen not containing disulfide-linked dimers; t, simulated values for secretion of type I* procollagen. The rate constants for secretion were calculated using values obtained by exponential peeling of curves for control type I procollagen (0), the patient’s type I** procollagen (O), the patient’s type I procollagen not containing disulfide-linked cul(1) dimers (A), and the patient’s type I* procollagen (*I.

the non-linked species, labeled proteins from various time points after the start of the chase were pepsin-treated and electrophoresed in the absence of 2-mercaptoethanol (Fig. 6s). Fluorograms were scanned by densitometry, and the results were plotted as the log of the per cent of type I procollagen remaining in the cells during the chase (Fig. 5). As was previously found for the secretion of labeled collagenous peptides by chick embryo tendon cells (30) and human skin fibroblasts (lo), at least two first-order rate constants were required to describe the kinetics of secretion of type I procollagen by control fibroblasts. Exponential peeling indicated that the two first-order rate constants were 3.2 x lo-’ min” and 1.8 X min”, respectively (Table I). These values deviate from the values of 1.9 x lo-’ min-’ and 2.7 X

min-’ previously reported for control human fibroblasts (10). In this context, it should be noted that the data developed here reflect the kinetics of secretion of type I procollagen, whereas the previously published values were based on the secretion of total collagenous peptides (10).

With the patient’s fibroblasts, the rate constants for the secretion of type I** procollagen, and of type I procollagen in which the d ( I ) chains were not disulfide-linked, were about the same as those obtained with the control fibroblasts (Table I). Also, the fraction of type I** procollagen secreted during the fast and slow phases closely corresponded to the values observed with control type I procollagen. With the patient’s fibroblasts, 93% of type I** procollagen was secreted in the fast phase described by the first rate constant. With control fibroblasts, 96% of type I procollagen was secreted in the fast

9060 Amino Acid Substitution in Osteogenesis Irnperfecta

TABLE I Kinetics for the secretion of normal and mutant type Zprocollagen by control and OZ fibroblnsts

Values for the rate constants and the fraction of type I procollagen secreted in each phase were calculated from the data in Fig. 5 as described under “Experimental Procedures.”

kl k? tLI for fast tH for slow Fraction secreted Fraction secreted phase phase in fast phase in slow phase

X I O P min” rnin“ rnin rnin 7% 76 Control

0 1 Type I procollagen 3.2 1.8

Type I** procollagen 3.1 1.9 Type I* and type I procollagen 3.5 1.8 Type I* procollagen 3.7 1.8

phase. From visual inspection of the fluorograms in Fig. 6S, it was apparent that a significant amount of type I procollagen was retained in the patient’s fibroblasts after the chase. Only 74% of the patient’s type I procollagen which did not contain al(1) dimers was secreted in the fast phase (Fig. 5 and Table I). The procollagen fraction consisted of normal trimers and trimers containing only one mutant pro-cul(1) chain. The ratio of the two species was 1:2 (Fig. 3C). This implies that the fraction of type I* procollagen secreted in the fast phase was even lower than 74%. Simulation of the secretion of the type I* species indicated that 63% of the patient’s procollagen, comprised of one normal pro-al(1) chain, one mutant pro- al(1) chain, and one pro-a2(1) chain, was secreted in the fast phase (Fig. 5 and Table I). Again, the rate constants for the secretion of the type I* species were essentially the same as those obtained with normal type I and the patient’s type I** procollagen.

DISCUSSION

The 0 1 patient studied here had a heterozygous defect for the synthesis of structurally altered pro-al(1) chains. Exam- ination of cyanogen bromide fragments demonstrated that the mutation resulted in the incorporation of a cysteine residue in fragment al(I)CB8, a fragment containing amino acid residues 124-402 of the al(1) chain (2).

Because human al(1) chains do not normally contain cysteine residues (2), the data suggested that the structural alteration is an amino acid substitution. They do not, however, definitively exclude the possibility of some other alteration in primary structure, since the evidence for an amino acid substitution is based entirely on the presence of disulfide- bonded al(1) dimers in SDS electrophoretic gels. Because the mutation falls beyond the reach of any of the available human pro-d(I) cDNAs (31), we were unable to employ nuclease S1 mapping analysis for further analysis of the structural alteration. Clearly, a precise definition of the defect will require cloning and direct sequencing of the affected allele, or alter- natively, determination of the amino acid sequence of the mutant al(1)CB fragment. However, by analogy to similar data developed on two other 01 variants, both of which contained a cysteine residue in cyanogen bromide fragment al(1)CBG (17, 25), it is reasonable to assume that the structural alteration described here is indeed an amino acid substitution.

