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Pepsin-solubilized collagens of guinea-pig dermis and dermal scar
454
Biochimica et Biophysica Acta, 365 (1974) 454--457 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA Report BBA 31184
PEPSIN-SOLUBILIZED COLLAGENS OF GUINEA-PIG DERMIS AND DERMAL SCAR
C.A. SHUTTLEWORTH and L. FORREST
Department o f Medical Biochemistry, University of Manchester, Medical School, Stopford Building, Oxford Road, Manchester, M13 9PT, and Department of Surgery, Manchester Royal Infirmary, Oxford Road, Manchester (U. K.) (Received June 20th, 1974) (Revised manuscript received August 23rd, 1974)
Summary Differential salt precipitation of pepsin-solubilized guinea-pig dermal collagen has enabled separation of two collagen species. Similar treatment with guinea-pig dermal scar collagen revealed the presence of only one collagen type. Differences were noted both in the subunit size of the collagen species isolated from dermal collagen and in amino acid composition. It is concluded that dermal collagen is composed of Type I and T y p e III collagens, and that thermal scar consists only of T y p e I collagen.
It has previously been shown that there are significant differences between the collagen in the dermis and that laid down in dermal scar tissues. The most significant chemical differences are in the types of cross-links [1,2 ] and in the degree of hydroxylation of the lysine residues [3]. Recently it has been shown that human skin contains t w o types of collagen molecules [4,5] Type I whose chain composition is [a, (I)] 2 a2 and Type III with chain composition [al (III)] 3 • We have examined guinea-pig dermis and dermal scar collagens in an attempt to isolate the collagen molecules associated with these tissues. Guinea-pig skin and scar collagen were prepared as previously described [1], the tissues were extracted with 1.0 M NaC1/0.05 M EDTA pH 7.5 at 4 ° C, washed with water and the residues freeze-dried. Portions of dermal collagen and dermal scar collagen were suspended in 0.1 M acetic acid (2 mg/ml) and incubated with pepsin for 24 h at 4°C (enzyme/substrate ratio 1/10). After
455 pepsin treatment any insoluble material was removed b y centrifugation (3000 X g for 20 min) and the solubilized collagen was precipitated from the supernatant b y the addition of NaC1 to a concentration of 0.9 M. The precipitated collagen was dissolved in 0.1 M acetic acid, dialysed against 0.1 M acetic acid and freeze dried. It was apparent that there were differences in the proportion of original tissue collagen that could be solubilized b y pepsin when skin and scar are compared, 90% of the dermal collagen was solubilized b y the above procedure in 24 h, b u t only 65% of the dermal scar tissue. Chung and Miller [ 4] used differential salt precipitation on pepsinsolubilized human collagen and were able to separate molecules with the chain compositions [~1 (I)] 2 ~2 and [~1 (III)] 3 ; w e have followed the same procedure. Pepsin-solubilized collagen samples were dissolved at 4°C in 1 M NaC1, 0.05 M Tris, pH 7.5 and the NaC1 concentration was increased in successive steps b y 0.1 M over the range 1.1 to 3.0 M. Using this procedure we obtained precipitates at 1.5 M NaC1 and 2.4 M NaC1 with the collagen solubilized from the dermis, b u t only a 2.4 M NaC1 precipitate when dermal scar tissue was used. The precipitates were dissolved in 0.1 M acetic acid, dialysed against 0.1 M acetic acid and freeze-dried.
ABSORBANCE0't 230nm
I L
/ 1 400
\
\/
~
,./ ~
600
',,
~'
800
EFFLUENT(ml)
Fig.1. A g a r o s e A 1 . 5 c h r o m a t o g r a p h y o f the p e p s i n - s o l u b i l i z e d c o l l a g e n m o l e c u l e s s e p a r a t e d b y differd e r m a l c o l l a g e n w h i c h precipitates a t 1.5 M NaC1, p H 7.5; . . . . . , d e r m a l collagen w h i c h p r e c i p i t a t e s at 2.4 M NaC1, P H 7.5; X - - X , d e r m a l scax c o l l a g e n w h i c h p r e c i p i t a t e s a t 2.4 M NaC1, p H 7.5.
