Collagen polymorphism: Isolation and partial characterization of α1(I)-trimer molecules in normal human skin

ARCHIVES

OF RI~CHEMISTRY

Vol. 192, No. 2, February,

Collagen

AND BIOPHYSICS

pp. 371-379,

1979

Polymorphism: Isolation and Partial Characterization al(I)-Trimer Molecules in Normal Human Skin’ JOUNI

Division

of Dermatology,

UITT02

Department of Medicine, Washington St. Tmis, Missouri fi:
University

School of’ Medicine,

Received July 25, 1978; revised August 28, 1978 Human skin has previously been shown to contain at least two genetically distinct types of collagen, type 1 and III. Here the presence of an additional form of collagen, al(I)trimer, is demonstrated. Skin collagen was solubilized by limited pepsin digestion and then fractionated by sequential precipitation with 1.5, 2.5, and 4.0 M NaCl at pH 7.4. The a-chain subunits of collagen were isolated by gel filtration and carboxymethylcellulose chromatography under denaturing conditions. The 1.5 and 2.5 M NaCl precipitates contained predominantly type I collagen with a chain composition of [al(I)],~uZ. In the 1.5 M precipitate a small amount of type III collagen was also recovered. In contrast, the 4.0 M NaCl fraction consisted almost exclusively of a-chains which on the basis of cyanogen bromide peptide mapping were shown to be identical with al(I). The amino acid composition of these chains was also similar to that of al(I), except that hydroxylysine was increased and lysinc was correspondingly decreased. The content of 3-hydroxyproline was also increased. These results suggest that the a-chains in al(I)-trimer are the same gene products as al in type I collagen, but that the co-translational or post-translational hydroxylation of lysyl residues is more extensive in al(I)-trimer. Estimation of the quantitative amounts of al(I)-trimer indicated that this collagen accounts for less than 5%~ of the total collagen in adult human skin. It is speculated, however, that al(I)-trimer collagen may play a role in the stability and tensile strength of normal human skin and other tissues, and defects in its biochemistry might be associated with diseases of connective tissue.

Collagen, the major extracellular component of the connective tissues, consists of a family of closely related but genetically distinct proteins. Four genetic types, types I to IV, are relatively well characterized [see (2-4)]. In addition, other genetic types of collagen, such as collagens consisting of distinct A and B chains, exist in the connective tissues; their biochemical identification and characterization are still in progress (5-7). 1 This work was supported in part by United States Public Health Service, National Institutes of Health Grants AM 12129 and AM 0’7284. A preliminary report of part of this work has been presented at the 35th Annual Meeting of the American Federation for Clinical Research, San Francisco, Calif., May 1, 1978 (1). ’ Recipient of Research Career Development Award 1 K04 AM 00455-01 from National Institutes of Health.

Each collagen molecule is composed of three polypeptide chains, a-chains, which are wound to a unique triple-helical conformation [see (3)]. Type I collagen is a heteropolymer in that it contains two identical polypeptides, Lul(I)-chains, while the third chain, a2, is genetically distinct. Type II, III, and IV collagens consist of three identical polypeptide chains (2, 3). In addition to the genetically distinct types of collagen, another molecular form of collagen, al(I)-trimer or type I-trimer, has been recently demonstrated (8- 13). The al(I)-trimer consists of three polypeptides which are genetically identical with al-chains of type I collagen. The ml(I)trimer collagen was originally isolated from a virus-induced tumor and its synthesis was observed in cultures of cells derived from such tumors (10, 12). The synthesis of al(I)371

0003-9861/79/020371-09$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

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JOUNI

trimer molecules has also been observed under various tissue culture conditions (8, 9, 13). It was suggested, therefore, that al(I)-trimer molecules may represent errors in the assembly of collagen polypeptides under tissue culture conditions or in malignant tumors, and these molecules may not be present in normal connective tissues. The present study reports on the isolation of al(I)-trimer collagen from normal human skin and on the partial characterization of this unusual molecular form of collagen. MATERIALS