Whether or not the defect is a substitution, it is apparent that coding sequences beyond the site of the mutation were in the correct phase, so that the mutant pro-otl(1) chains associated with normal pro-a([) chains and formed triple- helical procollagen. The presence of altered pro-al(1) chains in type I trimers had three consequences in terms of the characteristics of the procollagen molecules. There was post- translational overmodification of pro-a(1) chains, triple-heli-

22 387 96 4

22 367 93 20

7 392

19 74

389 26

63 37

cal trimers containing mutated pro-d(I) chains had a lower thermal stability than triple helices formed from normal pro- CY chains, and the kinetics of secretion of procollagen by the 01 fibroblasts were abnormal. By analogy with similar observations previously made on fibroblasts from other patients with 01 (9, 10, 13, 14, 17-19), the data developed here suggested that the structural alteration either interrupted the continuous triple-helical conformation of the protein, or in- terfered with the normal stabilizing effect of interchain hy- drogen bonding. Because the patient’s procollagen was efficiently processed by procollagen N-proteinase, we concluded that the native conformation of the N-proteinase cleavage site was not destroyed by slippage and mis-registration of Gly-X-Y-units on the amino-terminal side of the mutation, as was previously reported for three other 01 variants (9, 18, 19). The observed disruptive effect of the mutation on the conformation of the triple-helical domain was probably con- fined to the immediate vicinity of the mutation.

The heterozygous nature of the mutation resulted in the assembly of three kinds of triple helices, defined here as type I, type I*, and type I** procollagen, with a stoichiometry of about 1:2:1. An unexpected observation was that the T, value obtained for procollagen molecules containing only one mutant pro-al(1) chain was lower than the T,,, observed for procollagen molecules composed of two altered pro-al(I) chains (Fig. 3). Moreover, an alteration in the kinetics of secretion of procollagen by the patient’s fibroblasts, as re- flected by an increase in the fraction of procollagen secreted in the late phase (described by the rate constant k2), was only observed for the type I* procollagen species (Table I). The kinetics of secretion of the type I** species were essentially the same as those obtained with control type I procollagen. Consequently, procollagen containing only one altered pro- al(1) chain was more slowly cleared from 01 fibroblasts than procollagen composed of two disulfide-linked mutant chains (Fig. 6s). These corroborative observations indicated that the presence of a single cysteine residue in the triple-helical domain of the patient’s procollagen affected the conformation of that region to a larger extent than when an interchain disulfide bond was present in procollagen containing two mutant pro-al(1) chains. This phenomenon may reflect a compensatory stabilizing effect of the interchain disulfide bond on the conformation of the mutant procollagen. An alternative explanation would be that a disulfide bond between two altered pro-d(I) chains precluded the interaction of the mutant procollagen chain(s) with some other mole- cule(s). Because type I* heterotrimers contain only one mutant chain, about one-half of the patient’s assembled type I procollagen have a single reactive sulfhydryl group in the triple-helical domain. This implies that these heterotrimers could potentially interact more readily with other molecules. Such aberrant interactions would greatly distort the confor-


mation of the triple-helical domain of the patient’s procollagen containing only one altered pro-al(1) chain, and may have substantial implications in terms of the pathophysiology of the disorder.

The clinical features of the patient studied here were in many ways homologous to that of a variant described by Nicholls et ul. (25) . In both cases, the presence in collagen trimers of al(1) chains containing cysteine resulted in syn- dromes best classified as type I osteogenesis imperfecta. In contrast, the mutation reported by Steinmann et al. (17) produced a lethal disease. Although the molecular basis of the remarkable difference in clinical symptomatology is still un- clear, our data showed that an adverse effect of an amino acid substitution involving a cysteine residue on the thermal stability, processing, and secretion of type I procollagen molecules does not necessarily have to produce a lethal syndrome. In this context, it should be noted that the alteration reported by Steinmann et al. (17) was a new sporadic mutation against a background of an uncharacterized Marfanoid genotype. It is, therefore, very likely that the lethal variant studied by those investigators was a compound heterozygote. Indeed, compound heterozygosity has been demonstrated in another lethal variant of 01 (10). Because the role of the uncharacterized “Marfan” gene in modifying the characteristics of the lethal variant’s procollagen is unknown (17), a rigorous com- parison of the data obtained for the lethal variant and for the patient studied here may not be completely valid.