ential salt p r e c i p i t a t i o n f r o m d e r m i s a n d d e r m a l scar c o l l a g e n - - . ,
The collagen molecules isolated b y differential salt precipitation were dissolved in 1.0 M CaC12, 0.05 M Tris, pH 7.5 and chromatographed on a standardized column (3 cm × 150 cm) of Bio-Gel A1.5 (200--400 mesh) with 1.0 M CaC12,0.05 M Tris, pH 7.5 as the eluant. The elution profiles are shown in Fig.1. It was evident that the major portion of material precipitating at a NaC1 concentration of 1.5 M was composed of material with molecular weight of 300 000, similar to ~,-components. Polyacrylamide gel electrophoresis of this high molecular weight material in 5% sodium dodecylsulfate gels [6] in the presence of mercaptoethanol, revealed a single band with a molecular weight similar to ~-components. However, unlike the results of
456
Chung and Miller [ 4 ] , smaller molecular weight components were found, but they accounted for less than 20% of the total material. It can also be seen from Fig.1 that there is a difference in the subunit composition of the 2.4 M NaC1 precipitates when dermis and dermal scar collagen are compared. The material solubilized from dermal scar collagen contained a greater proportion of high molecular weight species than did the material isolated from dermal collagen. Whether these pepsin-resistant polymers seen in dermal scar collagen are related to the different types of cross-links found in scar collagen, or to the fact that the cross-links are located in pepsin-resistant regions is at present unknown. Amino acid analyses of the isolated collagens were obtained on a Jeol6AH automatic amino acid analyser. The amino acid composition of the 2.4 M NaC1 precipitates from skin and scar and the high molecular weight components isolated from the 1.5 M NaCl precipitate are shown in Table I. The material isolated from dermal collagen which precipitated at a NaCI concentration of 2.4 M was similar to that isolated from dermal scar collagen with the exception that the scar collagen contained more hydroxylysine than
TABLE I A M I N O A C I D C O M P O S I T I O N OF T H E C O L L A G E N F R A C T I O N S I S O L A T E D F R O M G U I N E A - P I G D E R M I S A N D D E R M A L S C A R , E X P R E S S E D AS A M I N O A C I D R E S I D U E S P E R 1 0 0 0 T O T A L AMINO ACID RESIDUES.
A m i n o acid
Hydroxyprolineq Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine ~-Cystine Valine Methio nine Isoleucine Leucine Tyrosine Phenylalanine Hydro x y l y s i n e Lysine Histidine Arginine
Skin 1.5 M
2.4 M
Scar 2.4 M
109 56 20 44 71 116 338 90 1.8 13.2 4.2 14.9 26 2 8.8 6.7 31.0 8.3 39
104 42 16 34 70 132 325 116 -20.8 4.6 10.4 22 1.3 11 6.2 30.5 4.0 50
109 48 17 37 77 128 327 100 -17.8 5.7 7.5 17.1 1.7 10.7 10.3 29.1 4.3 52
the skin collagen. Slight differences in composition are probably related to the presence of a contaminating glycoprotein associated with the scar collagen [7 ].
457 Apart from the presence of half-cystine in the material precipitating at a NaC1 concentration of 1.5 M other amino acid differences were noted between this material and that isolated by precipitating at 2.4 M NaC1. These include differences in alanine, valine, isoleucine and histidine, but unlike the results of Chung and Miller [4] the level of hydroxyproline was not elevated. It seems likely, however, that the material isolated from guinea-pig skin collagen is similar to the Type III collagen isolated from human tissues [4,5]. Differential salt precipitation of pepsin-solubilized guinea-pig dermal collagen has enabled isolation of two collagen species, which differ in amino acid and sub-unit composition. These results are in agreement with previously published information on human skin [4,5] and indicate that the dermis contains Type I collagen, containing two ~, and one ~2 chains, and Type III collagen containing three identical ~-chains held together by disulphide bonds. Similar treatment of dermal scar collagen failed to reveal the presence of Type III collagen and it is concluded that only Type I collagen is laid down during dermal repair. Scar tissue collagen is synthesized not by dermal fibroblasts but b y undifferentiated mesenchyme cells of local origin [8], and since scar tissue collagen does n o t contain Type III collagen it can be concluded that the synthesis of Type III collagen is n o t associated with the differentiation of mesenchyme cells into fibroblasts. Further work is being carried o u t to determine whether the synthesis of T y p e III collagen is associated with ageing of the fibroblast or to a different cell type. We are grateful to the Medical Research Council for financial support, and to Mrs J. Ward for expert technical assistance. References 1 Forrest, L., Sh uttlewo rth, C.A., Jackson, D.S. and Mechanic, G. (1972) Biochem. Biophys. Res. Commun. 46, 1776--1781 2 Jackson, D.S., Ayad, S. and Mechanic, G. (1974) Biochim. Biophys. Acta 336, 100--107 3 Forrest, L., Dixon, J. and Jackson, D.S. (1972) Conn. Tiss. Res. 1, 243--250 4 Chung, E. and Miller, E.J. (1973) Science 183, 1200--1201 5 Epstein, Jr, E.H. ( 1 9 7 4 ) J . Biol. Chem. 249, 3225--3231 6 Fttrthmayr, H. and Timpl, R. (1971) Anal. Biochem. 41, 510--516 7 Rajamaki, A. Kulonen, E. (1971) Biochim. Biophys. Acta 243, 398--406 8 Gzillo, H.C. (1963) Anm Sttrg. 157, 453--467
Biochimica et Biophysica Acta, 365 (1974) 454--457 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA Report BBA 31184
PEPSIN-SOLUBILIZED COLLAGENS OF GUINEA-PIG DERMIS AND DERMAL SCAR
C.A. SHUTTLEWORTH and L. FORREST
Department o f Medical Biochemistry, University of Manchester, Medical School, Stopford Building, Oxford Road, Manchester, M13 9PT, and Department of Surgery, Manchester Royal Infirmary, Oxford Road, Manchester (U. K.) (Received June 20th, 1974) (Revised manuscript received August 23rd, 1974)
Summary Differential salt precipitation of pepsin-solubilized guinea-pig dermal collagen has enabled separation of two collagen species. Similar treatment with guinea-pig dermal scar collagen revealed the presence of only one collagen type. Differences were noted both in the subunit size of the collagen species isolated from dermal collagen and in amino acid composition. It is concluded that dermal collagen is composed of Type I and T y p e III collagens, and that thermal scar consists only of T y p e I collagen.
