Eur. J. Biochem. 34, 91-96 (1973)

Effect of an Additional Peptide Extension of the N-Terminus of Collagen from Dermatosparactic Calves on the Cross-Linking of the Collagen Fibres

Allen J. BAILEY and Charles M. LAPIBRE

Agricultural Research Council, Meat Research Institute, Langford, Bristol, and Service de Dermatologie, HBpital de Bavibre, Universite de Libge

(Received Septembcr B/December 12, 1972)

Analysis of the highly disorganized fibres of dermatosparactic bovine skin revealed a marked decrease in the intermolecular cross-linking of the aldimine bond type. However, after removal of the additional peptides from the procollagen in solution with a crude procollagen peptidase preparation, normal fibres were formed on reprecipitation, and analysis of these fibres indicated the formation of normal reducible cross-links, These studies clearly demonstrate that one effect of the additional peptide extension is to inhibit the formation of both uniform fibres and fibres capable of producing the normal cross-links.

A recessive genetic defect in the connective tissue of Central and Upper Belgium cattle is characterised by an extremely fragile skin. The condition was reported some years ago [l] and is referred to as dermatosparaxis (torn skin). Microscopically, the collagen fibre bundles are extensively disorganized [3,2] and recent studies by LapiBre and collaborators [4,5] have demonstrated the presence of additional peptides a t the N-terminal end of the collagen a-chains. Collagen made of these elongated poly- peptides is procollagen, the precursor of the extra- cellular fibrillar collagen. Dermatosparactic procolla- gen a-chains are similar to the a-chain precursors isolated from fibroblasts or connective tissue cultures [6,7,8]. A specific enzyme, procollagen peptidase, is responsible for the removal of the additional peptides [9]. The lack of this enzyme activity is responsible for dermatosparaxis and may be the reason for the inability of the collagen to form stable fibres.

Dermatosparactic collagen extracted in neutral salt solutions forms opaque gels only very slowly when heated to 37 "C and the fibrils redissolve on cooling more rapidly than normal fibrils. These results suggest the inability of the molecules to cross- link, yet the dermatosparactic skin collagen is ex- tractable in lesser proportion than collagen from normal calf skin.

MATERIALS AND METHODS Preparation of Collagen Fibres

The dermatosparactic calves were obtained from the Department of Animal Genetics, University of Likge, Belgium. The animals were killed and their

tissues collected as described previously [4]. After removal of the hair, epidermis and subcutaneous fat, the dermis was cut into small pieces, homogenized and washed extensively with isotonic saline (0.9°/o, pH 7.4). Samples of tendon and articular cartilage were similarly washed extensively with saline (0.9 Ole, pH 7.4) and then homogenized.

Collagen was extracted from the dermis using 0.15 M NaC1, the solution centrifuged and the colla- gen precipitated in a fibrous form by dialysis against running tap water. The process was repeated three times, and the final precipitate used for electron microscopy and borohydride reduction.

Electron Microscopy Examination of the collagen fibrils by high-

resolution electron microscopy was carried out by negative staining with phosphotungic acid or silico tungstic acid using an AEI EM6 B microscope. A suspension of the fibres prepared as described above was mixed with an equal volume of silico tungstic acid ( 2 O / , wlv, pH 8.4) and deposited on a grid covered with a carbon-coated collodion film. After allowing the suspension to settle the excess liquid was removed and the grid allowed to dry [lo].

Reduction of Collagen Fibres The homogenized native intact fibres from the

skin, tendon and cartilage were reduced with tritiated potassium borohydride in carbonate-buffered saline. Fibres reconstituted by reprecipitation from solu-

92 Cross-Linking of Dermatosparactic Collagen Eur. J. Biochem.

tions of normal, dermatosparactic and procollagen- peptidase-treated dermatosparactic collagen were reduced under the same conditions.

After reduction the collagen was hydrolysed in 6 N hydrochloric acid and the amino acids and reduc- ed components separated on a Technicon Autoana-

lyzer using volatile buffers as described previously in detail [ 1 I].