In conclusion, the data generated here have two implications in terms of the nature and consequences of mutations that lead to the synthesis of structurally abnormal type I procollagen. One is that collagen mutations involving amino acid substitutions may be more prevalent than previously thought (3,4). A second implication is based on the observation that the presence in type I heterotrimers of one normal and one mutant pro-al(1) chain was more deleterious than the presence of two altered pro-al(1) chains. This suggests that procollagen from heterozygotes for the mutation may be more severely affected than procollagen from homozygotes, and as such may serve as a novel example of the concept of protein suicide (3, 4).

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MINIPRINT SECTION

Supplement to: THE MOLECULAR DEFECT IN AN AUTOSOMAL OOMINANT FORM OF OSTEOGENESIS

IMPERFECTA: Synthesis of Type I Procollagen Containing Cysteine in the Triple-

helical Domain of Pro-al(I) Chains

by U. Nancy de Yries and Uouter J. de Wet

EXPERIMENTAL PROCEDURES

The Clinical Phenotype - The patient was a 59-year old Caucasian female with a history of multiple bone fractures. On physical examination. she had blue sclerae, a short stature (138 cm), pectus carinatum. and thoracic kyphosco- liosis. X-rays showed generalized osteopenia, thinning of the calvarium, intra- cerebral calcifications in the frontal region, biconcave narrowing of the dorsal and lumbar vertebrae, bilateral acetabuli protrusio, as well as marked bowing of the femora, ulna, tibia, and fibula. NO Wormian bones were seen, and audiome- try revealed only slight conductive hearing loss. Also, no evidence for dentino- genesis imperfecta, joint hypermobility or skin changes, such as thinness, hyperextensibility, and fragility, was found. The extensive family history revealed an autosomal dominant mode of inheritance.' Based on the clinical symptomatology, the disorder is best classified as type I 01 (24).

Labeling of Fibroblasts and Analysis of Proteins and Peptides - Primary cultures of skin fibroblasts were established from biopsies of the patient and two unrelated controls. Skin fibroblasts were grown in 25-cm2 plastic culture flasks under standard conditions in Dulbecco's modified Eagle's minimal essential medium containing 10% fetal calf serum. Post-confluent cells, from passages 3 to 10, were fed for two days with the same medium containing 50 vglme sodium ascorbate, and then incubated at 37-C for varying times with 1.5 me of medium lacking fetal calf serum and containing 25 to 100 uCilmL of L-[4,5-3H]proline (14-32 Ci/mnole; Amersham or Commissariat a L'energie Atomique). To block the hydroxylation of procollagen chains in selected cultures, %a'-dipyridyl was added to the labeling medium to a concentration of 0.3 mM.

Following incubation, the medium was removed and a one-tenth volume of a stock solution of protease inhibitors was added to give final concentrations of 40 mM EDTA, 11 mM N-ethylmaleimide, 0.9 nM p-aminobenzamidine, 1.1 nM phenyl- methylsulfonylfluoride, 25 vglml of heat denatured soluble calf skin collagen (Cooper Biomedical. grade CLCS). and 0.1 M Tris-HC1 buffer, adjusted to pH 7.4 at room temperature. Proteins were precipitated at 4°C overnight with 176 mg/mi of a m n i u m Sulfate. The precipitates were recovered by centrifugation at 13000 x g for 2 h, and were dissolved at 4°C in 0.1 mP of cell homogenizing buffer which consisted of 0.27 M NaCl, 22 mM N-ethylmaleimide, 1.8 nM p-aminobenzamidine, 2.2 mM phenylmethylsulfonyl fluoride, 0.1% Triton X-100, 80 mM EDTA, and 200 pg/mL of heat denatured collagen in 0.27 M Tris-HC1 buffer adjusted to pH 7.4 at room temperature. For examination of the extracellular proteins by SDS-PAGE, a one-tenth volume of 20% SOS was added, the samples were heated in boiling water for 3 min, and were then dialyzed against a buffer which consisted of 1% SDS, 5% glycerol, and 0.001% bromophenol blue in 50 mM Tris-HCI buffer adjusted to pH 6.8 at room temperature. Electrophoresis was performed on polyacrylamide gels in the presence of SDS (32) and fluorography was performed according to standard procedures (33). For direct examination of cellular proteins by gel electrophoresis, the cell layers from 25-cm2 flasks were scraped in 0.5 me of the cell homogenizing buffer, homogenized. and prepared for SOS-PAGE as described previously (10).