AND METHODS

Sohbilizntion and isolation of human skin collugens. Samples of skin were obtained from healthy adult human subjects who underwent various surgical procedures. The subcutaneous tissue was carefully removed and the specimens were rinsed with cold (+4”C)O.15 M NaCl-0.05 M Tris-HCI, pH 7.5. The skin samples were then minced extensively with scissors in 0.5 M acetic acid (10 g tissue/l00 ml) and then homogenized with a Polytron mechanical tissue homogenizer at 4°C. Pepsin (Worthington, 2x crystallized) was added to a final concentration of 100 pg/ml, and the samples were incubated at 4°C for 16 h. At the end of the incubation, the homogenate was centrifuged at 30,OOOg for 60 min at 4°C. The pellet was dissolved in 0.5 M acetic acid and repepsinized and then centrifuged as above. The pooled supernatants were adjusted with cold 10.0 and 1.0 M NaOH solutions to pH 8.5, and they ivere then extensively dialyzed pH i.5. against 0.4 M NaC’l-0.1 M Tria-HCl. In order to partially separate various genetically distinct types of collagens. the solubilized collagen was fractionated by sequential precipitations with 1.5, 2.6, and 4.0 M NaCl. After the slow addition of solid NaCl to any given concentration, the samples were stirred for 24 h at 4°C and then centrifuged at 30,OOOg for 60 min. The pellet was dissolved in and dialyzed against 0.5 M acetic acid and then lyophilized. Chromntogrnphic proce&lres. Initial isolation of collagen a-chains from various salt precipitation fractions was achieved by gel filtration on 6% agarose under denaturing conditions. To prepare collagen samples for gel filtration, 50 mg of lyophilized sample was dissolved by stirring in 5 ml of 0.05 M Tris-HCl buffer, pH 7.5, containing 10 M urea, 20 mM disodium ethylendiaminetetraacetate, lOmM?v-ethylmaleimide, and 1 FM cu-toluenesulfonyl fluoride. The sample was heated for 5 min to 100°C. centrifuged at 18,000g for 10 min at 22”C, and then chromatographed on a 2.3 x 90-cm column of 6% agarose (Rio-Gel A-5m. ZOO-400 mesh, Bio-Rad Laboratories) eluted with 1 M CaCl,-0.05 M Tris-HCl, pH ‘i.5, at 22°C (14). In some samples Smercaptoethanol, in a final concentra-

JITTO tion of 2%, was added before heating. Fractions containing the protein peaks from the column were pooled, dialyzed extensively against distilled water, and lyophilized. The recoveries of the collagenous material in the gel filtration were in excess of 90%. In order to separate various a-chain subunits of collagen, samples of the lyophilized material were dissolved in 0.02 M sodium acetate, pH 4.8, containing 2 M urea. The samples were then heated for 5 min at 60°C and chromatographed on a 2.5 x 10.0.cm column of CM-cellulose” (CM-52, Whatman Biochemicals, Ltd.) equilibrated with 0.02 M sodium acetate, pH 4.8, and containing 2 M urea, at 45°C (15, 16). The proteins absorbed to the column were then eluted by a linear gradient formed with 300 ml of starting buffer consisting of 2 M urea in 0.02 M sodium acetate, pH 4.8, and 300 ml of limit buffer containing 0.1 M NaCl and 2 M urea in 0.02 M sodium acetate, pH 4.8. The total recoveries of the collagenous peptides varied from 82 to 94%. Other procedures. In order to examine the genetic types of the isolated a-chains, the polypeptides isolated by CM-cellulose chromatography were extensively dialyzed against distilled water at 4°C. lyophilized, and then subjected to cyanogen bromide cleavage, as described elsewhere (16, 17). Isolated proteins or cyanogen bromide peptides were examined by SDS-polyacrylamide slab gel electrophoresis using either 6 or 8% separating gels (16, 18, 19). The gels were stained with I’% Coomassie blue for 30 min and destained in a solution containing 10% methanol and 5%’ acetic acid. The peptide patterns \vere quantitated by scanning the gels at 560 nm with a Zeiss PM GK spectrophotometer with a scanning attachment. Hydroxyproline was quantitated in various samples either by using a specific calorimetric assay (20) or by amino acid analysis. The amino acid analyses were performed with a Beckman 1lY C amino acid analyzer using a single column and four buffer system. RESULTS

Solubilization of collagen by limited proteolytic digestion with pepsin resulted in the release of approximately 80 to 86% of the hydroxyproline-containing material from the total skin sample in four different experiments. Fractionation of the solubilized collagen by sequential salt precipitation demonstrated that most of the hydroxyproline-containing material was soluble in 1.5 M NaCl but could be precipitated with 2.5 M NaCl under neutral conditions (Table :I Abbreviations used: CM-cellulose, carboxymethylcellulose: SLIS, sodium dodecyl sulfate.

