Heterogeneity in the compositon of human collagen

ARCHIVES OF BIOCHEMIS'l'RY AND BIOPHYSICS

115, 95-101 (1966)

Heterogeneity in the Compositon of Human Collcqsn' E. K. PINE

AND

J. F. HOLLAND

Department of Medicine A, Roswell Park Memorial Institute, Blzgalo, New York Received November 15, 1965 The imino acid composition of established collagens (liver, skin, tendon) and newly synthesized collagens (tumor in liver, cirrhotic liver, and dermal scar) in the same and different individuals has been studied. Whereas the hydroxyproline levels of tendon collagen and that of collagen derived hom metastatic tumor nodules in liver of the same donor are similar, the proline levels were found to differ significantly, Thus isocollugens may exist in man, Hepatic metastasis collagen was identical to collagen elerived from normal and cirrhotic liver. Furthermore, the levels of both imino acids of skin and scar collagen are similar and resemble those of tendon collagen from the same donor, From these studies, it appears that the imino acid components of collagen derived from a diseased organ resemble those of collagen from the same normal organ. Both imino acid levels appear to remain constant during aging of collagen and are independent of the donor's age at the time of new collagen synthesis.

Since the establishment of the amino acid composition of mammalian collagen by Bowes and Kenton (1) and Neuman (2), the distinctiveness of this group of proteins has been recognized. Apart from their characteristically high content of glycine and proline and their low content of tyrosine, they uniquely contain hydroxyproline and hydroxylysine. Although in purified samples of adult mammalian collagen high concentrations of hydroxyproline and proline are always found independent of the collagen source (3), the constancy of the imino acid levels in these proteins during the lifespan of the individual has so far been less clearly demonstrated. Indeed, Briscoe et al, (4) found that in human lung collagen the hydroxyproline level increased with increasing age. The same correlation could be made for collagen derived from lung and tail tendon of 4- to 250-day-old rats (5). However, Boucek et ol. (6), who also studied collagen derived from human lungs, found that the

hydroxyproline content remained constant on aging. Similarly, Hall and Reed (7) found the hydroxyproline level of human skin collagen to remain constant with increasing age of the donors; and Boucek et al. (8) found no effect of age on the composition of newly synthesized human collagen obtained by biopsies of subcutaneous implants of Ivalon sponges in subjects of different ages. The studies cited so far were concerned with either the effect of aging on the hydroxyproline level of already established collagen (4-7) or the influence of age on the synthesis and composition of new collagen (8). We have now been able to combine these two types of observations in a single study, investigating, in the same individual, the effect of age on the imino acid composition of established collagen and on the com.position of collagen newly formed toward the end of life. Furthermore, we have extended previous studies on the imino acid composition of human collagens (9-11) to collagens extracted from scar tissue and normal and diseased liver. Although the imino acid composition of established and newly formed collagen remains constant with age, the

I This investigation was supported in part by American Cancer Society grant 1'-231 and Public Health Service research grant C 5834 from the National Cancer Institute.