Digestion of cellular or media proteins with pepsin and/or vertebrate collagenase (27) was carried out as described earlier (10). The skin fibroblast collagenase was a generous gift from Or. John Jeffrey, Division of Dermatology, Department of Medicine. School of Medicine, Washington University. St. LOUiS, Missouri. For cyanogen bromide peptide mapping, pepsinized or vertebrate Collage- nase-treated labeled media proteins were prepared for SDS-PAGE and separated in 5% polyacrylamide vertical slab gel strips measuring 150 mn x 12 mn x 1.3 mn. Following electrophoresis in the first dimension, the gel strips were rinsed with 70% formic acid, subjected to digestion with cyanogen bromide, and prepared for gel electrophoresis according to the procedures described by Barsh dl (34). SDS-PAGE in the second dimension was performed on 8-14% gradient polyacrylamide gels, and fluorograms were prepared.

Thermal Stability of Procollagen as Tested by Trypsin and Chymotrypsin Digestion - To examine the thermal stability of cellular procollagen from 01

and control fibroblasts, cells in 175-cm2 flasks were incubated for 3 h with medium containing 100 pcilme of L- k.5-3H]proline. The medium was replaced with phosphate-buffered saline and the cells were cooled at 2O0C for 20 min.

w . N. de Vries, G. S. Gericke, J. du Toit, J. Hamersma, J. OP't Unf~ and W. J. de Wet. manuscriDt in preparation.

The buffer was removed and the cells were scraped into 3.3 mL of Krebs improved Ringer I I medium containing 0.1% Nonidet P-40 and 10 nM EDTA. The cells were homogenized, imnediately cooled to 2D°C, and divided into 17 aliquots of 200 uL each. They were first incubated for 10 min at ten.oeratures ranging from 34 to 43'C, and then rapidly cooled to 20°C prior to the addition of a one-tenth volume of a stock solution containing 1 mg/ml trypsin (Cooper Biomedical, grade TPCK), 2.5 mg/mP chywtrypsin (Cooper Biomedical, grade CDI), and 2% Nonidet P-40 in 200 nM EDTA adjusted to pH 7.4. Digestion was continued for 2 min at 2OoC, and then terminated by the addition of soybean trypsin inhibitor (Cooper Biomedical, grade TRL) to a concentration of 0.5 mglmt. The samples were imnediately boiled for 5 min in the presence of 2% SDS, and prepared for SO$-PAGE on 44% gradient polyacrylamide gels as described above. Several fluorograms of the same gels were scanned on a Biorad densitometer.

Conversion of Procollagen to pC-collagen - For examination of the metabolic processing of type I procollagen, the following two-pronged approach was developed. Fibroblasts were fed fortwo days in the presence of sodium ascorbate as described above. The unlabeled medium from control flasks were pooled and stored on ice. 01 and control fibroblasts were pulse-labeled for 3 h with 100 vCi/d of L- k,5-3H]proline. To initiate the chase, the labeled media were replaced with chase medium consisting of unlabeled medium, previously obtained from control cultures and supplemented with 10 mM unlabeled proline. Different flasks were chased for 4, 8, 16 and 24 h. After the chase, the chase medium was prepared for direct examination by SOS-PAGE as described above. Whilst this was in progress, the pooled pulse-labeled medium from either 01 or control fibroblasts were combined with an equal volume of unlabeled medium. previously obtained from control cultures, and incubated at 37-C. Samples were removed at the times indicated and processed for direct examination by SDS-PAGE. Fluoro- grams were scanned to quantitate the per cent conversion of pro-al(I) to PC-al(1) (19).

Kinetics of Secretion of Procollagen - To examine the kinetics of secretion of control and mutant type I procollagen, pulse-chase experiments were carried out according to a modification of the procedures described previously (10.20). Fibroblasts in 25-cm2 flasks were labeled with 100vCi/mL of L-~.5-3H]proline for 3 h. To initiate the chase, the labeled medium was replaced with 1.5 ml of chase medium. The chase medium consisted of fresh Dulbecco's modified Eagle's minimal essential medium containing 50 uglml of sodium ascorbate and 10 nM proline. Dul,ing the chase. the medium was repeatedly replaced with 1.5m.i of fresh chase medium at the times indicated. The different aliquots of chase medium were centrifuged for 3 min at 18000 x g. and then subjected to a m n i u m sulfate precipitation as described above. The precipitates were resuspended in 50 u1 of cell homogenizing medium, digested with pepsin, and prepared for SDS-PAGE. After the final chase period. the cell layers were homogenized in 250 ul of cell homogenizing medium, digested with pepsin, centrifuged for 5 min at 18000 x g, and subjected to amnonium sulfate precipitation. The precipitates were resuspended in 100 u l of cell homogenizing medium and prepared for SOS-PAGE. The different samples were electrophoresed on 44% gels, and fluorograms were scanned to quantitate the fraction of type I procollagen remaining in the Cells at various time points during the chase. The amount of type I PrOCOllagen in the fibroblasts at time zero was taken as 100%.