TYPE

I-TRIMER

COLLAGEN

I). About 24% of the hydroxyprolinecontaining material precipitated in 1.5 M NaCl, while another 5% of the material was soluble in 2.5 M NaCl but could be precipitated with 4.0 M NaCl. The supernatant, after 4.0 M NaCl precipitation, contained less than 2% of the total hydroxyprolinecontaining material and was not examined further. Amino acid analyses of the 1.5, 2.5, and 4.0 M NaCl precipitates indicated that over 90% of the protein consisted of collagen. Repepsinization of the precipitated material before denaturation did not change the elution pattern of the protein in subsequent chromatographic steps (see below), indicating that the collagen a-chains isolated by the differential salt precipitation were in native triple-helical conformation. In order to isolate a-chains in the 2.5 M NaCl precipitate, a sample was chromatographed on an agarose column under denaturing conditions. A major fraction of the protein eluted in the position of a-chains with an apparent molecular weight of 94,000 (Fig. 1A). Peaks eluting in /3- and y-chain positions as well as larger aggregates eluting near the void volume of the column were also noted. Examination of the polypeptides larger than a-chains by amino acid analysis demonstrated that these peaks consisted of collagen. Also, rechromatography of the individual peaks in the presence TABLE

I

RELATIVE DISTRIBUTION OF TYPE I, TYPE III, AND al(I)-TRIMER COLLACENS IN HUMAN SKIN”

Amount NaCl fraction

Total

Type I

Type III 3.0

1.5 M

6.1

3.1

2.5 M 4.0 M

18.0 1.2

18.0

Total

25.3

21.3

(100.0%)

of collagen (mg)

0.2 (84.1%;)

al(I)-Trimer

-

1.0

3.0

1.0

(11.9%)

(4.0%))

” Starting material: 100 mg wet wt of skin. h The amounts are calculated on the basis of hydroxyproline assay and the genetic types estimated from the ratio of nl(I):aB:al(III) in CM-cellulose chromatographg.

IN HUMAN

07t

373

SKIN

n

FIG. 1. Isolation of o-chains by gel filtration from the 2.5 and 4.0 M NaCl precipitates. Human skin collagen, solubilized by limited pepsin proteolysis, was fractionated by successive precipitations with 1.5, 2.5, and 4.0 M NaCl at pH 7.4. The protein in the 2.5 and 4.0 M NaCl precipitates was prepared for gel filtration, as described under Materials and Methods, and chromatographed on a 2.5 x 90-cm column of 6’7%agarose eluted with 1 M CaCl,-0.05 M Tris-HCl, pH 7.5. The void volume of the column (Vo) and the elution positions of type I collagen u-, p-. and y-chains are indicated in the chromatogram. (A) Elution pattern of protein in 2.5 M NaCl precipitate. (B) El&ion pattern of protein in 4.0 M NaCl precipitate.

of 2-mercaptoethanol did not change their elution position. It is concluded, therefore, that, in addition to the a-chains recovered in the 2.5 M NaCl precipitate, pepsinization solubilized larger collagen aggregates which contained covalent interchain crosslinks but which were not held together by interchain disulfide bonds.

