aging and cross-linking of skin collagen

Vo1.152, No. 2,1988

April 29,1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 898-903

A G I N G A N D C R O S S - L I N K I N G O F SKIN C O L L A G E N

Mitsuo Y a m a u c h i ~ David T. Woodley @ and Gerald L. Mechanic*

@ +CB #7455, Dental Research Center, Department of Dermatology, and

~Dental Research Center and Department of Biochemistry University of North Carolina at Chapel Hill

Chapel Hill, NC 27514-7455

Received March II, 1988

This report represents a clear demonstration of a cross-link in collagen whose abundance is related to chronological aging of an organism. Recently its structure was identified as histidinohydroxylysinonorleucine. Quantification of the cross-link in various aged samples of bovine and human skin indicate that it rapidly increases from birth through maturation. Subsequently, a steady increase occurs with aging, approaching 1 mole/mole of collagen. This compound seems to be related to the relative proportions of soluble to insoluble collagen from skin in neutral salt, dilute acid, and denaturing aqueous solvents (higher concentration in the insoluble portion). It is absent from other major collagenous tissues such as dentin, bone and tendon. ® z988 Aoadem~c Press, Zno.

The covalent intermolecular cross-links between collagen molecules in macromolecular fibrils are

essential in providing connective tissue matrices with their stability and physicochemical properties. A

number of cross-linkng compounds have been isolated from various collagenous tissues and their

structures have been identified (1). The majority are iminium compounds and were isolated as their

NaBH4-reduced products. The reduction is performed under mild conditions and render the products

stable to acid hydrolysis. Recently two naturally occuring non-reducible, mature, stable collagen cross-

links have been isolated and characterized (proven by isolation of cross-linked peptides from collagen)

(2,3). Pyridinoline, a well characterized non-reducible fluorescent cross-linking compound, is present in

variety of connective tissues such as cartilage, bone. dentin, achilles tendon,and ligament (4). However,

this widely distributed cross-link is completely absent from skin collagen which is one of the most

abundant connective tissue in the body (4). Histidinohydroxylysinonorleucine (HHL) was recently

isolated from an acid hydrolysate of mature bovine skin collagen and its structure was determined (3).

Amino acid and peptide sequence analyses of three-chain peptides cross-linked by HHL isolated from a

tryptic digest of unreduced 6 M guanidine-HC1 insoluble mature bovine skin collagen unequivocally

identified its fibrillar molecular locus in skin collagen (5,6). It was also found that the HHL peptides

+ To whom all cor respodence should be addressed

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved. 898

Vol. 152, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

were only derived from type I collagen fibrils (6). This communication describes the quantitative

significance of H H L in collagen using samples of skin from a variety of chronologically aged bovine

animals and humans. The relation of H H L content to insolubility of skin collagen was also studied.

The results indicated that I-IHL is the major non-reducible stable cross-link in skin collagen in both

species and is a true age-related cross-link.