The identity of the reduced components was confirmed by comparison with authentic standards using the extended basic column of the Beckman amino acid analyser and high-voltage electrophoresis.

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Fig. 1. Distribution of tritium radioactivity of acid hydrolysates of reduced collagen. (A) Normal calf skin; (B) dermatosparactic calf skin ; (C) dermatosparactic tendon and (D) dermatosparactic cartilage. (-) Normal ; (. . . . . .) dermatosparactic. Shaded areas indicate markers. Nle(0H) = hydroxynorleucine; Lys(0H)-Nle = hydroxylysylnorleucine; Lys(OH),-Nle = dihydroxy-

1 ysylnorleucine

Fig. 2. Electron micrographs of collagen fibrils from various tissues of normal and dermatosparactic calves negatively stained with phosphotungstic acid. (A) Collagen fibres (40000 x ) from normal bovine skin. The individual fibres are highly regular and of uniform diameter throughout. (B) Dermatosparactic calf skin (40000 x ). Note the twisted ribbon-like nature of the fibre. (C) Dermatosparactic calf skin (40000 x ). Note the irregular interweaving of the narrow fibres in contrast to the

highly regular individual fibres of uniform diameter from normal skin collagen. (D) Dermatosparactic calf tendon fibres (40000 x ). The abnormal fibres (top fibre) are not as disordered as dermatosparactic skin fibres and a high pro- portion of normal fibres (bottom fibre) are present. @) Dermatosparactic calf cartilage fibres. Cartilage contains only a small proportion of slightly disordered fibres (bottom

fibre)

Vol. 34, No. 1, 1973 A. J. BAILEY and C. M. LAPIBRE 93

Fig. 2 B-E

Eur. J. Biochem. 94 A. J. BAILEY and C. M. LAPI~RE : Cross-Linking of Dermatosparactic Collagen

Acrylamide-Gel Electrophoresis The denatured collagen from normal, dermato-

sparactic and procollagen-peptidase-treated dermato- sparactic collagen were analyzed for polypeptide composition using dodecylsulphate acrylamide gel electrophoresis. Electrophoresis was carried out using a flat-bed acrylamide gel (7.50/,), both gel and elec- trode buffers a t pH 8.5 as described previously [12]. The gel slab was sliced to remove the top and bottom faces and the centre section stained with coomassie blue.

Incubation of Procollagen with Procdlagen Peptidase

A crude enzyme preparation was made as clescrib- ed by Lapiere et al. [9]. About 5 g of normal calf skin was homogenized in 0.9O/, saline (10 ml) a t 4 "C using a Polytron homogenizer (Northern Media Supply Ltd, Hull, U.K.). The homogenate was stirred for 2 h at 4 "C then centrifuged at 40000 x g for 20min and the supernatant fluid used as a crude enzyme extract. Cleavage of the additional peptides, whilst retaining both the helical structure and the normal N-terminal telopeptides, was achieved by incubation of a solution of acetic-acid-extracted dermatosparactic collagen (5 ml, 1 mg/ml) with the crude procollagen peptidase preparation (5 ml) con- taining 0.5 M calcium acetate. The incubation was carried out a t 26 "C and pH 7.2 for 5.5 h. After in- cubation 0.15 M Tris buffer (5 ml) adjusted to pH 2.5 with 25O/, trichloroacetic acid was added and the mixture stirred for I h a t 4 "C. The solution was then centrifuged for 20 min at 40000 x g and an aliquot of the supernatant analyzed by acrylamide gel electro- phoresis (Fig.4). The remainder of the solution was precipitated to form native-type fibres.