374

JOUNI

To analyze the composition of the a-chains isolated by gel filtration from the 2.5 M NaCl precipitate, a sample was chromatographed on CM-cellulose under denaturing conditions. The a-chains resolved into two major peaks corresponding to c-ul-and c-w2-chains of human collagen (Fig. 2A). The ratio of cJ/c~2 in this material, as estimated from the elution pattern, was approximately 1.9-2.3:1. This observation suggests that essentially all a-chains recovered in the 2.5 M NaCl precipitate were derived from type I collagen. Further examination of the isolated al-chains by cyanogen bromide peptide mapping demonstrated that these polypeptides yielded peptide patterns typical of al-chains of human type I collagen (Fig. 3A). Samples from the 4.0 M NaCl precipitate were also chromatographed on the agarose

0

0.1

02

03

04

ELUTION VOLUME

0.5

'0

IO

20

30

MIGRATION

40

50

60

(mm)

FIG. 3. Cyanogen bromide peptide mapping of a-chains isolated from the 2.5 and 4.0 M NaCl fractions by gel filtration and CM-cellulose chromatography. The polypeptides eluting in CM-cellulose chromatography in the al-chain position were dialyzed, lyophilized, and subjected to digestion with cyanogen bromide, as described under Materials and Methods. The resulting peptides were examined by SDS-polyacrylamide slab gel electrophoresis using 8% separating gels. The gels were stained with Coomassie blue and the peptide patterns were quantitated by scanning, as indicated in the text. The electrophoretic positions of characteristic marker peptides derived from human al(I)-chains, as well as the mobility of bromophenol blue (dye front), which was added to the samples, are indicated by arrows. (A) Cyanogen bromide peptide pattern of al-chains isolated from the 2.5 M NaCl precipitate. (B) Cyanogen bromide peptide pattern of al-chains isolated from the 4.0 M NaCl precipitate.

06

(11

2. CM-Cellulose chromatography of a-chains isolated by gel filtration from the 2.5 and 4.0 M NaCl precipitates. The polypeptides eluting in the o-chain position on agarose chromatography (see Fig. 1) were extensively dialyzed, lyophilized, and prepared for CM-cellulose chromatography, as described under Materials and Methods. The samples were chromatographed on CM-cellulose equilibrated with 0.02 M sodium acetate, pH 4.8, containing 2 M urea, as described in the text. The polypeptides were eluted with a linear gradient of O-O.1 M NaCl in 0.02 M sodium acetate, pH 4.8, containing 2 M urea. The elution positions of al(I)- and cr2-chains of human type I collagen are indicated in the chromatograms. (A) Elution pattern of the a-chains isolated from the 2.5 M NaCl precipitate. (B) Elution pattern of the a-chains isolated from the 4.0 M NaCl precipitate. FIG.

UITTO

column. In this material, a large fraction of the protein chromatographed in the a-chain position, although larger aggregates were also seen (Fig. 1B). Examination of the isolated a-chains by CM-cellulose chromatography demonstrated that most of the protein eluted in the same position as al(I) and a much smaller peak in the aZchain position was seen (Fig 2B). The ratio of (~lla2 chains in eight different preparations varied from 5.6:l to as high as 2O:l. Similar values were obtained when the al/a2 ratios were estimated by SDS-polyacrylamide slab gel electrophoresis. Also, amino acid composition of the a-chains from the gel filtration column resembled more the com-

TYPE

I-TRIMER

COLLAGEN

position of al(I) than that of type I collagen. Cyanogen bromide cleavage of the peptides eluting in the position of al-chains in CMcellulose chromatography yielded a peptide map which was identical to a map derived from the al(I)-chains recovered in the 2.5 M NaCl precipitate (Fig. 3B). On SDSpolyacrylamide slab gel electrophoresis the isolated al-chain from the 4.0 M NaCl precipitate migrated as a single peak in the same region as al(I) from type I collagen, and no evidence of larger polypeptides, such as B chains of A-B collagen (5-7) or al(W)-chains, was noted. Since the collagen molecules in the 2.5 and 4.0 M NaCl fractions were precipitated as native, triplehelical molecules under nondenaturing conditions, it is clear that most of the a-chains in the 4.0 M NaCl fraction were derived from collagen molecules which consist of three identical al(I)-chains. The al(I)-chains isolated in the 2.5 and 4.0 M NaCl precipitates were partially characterized by amino acid analyses. Examination of the amino acid composition demonstrated that most of the amino acids were present, within the limits of experimental accuracy, in the same relative proportions in al(I)-chains from both sources (Table II). An exception was hydroxylysine; the relative amount of this amino acid in al(I)-chains isolated from the 4.0 M NaCl fraction was almost twice as high as its content in al(I)-chains from the 2.5 M NaCl precipitate. The values for lysine were correspondingly decreased in the 4.0 M NaCl fraction, so that the total number of hydroxylysine plus lysine residues was the same in al(I)-chains from both sources. Amino acid analyses also indicated that the content of 3-hydroxyproline was increased in al(I)-chains isolated from the 4.0 M NaCl precipitate (Table II). The value 2.1 * 0.3 residues per 1000 (mean & SD of four determinations) in al-chains of the 4.0 M NaCl precipitate is significantly different (P < 0.05) from the corresponding value, 1.1 ? 0.2, in the 2.5 M NaCl fraction. No significant differences were noted in the relative contents of 4-hydroxyproline or proline in the al(I)-chains isolated from the 2.5 and 4.0 M fractions. In further experiments the a-chains in