Materials and Methods

Biopsies of normal bovine skin were obtained from the neck region of various aged prenatal and postnatal animals (from 3 month-old embryo to 9 year-old animal) at slaughter house or veterinary school. Af te r removal of fat and hair, samples were stored at -90 °C. Normal human skin samples were obtained from thigh (some from abdomen and foreskin) of various aged humans (from 7 day-old to 89 years of age). Af ter removal of adhering tissues, samples were suspended in 0.5 M potassium bromide overnight at 4 °C to separate the epidermis from dermis. The dermis was then washed with cold water and stored at -90 °C until use. Samples were pulverized under liquid N 2 by Spex Freezer Mill, washed with cold 0.02 M sodium phosphate buffer, pH 7.4 and distilled water, and lyophilized. Approximately 5 mg of each sample was hydrolysed with 6 N HC1 for 24 hours at 115 °C. The hydrolysates were then evaporated and the residue were dissolved in 1 mt of water. An aliquot of the hydrolysate was subjected to the amino acid analyzer (Varian 5560 liquid chromatography, AA911 column, Interaction) (3) to determine Hyp content. H H L content was determined by amino acid analyzer directly and/or after gel fil tration by P-2 column. In the latter case, an aliquot of hydrolysate of the known Hyp content was applied to the standardized P-2 column (15 X 55 era, -400 mesh) equilibrated with 0.1 M acetic acid to remove the bulk of amino acids (modified from our recent report) (3). Fractions which encompassed the elution position of standard H H L were quantitatively transferred to a test tube and dried under reduced pressure in a vacuum centrifuge (Savant Instruments Inc.). Af ter each sample was dissolved in an exact amount of distilled water, an aliquot of the sample corresponding to 300 nM Hyp of the original hydrolysate was applied to the amino acid analyzer. H H L content was quantified based on its ninhydrin color factor obtained from the amino acid composition of apparently pure H H L containing peptides (5,6). Five g. of 2 year old bovine skin collagen was prepared as described above and subjected to a sequential extraction using various solvents. Each extraction was performed for 2 days at 4 °C. H H L was assayed in each extract starting with the 1 M NaC1, 0.05 M Tris-HCl buffer pH 7.4 extract (newly synthesized collagen), then first 1% acetic acid extract(I), second 1% acetic acid (II), first 3% acetic acid (I), second 3% acetic acid (II) etc. Af ter 7.5 % acetic acid extraction, each extract contains successively earlier synthesized collagen, the residue was extracted with 6 M guanidine-HC1 (Gu-HC1). Both supernatant (6 M Gu-HC1) and residue (RES) from the 6 M Gu-HC1 extract were analyzed. Each was assessed as described above.

Results

Figure 1 D,E.and F depict examples of the amino acid analysis profiles obtained between Tyr

and Lys from different aged human skin (1.5 months, 43 and 78 year old) after gel filtration on the P-2

column. Elution patterns of hydrolysates of elastin, Fig 1A (elastin kindly provided by Dr. C.Franzblau

of the Boston University), purified HHL cross-linked peptide Fig 1B (5,6) and HHL standard, Fig 1C (3)

in this region are also shown. In the case of human skin, two ninhydrin positive peaks which elute after

H H L were consistently observed on the chromatograms (see figure). Based on their elution positions

using two different gradient systems they were identified as isodesmosine and desmosine. Increased

amounts of these cross-links may reflect accumulation of mature dermal elastin with age (7). These

peaks were less significant in the bovine skin samples.

The results for the quantification of H H L from various aged skin samples are presented in Figs.

2 (bovine) and 3 (human). Similar curves for its increase with chronological age were observed for each

specie. In the case of bovine (Fig.2), little if any H H L was present in early embryonic development (3-5

899


,r• A IDE | Lys T y r - , . - P h e ID~ ES NH~

,

His B

D

/ I I I I / 6 0 7 0 8 0 9 0

Elution Time (rain)

Figure 1. HHL cross-link analyses, see Methods for details A) elastin, B) purified HHL containing tryptic peptide (5,6), C) HHL standard (3), D) 1.5 month human skin, E) 43 year old human skin, F) 78 year old hyman skin. Prior to the determination of HHL, an aliquot of the complete collagen hydrolysate, from each sample, of known Hyp content was applied to the standardized P-2 column (11) to remove the bulk of amino acids. The fractions encompassing the elution position of standard HHL, from each, were pooled, lyophilized and subjected to the amino acid analysis.each human skin sample containing 300 nmole Hyp was applied to the amino acid analyzer (slightly modified gradient system of our report, see ref.3).