95

9G

PINE AND HOLLAND

composition differs in the same individual with the organ from which it is derived. These collagens may thus be considered isoproteins. METHODS All tissues were collected at necropsy. As aources of established collagen we chose Achilles tendon, skin, and liver; and as sources of newly synthesized collagen, tumor metastasized to liver, cirrhotic liver, and scar tissue. Tumor nodules were easily separated from normal liver because of their obviously different macroscopic appearance. Necrotic tumor masses were not used. Cirrhotic livers were obtained from subjects with histories of excessive alcoholic intake and were grossly and microscopically distinct from normal liver. Since normal liver is a collagen-poor tissue, significant contamination of tumor or cirrhotic liver collagen by normal liver collagen was considered unlikely. After careful dissec Lion all tisaues except normal and cirrhotic livers were soaked in isotonic NaCI for two 24-hour periods and then defatted with a mixture of chloroform and methanol (2: 1 volume for volume) according to the method of Popenoe and Van Slyke (12). After drying, the samples were weighed and minced once more, and the insoluble collagen was isolated by alkali treatment and converted to gelatin, all according to the method of Jackson (13). After separation of the nongelutinous from the gelatinous proteins by precipitation with 5% trichloroacetic acid (TCA), in which the gelatinous proteins are soluble, the precipitate was discarded. Subsequently, the TCA was eliminated by exhaustive extraction with ether. Finally, the gelatin was precipitated by acetone as described by Jackson. The precipitate was dissolved in the smallest possible amount of warm, distilled water, eli alyzed overnight against two changes of one thousand times its volume of distilled water, and lyophilized. For reisolation, the lyophilized samples were again dissolved in hot distilled water and the solution was made 5% with respect to TCA. Usually no precipitate formed. Nevertheless, the solution was filtered through an F-fl'itted glass filter, the TCA was removed by ether, and the gelatin was precipitated and lyophilized as described above. For normal and cirrhotic livers a preliminary separation of fiberlike material from liver parenchyma was carried out according to the method of Sulitzeanu et al. (14), substituting 6-10 washings of the homogenate in ice-cold physiologic saline for the 21 washings in borate saline described in their paper. After defatting in the chloroform-methanol mixture, the collagen was iso-

lated from this washed and defatted material by the method of Jackson. All gelatin samples were heated at 104-lOGo to constant weight and stored in the desiccator over P 206. One 01' two portions of each sample were weighed and solubilized separately in 1 ml G N HCI per 10 mg protein. They were then hydrolyzed by autoclaving at 15 pounds pressure for 15-18 hours ill closed glass vials. Aut oclaving was found not to destroy proline, hydroxyproline, or glycine. After autoolaving, the Hel was removed by evaporation on the water bath, and the residues were dissolved in water and analyzed in duplicate at 2 or 3 dilution levels for hydroxyproline by the method of Bergman and Loxley (15), for proline by the method of Sarid ei ai. (Hi), and for nitrogen by the method of Koch and McMeekin (17). Tyrosine was determined after alkaline digestion of the protein by the method of Bernhart (18) as well as chromatographically. Glycine was determined chromatographically. These methods proved as reproducible as indicated by their original authors. For chromatography, samples of known hydroxyproline content were dried on the water bath, and the residue, redissolved in buffer, was put on a Dowex 50 Chromobeacls column, 6 mm diameter X 133 em height, and developed at 60°. This column was part of an automatic amino acid analyzer (Technicon Chromatography Corp., Chauncey, New York). Amino acids were eluted by means of a continuous pH and salt gradient devised by the manufacturer over a Ii-hour period. The gradient is established by sodium citrate buffers varying in pH from 2.75 to 6.0 and in sodium concentration from 0.2 to 2.4 N. The equipment had previously been calibrated with known concentrations of specific amino acids. Norleucine was included as an internal standard in each run." RESULTS

After investigating several methods for their adequacy in separating insoluble collagen from a heterogeneous protein mixture, we found that the modified Jackson procedure described in the previous section suited our needs the best. To determine the reproducibility of the method, we performed 5 replicate isolations from the least homogeneous tissue under study, namely, tumor metastasized to the liver. The reproducibility of the isolations was measured in terms 2 We are grateful to Dr. Allan Grossberg, in whose laboratory the chromatographic analyses were performed.

97

IMINO AOID OOMPOSITION OF lIUMAN OOLLAGENS

TABLE I QUANTITATIVE INDIeES FOR DETEHMINING THE PUIUTY OFINSOLUBLE COLLAGEN DERIVED FROM VARIOUS TISSUES, AFTER ONE 'ro FOUR ISOLATIONS" Tissue

Normal Iiver

Tumor ill li Vel'