It has previously been shown that there are significant differences between the collagen in the dermis and that laid down in dermal scar tissues. The most significant chemical differences are in the types of cross-links [1,2 ] and in the degree of hydroxylation of the lysine residues [3]. Recently it has been shown that human skin contains t w o types of collagen molecules [4,5] Type I whose chain composition is [a, (I)] 2 a2 and Type III with chain composition [al (III)] 3 • We have examined guinea-pig dermis and dermal scar collagens in an attempt to isolate the collagen molecules associated with these tissues. Guinea-pig skin and scar collagen were prepared as previously described [1], the tissues were extracted with 1.0 M NaC1/0.05 M EDTA pH 7.5 at 4 ° C, washed with water and the residues freeze-dried. Portions of dermal collagen and dermal scar collagen were suspended in 0.1 M acetic acid (2 mg/ml) and incubated with pepsin for 24 h at 4°C (enzyme/substrate ratio 1/10). After
455 pepsin treatment any insoluble material was removed b y centrifugation (3000 X g for 20 min) and the solubilized collagen was precipitated from the supernatant b y the addition of NaC1 to a concentration of 0.9 M. The precipitated collagen was dissolved in 0.1 M acetic acid, dialysed against 0.1 M acetic acid and freeze dried. It was apparent that there were differences in the proportion of original tissue collagen that could be solubilized b y pepsin when skin and scar are compared, 90% of the dermal collagen was solubilized b y the above procedure in 24 h, b u t only 65% of the dermal scar tissue. Chung and Miller [ 4] used differential salt precipitation on pepsinsolubilized human collagen and were able to separate molecules with the chain compositions [~1 (I)] 2 ~2 and [~1 (III)] 3 ; w e have followed the same procedure. Pepsin-solubilized collagen samples were dissolved at 4°C in 1 M NaC1, 0.05 M Tris, pH 7.5 and the NaC1 concentration was increased in successive steps b y 0.1 M over the range 1.1 to 3.0 M. Using this procedure we obtained precipitates at 1.5 M NaC1 and 2.4 M NaC1 with the collagen solubilized from the dermis, b u t only a 2.4 M NaC1 precipitate when dermal scar tissue was used. The precipitates were dissolved in 0.1 M acetic acid, dialysed against 0.1 M acetic acid and freeze-dried.
ABSORBANCE0't 230nm
I L
/ 1 400
\
\/
~
,./ ~
600
',,
~'
800
EFFLUENT(ml)
Fig.1. A g a r o s e A 1 . 5 c h r o m a t o g r a p h y o f the p e p s i n - s o l u b i l i z e d c o l l a g e n m o l e c u l e s s e p a r a t e d b y differd e r m a l c o l l a g e n w h i c h precipitates a t 1.5 M NaC1, p H 7.5; . . . . . , d e r m a l collagen w h i c h p r e c i p i t a t e s at 2.4 M NaC1, P H 7.5; X - - X , d e r m a l scax c o l l a g e n w h i c h p r e c i p i t a t e s a t 2.4 M NaC1, p H 7.5.