0.7

~ 0.6-

0 .5-

~o4

0 0.3-

~ 0 . 2 - ..1 0 ~; 0.1-

®

/ . ./

o , 2 ~ .~ ~ A G E ( Y E A R )

0.7-

0.6-

0.5-

0.4-

® 0 . 3 -

i 0.2-

~ 0.1-

®o ~ i I i i i I i i i 10 20 30 40 50 60 70 80 90

Age (Year)

Figure 2. Increase of HHL with development, maturation and aging in bovine skin. HHL and Hyp were determined by amino acid analysis using ninhydrin. The content of HHL is expressed in mole/mole of collagen based on a value of 300 residues of Hyp per mole of collagen.

Figure 3. Change in the content of HHL with development,maturation and aging in human skin. Samples were mainly obtained from thighs. HHL content is expressed in a same manner as Fig.2.

900


c 0.5-

== 0.4" 0

0 o

1M 1%(I) II 3% II 5% II 7.5% 6M NaCI AcOH (I) (I) G-H

RES

Figure 4. HHL content and extractability of 2 year old bovine skin collagen. Each extraction was performed for 2 days at 4 C. HHL was assayed in each extract starting with the 1 M NaCI, 0.05 M Tris-HCl pH7.4 extract, then first 1% acetic acid extract (I), second 1% acetic acid extract (II), first 3% acetic acid extract (I), second 3% acetic acid extract (II) etc. After 7.5% acetic acid extraction, the residue was extracted with 6 M guanidine-HCl. Both supernatant (6M G-H) and residue (RES) were analyzed.

m o n t h old embryo, <0.02 mole/mole of total collagen). However, f rom the middle of gestation period (7

mon th fetus) th rough post nata l matura t ion (up to 4-5 year old), H H L markedly increased with age and

a t ta ined a value of approximately 0.6 mole/mole of total collagen. Af te r this stage a small but

cont inuous increase with aging was observed. In the case of human (Fig.3), H H L content was 0.03

(abdomen)-0.12 mole (foreskin)/mole of collagen in the new born skin and then showed a rapid increase

with age which reached a concent ra t ion of 0.65 moles/mole of total collagen in the sample of 89 year old

human.

Figure 4 shows the H H L content in the various extracts using 2 year old bovine skin. In the 1 M

NaC1 extract (newly synthesized collagen), little or no HHL was found (<0.02 mole/mole of collagen).

Apprec iab le increases in H H L concent ra t ion are evident as the earlier synthesized collagen is extracted

(successive acetic acid extracts). The highest concent ra t ion of H H L (0.46 mole/mole of collagen) was

found in the most insoluble port ion (6 M Gu-HC1 residue) of the skin.

Various aged normal h u m a n achilles tendon (20 to 70 year old), fetal as well as post natal mature

bovine bone and dentin, mature bovine carti lage were analysed for H H L but little or none (<0.02

mole/mole of collagen) was found.

Discussion

The structure of the major nonreducible stable t r i funct ional cross-link,

h is t id inohydroxylys inonor leucine (HHL), was recently identified af ter isolation f rom mature bovine skin

collagen (3). Its structure, as well as in vitro incubat ion studies (embryonic skin) strongly indicate that it

is formed by a condensat ion between dehydro-hydroxylysinonorleucine (deH-HLNL, iminium cross-link

between Lys ald and Hyl) and the imidazole C-2 carbon atom of His (3). The molecular locus of HHL in

collagen fibri ls was identified to be etl(I) Lysald-16 C al(I), Hyl-87 and a2(I),His-92 of type I collagen (5,6).

In this communica t ion we demonstra te tha t H H L content shows a cont inuous increase throughout

901


chronological aging in both bovine and human skin collagen and does not diminish with time (Fig 2 and

3). The patterns undoubtedly demonstrate that H H L is a true age-related cross-link in skin collagen in

both human and bovine. In both species, HHL content reached 0.6-0.7 moles/mole of collagen in aged

skin. Since type I collagen represents 80-85% of total collagen in mature dermis (8), and H H L was only

found in type I collagen (6), this strongly indicates a level of approximately 1 mole HHL/mole of type I

collagen in skin from aged organisms. In addition to the age-related changes, its abundance seems to be

related to the relative insolubility of the skin collagen (Fig 4). It is well known that the solubility of

skin collagen diminishes with maturation and aging in human (9) and bovine (10). The latter authors

also reported that the concentration of deH-HLNL decreases with age. Based on their results they

suggested the presence of unknown nonreducible stable mature cross-links were replacing the reducible

iminium labile cross-links during maturation and aging. H H L was demonstrated to form by the

condensation of His and deH-HLNL (3). The latter can partially explain previous observations

concerning the disappearance of deH-HLNL during maturation and aging.