Sample

Hydroxyproline Proline Glycine Tyrosine

A A A A

104.7 117.8

Hydroxyproline

A

103.5 105.9 125.0 134.5 342.7

4

B Proline Glycine Tyrosine

Cirrhotic liver

Hydroxyproline Proline Glycine Tyrosine

Tendon pool

Isolation number

Amino acid residues/lOOO residues lJ

Hydroxyproline Proline Glycine Tyrosine

A B A B A A B A B A A A B A B B A B

113.1 128.5 330.8

113.1 126.1 334.4

105.9 101.2 129.7 122.6 353.4

110.7 104.7 130.9 126.1

3.45 108.3 105.9 135.7 126.1 351.1

323.7 2.61 102.3 109.5 109.5 121.4

101.2 100.0 113.1 117.8

107.1 101.2 123.8 1Hl.G 329.(i 3.68

100.0 98.8 114.2 111.9 292.7 3.92

86.9 92.8

94.0 96.4 142.8 141.6

100.0'

138.0

148.8 352.2

4.64 4.04

4.04 4.04

a Collagen was extracted from various sources and was purified 3 01' 4 times j aiter each purification one or two samples were analyzed in duplicate for the listed amino acids. b See footnote', Table II. c 15% KCI was the precipitating agent.

of the proline and hydroxyproline content of each isolate. The proline residues of the 5 isolates ranged from 122.3 to 133.0 residues/WOO residues, with a mean of 126.4 ± 4.27 SD; the hydroxyproline residues ranged from 105.6 to 114.0 residues/1000 residues with a mean of 108.2 ± 3.45 SD. Although the hydroxyproline content of these isolated gelatin samples compared favorably with the hydroxyproline content of a sample of known purified calf skin gelatin (Eastman Kodak) (110.8 residues/WOO residues), repeated additional solubilizations and reprecipitations were performed in an attempt to increase the purity of the samples. The experiment, summarized in Table I, was car-

ried out as follows; After the first isolation of collagen as gelatin, one or two independent samples designated A and B were weighed out and hydrolyzed, and their nitrogen, proline, hydroxyproline, glycine, and tyrosine levels were determined. The bulk of the sample was then once more solubilized in 5 % TeA and treated as described under Methods. After lyophilization of this second isolate, two independent samples were again weighed out and hydrolyzed, and their imino acid content was determined. The siolation was repeated in this manner for a total of 4 times. I t may be seen in the table that, except for collagen extracted from normal liver,

98

PINE AND HOLLAND TABLE II

COMPARISON OF THE IMINO ACIJ) LEVELS OF COLLAGENS ISOI,ATED FROM ES1'ABLISHED AND NEWLY SYNTHESIZED TISSUES IN 'l'HE SAME DONOR

Residues amino acid/1000 total residues' --~~-~-

Age Subject (years)

-PIG

30 60 64 77

C

P2 A PaG

78 63

N A

(i\)

7(j

F

Tendon

Primary tumor

I Ca of cervix Ca of colon Reticulum cell ca Ca of endometl'iuffi Adenoca of lung Ca of cervix Ca of bronchus Ca of bladder Mean ± SD

j

Metastatic tumor

Scar

Skin

,-'

Hydroxyproline

Proline

97.5 101.1 95.2 104.8

132.0 163.0 145.0 163.0

98.9 144.0 106.0 165.2 98.9 162.0 157.0 101.1 100.4/' 153.9b ±3.61 ±12.1O

Hydroxyproline

roe. 0 101.1 96.4 107.0 103.5

102.8 b ±4.25

Proline

--

Hydroxyproline

Proline

107.0 112.0 123.8 114.3 ±8.lJ3

152.2 07.5 148.9 I 115.2 155.0 I 119.0

--- - - - ,---

Hydroxy-

Proline

proline

123,8 125.0

lULO 119.0 110.9

119.6" ±5.55

152.0 1110.6 ±3.05 ±9.38

I

147.5 15(j.O 15:3.5 153.2 ;:/:4.91

All analyses were made on collagens isolated two times except for samples from donors 1', and 1'3. Collagen samples from donors 1', and 1'3 excluded from statistical computations. • Data were originally calculated as mg amino acid N /100 mg collagen N and were converted by multiplying this unit by: a

b

91.5 (mean residue wt) 14 (atomic wt. N)

-

18.2 (% total N in x10

collagen).