ential salt p r e c i p i t a t i o n f r o m d e r m i s a n d d e r m a l scar c o l l a g e n - - . ,
The collagen molecules isolated b y differential salt precipitation were dissolved in 1.0 M CaC12, 0.05 M Tris, pH 7.5 and chromatographed on a standardized column (3 cm × 150 cm) of Bio-Gel A1.5 (200--400 mesh) with 1.0 M CaC12,0.05 M Tris, pH 7.5 as the eluant. The elution profiles are shown in Fig.1. It was evident that the major portion of material precipitating at a NaC1 concentration of 1.5 M was composed of material with molecular weight of 300 000, similar to ~,-components. Polyacrylamide gel electrophoresis of this high molecular weight material in 5% sodium dodecylsulfate gels [6] in the presence of mercaptoethanol, revealed a single band with a molecular weight similar to ~-components. However, unlike the results of
456
Chung and Miller [ 4 ] , smaller molecular weight components were found, but they accounted for less than 20% of the total material. It can also be seen from Fig.1 that there is a difference in the subunit composition of the 2.4 M NaC1 precipitates when dermis and dermal scar collagen are compared. The material solubilized from dermal scar collagen contained a greater proportion of high molecular weight species than did the material isolated from dermal collagen. Whether these pepsin-resistant polymers seen in dermal scar collagen are related to the different types of cross-links found in scar collagen, or to the fact that the cross-links are located in pepsin-resistant regions is at present unknown. Amino acid analyses of the isolated collagens were obtained on a Jeol6AH automatic amino acid analyser. The amino acid composition of the 2.4 M NaC1 precipitates from skin and scar and the high molecular weight components isolated from the 1.5 M NaCl precipitate are shown in Table I. The material isolated from dermal collagen which precipitated at a NaCI concentration of 2.4 M was similar to that isolated from dermal scar collagen with the exception that the scar collagen contained more hydroxylysine than
TABLE I A M I N O A C I D C O M P O S I T I O N OF T H E C O L L A G E N F R A C T I O N S I S O L A T E D F R O M G U I N E A - P I G D E R M I S A N D D E R M A L S C A R , E X P R E S S E D AS A M I N O A C I D R E S I D U E S P E R 1 0 0 0 T O T A L AMINO ACID RESIDUES.
A m i n o acid
Hydroxyprolineq Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine ~-Cystine Valine Methio nine Isoleucine Leucine Tyrosine Phenylalanine Hydro x y l y s i n e Lysine Histidine Arginine
Skin 1.5 M
2.4 M
Scar 2.4 M
109 56 20 44 71 116 338 90 1.8 13.2 4.2 14.9 26 2 8.8 6.7 31.0 8.3 39
104 42 16 34 70 132 325 116 -20.8 4.6 10.4 22 1.3 11 6.2 30.5 4.0 50
109 48 17 37 77 128 327 100 -17.8 5.7 7.5 17.1 1.7 10.7 10.3 29.1 4.3 52
the skin collagen. Slight differences in composition are probably related to the presence of a contaminating glycoprotein associated with the scar collagen [7 ].
457 Apart from the presence of half-cystine in the material precipitating at a NaC1 concentration of 1.5 M other amino acid differences were noted between this material and that isolated by precipitating at 2.4 M NaC1. These include differences in alanine, valine, isoleucine and histidine, but unlike the results of Chung and Miller [4] the level of hydroxyproline was not elevated. It seems likely, however, that the material isolated from guinea-pig skin collagen is similar to the Type III collagen isolated from human tissues [4,5]. Differential salt precipitation of pepsin-solubilized guinea-pig dermal collagen has enabled isolation of two collagen species, which differ in amino acid and sub-unit composition. These results are in agreement with previously published information on human skin [4,5] and indicate that the dermis contains Type I collagen, containing two ~, and one ~2 chains, and Type III collagen containing three identical ~-chains held together by disulphide bonds. Similar treatment of dermal scar collagen failed to reveal the presence of Type III collagen and it is concluded that only Type I collagen is laid down during dermal repair. Scar tissue collagen is synthesized not by dermal fibroblasts but b y undifferentiated mesenchyme cells of local origin [8], and since scar tissue collagen does n o t contain Type III collagen it can be concluded that the synthesis of Type III collagen is n o t associated with the differentiation of mesenchyme cells into fibroblasts. Further work is being carried o u t to determine whether the synthesis of T y p e III collagen is associated with ageing of the fibroblast or to a different cell type. We are grateful to the Medical Research Council for financial support, and to Mrs J. Ward for expert technical assistance. References 1 Forrest, L., Sh uttlewo rth, C.A., Jackson, D.S. and Mechanic, G. (1972) Biochem. Biophys. Res. Commun. 46, 1776--1781 2 Jackson, D.S., Ayad, S. and Mechanic, G. (1974) Biochim. Biophys. Acta 336, 100--107 3 Forrest, L., Dixon, J. and Jackson, D.S. (1972) Conn. Tiss. Res. 1, 243--250 4 Chung, E. and Miller, E.J. (1973) Science 183, 1200--1201 5 Epstein, Jr, E.H. ( 1 9 7 4 ) J . Biol. Chem. 249, 3225--3231 6 Fttrthmayr, H. and Timpl, R. (1971) Anal. Biochem. 41, 510--516 7 Rajamaki, A. Kulonen, E. (1971) Biochim. Biophys. Acta 243, 398--406 8 Gzillo, H.C. (1963) Anm Sttrg. 157, 453--467