RESULTS AND DISCUSSION The formation of the intermolecular crosslinks

in collagen fibres requires a precise alignment of the molecules in the fibre. The lysine-derived aldehyde precursors (allysines) are located in the N- and C- terminal tclopeptide regions and these rea,ct with specific hydroxylysine residues in the helical body of the molecule to form aldimine bonds [13,14,15]. Similarly the intramolecular aldol crosslinks derived from two allysine residues can react further with specific residues on adjacent molecules to produce the multifunctional cross-link [ 13,161 designated Fr. C (Fig.1). The disorganization of the dermato- sparactic collagen fibres, clearly evident in the electron micrographs (Fig.2B and C, and 3A) suggests that the crosslinking would be inhibited, a t least partially, due to incorrect spatial relationship of the allysine and the intramolecular aldol with respect to the reactive groups on adjacent molecules.

Both the precursor allysine and the two aldimine cross-links were detected after tritiated borohydride reduction to give hydroxynorleucine [Nle( OH)]- hydroxylysylnorleucine [Lys(OH)-Nlel and Fr. C respectively (Fig. 1 A). As anticipated, analysis of the dermatosparactic skin confirmed a decrease in intermolecular crosslinking of the aldimine bond type, the major reducible components being allysine and the intramolecular allysine aldol (Fig. 1 B). The aldol crosslink within the molecule would not be expect,ed to be affected, and the increased amount of its acid degradation product (D, Fig. 1 B) reflects its inability to form an intermolecular crosslink with a reactive compound on an adjacent molecule i . e . Fr. C (Fig. 1 A).

Examination of the dermatosparactir: tendon and cartilage collagen fibres by electron microscopy demonstrated that only a small proportion of the fibres were disorganized (Fig. 2D and E). Similarly, analysis of the reducible components in the fibres revealed only a slight reduction in the proportion of reducible crosslinks, about 20°/, in the case of tendon, but the change in the cartilage was not significant (Fig. 1 C and D). These findings are con- sistent with the lower proportion of precursor types of a-chains (f Z O O / , ) in dermatosparactic tendon and its near absence in cartilage (& 2 O / , ) as determin- ed by acrylamide gel electrophoresis.

After removal of the additional peptides with the crude procollagen-peptidase preparation it is evident. from the acrylamide gel electrophoresis that the enzyme has no effect on the proportion of the /I component (Fig. 4), clearly demonstrating that this peptidase only removes the additional peptide and leaves intact the normal N-terminal telopeptide containing the crosslink precursor. These molecules should therefore produce normal fibres capable of forming the typical aldimine crosslinks. Indeed fibres reconstituted from the enzyme-treated dermato- sparactic collagen revealed normal fibres on examina- tion by electron microscopy (Fig. 3 B). Reduction of the reprecipitated fibres from the enzyme-treated dermatosparactic collagen with borohydride produces an elution pattern identical to that of reprecipitated fibres from a normal calf collagen (i.e. similar to Fig. 1 A) demonstrating that the additional peptide is responsible for the lack of formation of reducible intermolecular crosslinks. The effect of the additional peptidc on the dermatosparactic skin collagen appears to prevent the filaments binding t o form stable thick fibres. The periodicity evident in the fine fibrils shows that the molecules retain their ability to aggregate and presumably when sufficient molecules have aggregated the effect of the polar additional peptides along the fibre length is to disrupt the specific charge distribution along them and prevent further aggrega- tion.

The disorganized collagen fibres of the dermato- sparactic skin clearly contain less intermolecular

Fig. 3. Electron micrographs of reprecipitated collagen fibrils. (A) Disorganized fibrils reprecipitated from, a 0.15 M NaCl extract of dermatosparactic collagen (40000 x ). As in the native dermatosparactic fibres from skin the narrow fibrils have not aggregated properly to form smooth fibres of uniform

diameter. (B) Normal fibres reprecipitated from a solution of dermatosparactic collagen after treatment with the pro- collagen peptidase to remove the additional N-terminal peptide (I20000 x ). The fibres formed are extremely regular

in appearance and of uniform diameter

crosslinks and therefore its collagen might be expect- ed to be more extractable, whereas in fact the reverse is true. However, although it has been demonstrated that inhibition of the formation of crosslinks, e.g. by lathyrogens, results in both a fragile skin and an extensively soluble collagen, the relationship between reducible crosslinks and solubility is not clear. The aldimine crosslinks present in skin are labile intermediates and do not contribute to the insolubility although their subsequent stable forms might be expected to do so; a t the present time it is not possible to correlate solubility and crosslinking.