Recently a substance designated as Compound M has been reported to be related to collagen

maturation (14). It was found to be present in bone, tendon and skin of various animals. The molecular

weight of this yet unidentified material is similar but not identical to HHL. We could not find HHL in

tendon and bone.

The cross-linking theory is one of the popular current theories of biological aging (11). Previous

studies have suggested that the cross-linking of collagen may not only be important for optimum

function but also may be a principal mechanism regulating the rate of in vivo catabolism (12). The cross-

linking of collagen begins almost immediately after fibrillogenesis with the formation of covalent

intermolecular iminium bonds. It was demonstrated that the introduction of 0.1 residue of iminium

cross-link per mole of collagen, into fibrils devoid of cross-links, imparts a 2 to 3 fold resistance to

degradation by mammalian collagenase (13). The intermolecular cross-link interactions, are progressive.

time-dependent post-translational processes, that in effect probably slow collagen turn over. Therefore

more of these interactions may take place in the earlier synthesized collagen fibrils that may escape

catabolism. The time-dependent changes in cross-link formation may not be fundamental factors in the

aging process, however they reflect an important aspect of aging. The progressive formation of stable

collagen molecule networks by the continuous increase in stable cross-link content as reported here may

serve to further slow protein turnover and might eventually have deleterious consequences in the aging

organism.

Acknowledgments: This work was supported by NIH Grants DE 08522, A R 19969, AR 30587 and NASA Grant N A G 2-181. We thank Dr. M. Henmi for initial analyses, and Ms. C. Bunch for technical assistance.

902


References

1. Tanzer, M.L. Cross-linking. In Biochemistry of Collagen (eds. G.N.Ramachandran and A.H. Reddi New York, Plenum Press) p.137-162

2. Fujimoto, D, Akiba, K. and Nakamura, N. (1977) Biochem. Biophys. Res. Commun. 76, 1124-1129 3. Yamauchi, M, London, R.E, Guenat, C, Hashimoto, F. and Mechanic, G.L. (1987) J. Biol. Chem.

262, 11428-11434 4. Eyre, D.R, Paz. M.A. and Gallop, P.M. (1984) Ann. Rev. BiocherrL 53, 717-748 5. Yamauchi. M, Noyes, C, Kuboki, Y. and Mechanic, G.L. (1982) Proc. Natl. Acad. Sci. USA 79, 7684-

7688 6. Mechanic, G.L, Katz, E.P, Henmi, M, Noyes, C. and Yamauchi, M. (1987) Biochemistry 26, 3500-

3509 7. Pearce, R.H. and Grimmer, B.J. (1977,) L~Inve.st Derma.to158, 347-361 8. Epstein, E.H,Jr. (1974) J. Biol. Chem. 249, 3225-3231 9. Bakerman, S. (1962) Nature 196, 375

10. Robins, S.P, Shimokomaki, M. and Bailey, A.J. (1973) Biochern. J. 131, 771-780 1L Hayflick, L. (1985) Exp. Geront. 20, 145-159 12. Harris, E.D. and Farrell, M.E. (1972) Biochim. Biophys. Acta, 278, 133-141 13. Vater, C.A, Harris, E.D,Jr. and Siegel, R.C. (1979) Biochem. J. 181, 639-645 14. Barnard, K, Light, N.D, Sims, T.J. and Bailey, A.J. (1987) Biochem. J, 244, 303-309

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aging and cross-linking of skin collagen

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