Mean residue weight n.nd total N values as obtained by Eastoe for human collagen (9).

the imino acid and glycine levels do not increase significantly with successive isolations, nor was there an appreciable decrease in the tyrosine levels. Thus, no appreciable increase in purity was achieved by repeated precipitations except in the extraction of collagen from normal liver. We believe that in this case the low amino acid values observed in the first isolation are due to initially large quantities of liver used, which appeared to make the separation of collagenous from noncollagenous proteins less efficient. Therefore, in all subsequent isolations we subdivided the tissue into small portions of 3 gm or less, which allowed good separation on the first isolation. As previously, the variations in the amino acid levels from one isolation to the next reflect the sum of the procedural errors. On the basis of these data, all other collagen samples were subj ected to initial extraction followed by one reisolation, To study the effect of age on the imino

acid level of collagen of already established and of newly synthesized tissue in the same donor, we compared collagen extracted from tendon and skin with collagen extracted from tumor and scar. We selected tendon and skin as examples of established tissues because they have been shown to have very little turnover in the adult rat (19,20). On the other hand, it was estimated for the tumors, and documented for the scars, that these tissues were synthesized recently, no more than 5 years before death. The collagens extracted from the various tissues of the same donor were processed simultaneously under identical conditions; their imino acid levels have been tabulated in Table II. Examination of the data for the first five donors listed in the table shows that for each individual donor there is no significant difference between the hydroxyproline levels of tendon and tumor collagen, although the approximate age at which the tumors were

99

IMINO ACID COMPOSITION OF HUMAN COLLAGENS TABLE III

AVERAGE IMINO ACID LEVELS OF HUMAN COLLAGENS DERIVED FROM VAR10US TISSUES AND ORGANS"

Residues amino add/fOOD total residues d Tissues

Age of collagen

Hydroxyproline (mean ± SD)

Proline (mean ± SD)

Tendon" (6)b Skin" (3) Scar' (3) Liver Normal (1) Tumorous' (3) Cirrhotic (2)

Established Established Newly synthesized

100.4 ± 3.61 1l0.6 ± 9.38 114.3 ± 8.(j3

153.9 ± 12.10 153.2 ± 4.91 152.0 ± 3.05

Established Newly synthesized Newly synthesized

113.1 102.8 ± 4.25 103.5 ± 1.36

124.0 119,6 ± 5.55 120.0 ± 5.95

All analyses were made on collagens isolated two times. Number of separate donors. , Same data as Table II. d See footnote', Table II. a

b

synthesized varied from about 25 years (for donor PI) to about 73 years (for donor P3)' Similarly, no statistically significant difference could be found when the hydroxyproline levels of the tendon collagens were compared as a group with the hydroxyproline levels of all the tumor collagens (p < 0.1, Table III). However, the proline levels of these same collagens differ significantly from each other (p < 0.01, Tables II and III), although, like the hydroxyproline levels, they appear independent of the age at which the collagen synthesis took place. On the other hand, the individual differencesin both the hydroxyproline and proline levels of tendon, skin, and scar collagens contributed by the last three donors in Table II are insignificant. The hydroxyproline level of skin collagen (an established tissue) appears to increase with age; because of the small number of samples examined, this finding cannot be interpreted. The difference between the proline content of collagen derived from two kinds of newly synthesized tissues, tumor and scar, is significant (p < 0.02, Table II). Inthis instance, the statistical comparison. was made between the difference in proline levels of tendon and tumor collagen on the one hand, and tendon and scar collagen on the other. Since neither the anatomical location 1101' the histological character of the primary tumors appeared to affect the imino acid levels of the collagens in their hepatic metastases, we decided to examine the composi-

tion of the target organ, namely, liver collagen. It may be seen from Table III that the imino acid levels for all liver collagens are similar, be they derived from normal, tumorous, 01' cirrhotic liver. Furthermore, the proline levels of all liver collagens differ significantly from the proline levels of that group of collagens which was isolated from tendon, skin, and scar. There are then two possible explanations for the observed similarity of the imino acid levels of tumors, which cannot be distinguished by our experiments: either the levels are determined by the stroma of the host organ which in our experiments is liver, or by the tumor per se and are similar for all tumors. DISCUSSION