Whatever is the primary role of the additional peptides during the biosynthesis of the collagen molecule, it is now clear that their effect on the collagen is to inhibit the formation of both uniform fibres and fibres capab1e Of forming the

Fig.4. Dodecylsulphete acrylamide-gel patterns of ( A ) normal calf-skin collagen, (B) dermatosparactic calf collagen and (c) procollagen-peptidase-treated dermatosparactic collagen. The dermatosparactic collagen contains a higher proportion of

p-a than a-chains crosslinks.

96 A. J. BAILEY and C. M. LAPI~RE : Cross-Linking of Dermatosparactic Collagen Eur. J. Biochem.

We are grateful to Mr T. J. Sims for skilful technical assistance and to Mr C. A. Voyle for the electron niicro- graphs.

REFERENCES 1. Hslnset, R. & Ansay, &I. (1967) Ann. Med. Vet. 7 , 451. 2. O’Hara, P. J., Read, W. K., Romane, W. M. & Bridges,

3. Simar, L. J. & Betz, E. H. (1971) Hoppe-Seyler’s 2.

4. Lenaers, A., Ansay, M., Nusgens, B. V. & Lapiire, Ch. M.

C. H. (1970) Lab. Invest. 23, 307.

Physiol. Chem. 352, 13.

(1971) Eur. J . Biochem. 23, 533-543. 5. Stark, M., Lenaers, A., LapiBre, Ch. M. & Kuhn, K.

6. Lavman. D. L.. McGoodwin, E. B. & Martin. G. R. (1971) F E B S Lett. 18, 225.

(“1971)’Proc. Natl. Acud. Sci. U . S. A. 68, 454. ‘ 7. Bellamy, G. & Bornstein, P. (1971) Proc. Natl. Acad. Sci.

U . S. A. 68, 1138. 8. Dehm, P., Jimenez, S. A., Olsen, B. R. & Prockop, D. J.

(1972) PTOC. Natl. Acad. Sci. U . S. A. 69, 60. 9. LapiBre, Ch. M., Lenaers, A. & Kohn, L. D. (1971) Proc.

Natl. Acad. Sci. U . S. A. 68, 3054.

10. Tromans, W. J., Horne, R. W., Gresham, G. A. & Bailey, A. J. (1963) 2. Zellforsch. Mikrosk. And. 58, 798.

11. Bailey, A. J., Peach, C. M. & Fowler, L. J. (1970) Bio- chem. J . 117, 819.

12. Sykes, B. C. & Bailey, A. J. (1971) Biochem. Biophys. Res. Commun. 43, 340.

13. Bailey, A. J., Peach, C. M. & Fowler, L. J. (1970) I n Chemistry and Molecular Biology of the Intercellular Matrix (Balazs. E. A.. ed.) Vol. 1. D. 304. Academic

I , , I

Press, New York. 14. Mechanic, G., Gallom P. &;I. & Tanzer, M. L. (19711

\ I

Biochem. Biophys. k e s . Commun. 45, 644.

Res. Commun. 45, 1416.

Biophys. Res. Commun. 39, 176.

15. Davis, N. R. & Bailey, A. J. (1971) Biochem. Biophys.

16. Kang, A. H., Faris, B. & Franzblau, C. (1970) Biochem.

A. J. Bailey Agricultural Research Council, Meat Research Institute, Langford, Bristol, Great Britain, BS18 7DY C. M. Lapiire Service de Dermatologie, HBpital de BaviBre, Boulevard de la Constitution 66, B-4000 Liige, Belgium