The data 0 1 , type I procollagen in the medium and cell layer were used to calculate the rate constants as described earlier (10.30) from the relationship,

T = ~ e - k l t + 6e-k~' where T is the per cent labeled type I procollagen remaining in the fibroblasts at the different chase times, A and B are constants which reflect the fraction of type I procollagen secreted in the fast (A) and slow (0) phases. k1 and k2 are first order rate constants, and t is chase time. .The data on the secretion of normal type I procollagen by control fibroblasts, and on the Secretion Of

the type I and type I* species by the patient's fibroblasts at the various chase times were used to simulate the secretion of type I* from the relationship.

I* = 312 [( l/jI + 2/31* ) - ' /gI]

where I* i s the simulated per cent labeled type I* procollagen remaining in the fibroblasts at the different chase times, (l/3 I + '13 1') the per cent labeled type I and type I* procollagen in the patient's fibroblasts. and 1 i s the per cent type I procollagen remaining in control fibroblasts at the indicated chase times.


T m X

+ - a

t Diagonal SOS-polyacrylamide gel electrophoresis of pepsin-digested media proteins. The atient's fibroblasts were labeled by incubation with 50 UCilmL of [3Hjpr~line for 4 h. After digestion with pepsin, labeled media proteins were electrophoresed in the first dimension under nonreducing conditions in 5% polyacrylamide gel strips. Electro- phoresis in the second dimension was performed in 4 to 0% gradient of polyacrylamide in the presence of 2-mercaptoethanol (+SH).

A 34353637 38 39 40 41 42 43OC I I I I I I I I I I

Ial(IIU13-

X-

1 2 3 4 5 6 I 8 9 X)tl1213141516

Thermal stability of triple-helical trimers o f procollagen containing normal and altered pro-al(1) chains. Control and 01 fibroblasts were incubated for 3 h with p'ijproline as described in "Experimental Procedures". The cell homgenates were incubated at the temperatures indicated for 10 min, and then they were rapidly cooled to 20°C and digested with chymotrypsin and trypsin. A. SOS-polyacrylamide gel electrophoresis of cellular proteins from control fibroblasts. E, SDS-polyacrylamide gel electrophoresis of cellular proteins from the patient's fibroblasts. An additional band ( X ) was occasionally observed in pepsin-digested nonreduced samples from control and 01 Cells.

A 0 2 4 8 1 6 2 4 8 16 CPCP CP C P C P C P C P C P "" " - -

Clearance of laneledprotein material from fibroblasts. Control (C) and the patient's (PI fibroblasts were labeled with [3dproline for the times indicated. Cell homogenates (A) and media proteins ( 6 ) were digested with pepsin and then examined by SDS-polyacrylamide gel electrophoresis in the absence of reducing agents.

SDS-polyacrylamide gel electrophoresis of media proteins secreted by control (C) and the patient's (P) fibroblasts. Fibroblasts were labeled with L3tjproline for the times indicated. Media proteins were reduced and then examined by SDS-polyacrylamide gel electrophoresis without prior digestion with pepsin. Lanes 1-5 were longer exposed than lanes 6-8.


A P C

0 4 8 1624 0 4 E 1624

B P C "

4 8 16 24 4 8 16 24

- proalill

- proa2l11

SDS-electrophoretograms of media proteins after exposure to endogenous procollagen N-proteinase for the times indicated. Control (C) and the patient's (P) fibroblasts were pulse-labeled by incubation with 100 uCi/M Of L3dproline for 3 h. Pulse-labeled media and cell layers were then incubated with chase medium as described in "Experi- mental Procedures". A, labeled media proteins incubated with chase medium and then electrophoresed under reducing conditions. E, media proteins secreted by pulse-labeled fibroblasts during the chase and then electrophoresed under reducing conditions. 2, labeled media proteins incubated with chase medium and then electrophoresed under nonreducing conditions.

Fig. 65

M M

2 3 4 6 C chasetime(h) 53 '13 1 2 3 4 6 C

Kinetics of secretion of type I procollagen. Control @eft pme2)and the patient's(right panet) fibroblasts were pulse-labeled and then chased as described under "Experimental Procedures". Media proteins from the indicated successive chase periods (M) and cell layer proteins from the final chase period (C) were pepsin-digested and then electrophoresed under nonreducing conditions.

of biological vol. no. of july 5, pp. 9056-9064, …0 1986 by the american society of biological...

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