Although there is an imino acid difference with respect to collagens extracted from various tissues, this difference appears to be independent of both the age at which new collagen synthesis takes place and the age of the established collagen, as reflected by the age at death of the donor. Another indication of the independence of composition and age may best be seen in Figure 1 where we have plotted hydroxyproline levels of established collagen derived from Achilles tendon of 28 donors against the age at death of the donors. This finding is valid, although a systematic error due to the single isolation of all tendon collagens led to lower hydroxyproline values than reported in

100

PINE AND HOLLAND

to the preparation of the collagen samples or to the differences in assay procedure. Ino deed we found repeatedly that the values for o o 00 o proline of samples analyzed by the method • ~ •• L • • o. of Sarid (IG) were between 8 and 12 % higher • MALE than the values obtained for the identical o FEMALE samples by the amino acid analyzer. Such I I I o 40 80 20 60 discrepancies in values due to the use of AGE IN YEARS different methods are by no means uncomFIG, 1. Hydroxyproline levels of insoluble colmon and fall within the usually accepted lagens derived from tendons of donors of various range. ages. All collagens were isolated once. Differences in the composition of collagens from different tissues of the same species Tables II and III. Our results indicate that have been shown previously with respect collagens extracted from all tissues examined, to proline aswell as hydroxylysine and lysine. with the possible exception of skin collagen, Thus, in humans the proline level of collagen maintain the same imino acid levels through- extracted from kidney reticulin is lower than out the life of the individual, but the abso- that of collagen extracted from bone or tenlute values of these levels may differ from don; but the level of hydroxylysin~ is lo.w each other. Although several of the scar and in collagen extracted from bone, higher in tumor collagens were newly synthesized at collagen extracted from tendon, and the similar ages toward the end of life, their highest in renal reticulin (9, 10). Although imino acid profiles are dissimilar, each reflect- at present nothing is known about the ing its own tissue specificity. Thus newly functional significance of these variations synthesized collagen in liver disease (meta- in the amino acid pattern of collagens destatic tumor and cirrhosis) is similar to nor- rived from different tissues of the same mal liver, and newly synthesized c.ollagen species, such variations in the composition in skin (scar) is similar to normal skin (Ta- of proteins with apparently identical funcble III). tions and derived from various tissues of It seems unlikely that the hydroxyproline the same individual (isozymes) have been levels of skin collagen increase with age, described. To our knowledge, the present though our results do not exclude this possi- work provides the first evidence that there bility. It should be emphasized, however, exists a heterogeneity of the composition that the constancy of the imino acid levels of collagen within the same individual; such with age is entirely compatible with age- proteins might be termed "isoproteins." dependent changes in the physical properties of collagen: Verzar and Willenegger (21) ACKNOWLEDGMENTS found that after heating tissues for 10 minWe are indebted to Dr. Henry Hosley for his nutes at 65°, aged collagen released into active participation in the early studies; to Dr. solution a much lesser part of its total pep- Paul Sheehe for statistical consultations; to Drs. tide-bound hydroxyproline than did newly H. James Wallace, Jr. and Salman Gailani for obtaining the speoimens; and to the Pathology Desynthesized collagen. Our observations agree with the previous partments ofRoswell Park Memorial Institute and finding of Fleischmajer et al. (10) that the the Veterans Administration Hospital for making imino acid levels of skin and tendon col- suitable specimens available. Mrs. Elizabeth Carter, Miss Joyce Pfeuffer, lagen are similar. Our absolute values for Mrs. Anita Jenkins and Miss Sandra Anthony gave the mean of the hydroxyproline and proline valuable technical assistance during various stages levels of insoluble collagen extracted from of this study. skin are each 18 % higher, and the values REFERENCES for the mean of the hydroxyproline and proline levels from tendon are 8 and 18 % higher, 1. BOWES, J. H., AND KENTON, R. H., J. BioI. respectively, than the previously reported Chern. 43, 358 (1948). levels (9, 10). These differences may be due 2. NEUMAN, R. E., Arch, Biocheni, 24, 289 (1949).

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I

IMINO ACID COMPOSITION OF I-WMAN COLLAGENS

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POl'ENOE ,