IN HUMAN

375

SKIN TABLE

AMINO

ACID

ANALYSIS

II OF al(I)-CHAINS

IN 2.5 AND 4.0 M NaCl FRACTIONS

Relative Amino acid 3Hydroxyproline” 4-Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Lysine Histidine Arginine

2.6 M NaCl 1.1 91.9 16.3 20.6 36.2 62.8 131.0 320.1 115.6 21.6 7.3 9.6 26.4 2.3 12.8 4.6 31.9 3.9 54.7

amount”

4.0 M

NaCl

2.1 95.9 4.X-1 18.0 37.3 66.6 126.1 326.2 127.1 18.3 7.6 9.5 24.1 1.9 11.9 9.6 25.2 3.6 50.0

’ Residues per 1000; the values are means of four analyses from three different preparations. ’ The estimates of 3-hydroxyproline were based on standards derived from bovine lens capsule collagen kindly provided by Dr. Peter Dehm, Connective Tissue Research Institute, University of Pennsylvania.

the 1.5 M NaCl precipitate, which is expected to contain type III collagen, were isolated by gel filtration on agarose (Figs. 4A and B). Since type III collagen consists of a-chains which are linked by interchain disulfide bonds (4, 21), the y-region of the chromatogram obtained without reduction was pooled and rechromatographed after reduction with 2-mercaptoethanol. Part of the protein which initially eluted as a material with an apparent molecular weight of about 285,000 was now recovered in the a-chain position with an apparent molecular weight of 94,000. Examination of these a-chains by CM-cellulose chromatography revealed a major protein peak eluting in the position intermediate between al(I) and c~2 of human skin type I collagen, while the a-chains obtained in the initial chromatography without reduction consisted of cul-

376

JOUNI

FIG. 4. Isolation of a-chains from the 1.5 M NaCl precipitate by gel filtration. The material recovered in the 1.5 M NaCl precipitate of pepsin-solubilized human skin was chromatographed on 6%, agarose without reduction (-MSH), as described in the text and legend to Fig. 1. The polypeptides eluting in the y-chain region were then pooled, concentrated, and rechromatographed after reduction with 2-mercaptoethanol (+MSH). (A) Elution pattern of the protein recovered in the 1.5 M NaCl precipitate. (B) Rechromatography, after reduction with 2-mercaptoethanol, of the material eluting in the y-chain region indicated by the horizontal bar in (A).

UITTO

FIG. 5. Carboxymethylcellulose chromatography of the a-chains isolated from the 1.5 M NaCl precipitate by gel filtration either without reduction or after reduction with 2-mercaptoethanol. The elution positions of human al(I)-, cu2-, and cul(III)-chains are indicated in the chromatograms. (A) Elution pattern of a-chains isolated from the 1.5 M NaCl precipitate by gel filtration without reduction (Fig. 4A). (B) Elution pattern of a-chains which initially chromatographed in the y-chain region (see Fig. 4A), but which upon rechromatography after reduction with S-mercaptoethanol were recovered in the a-chain position (Fig. 4B).

collagens in normal human skin. The results, in accordance with previous publications (4, 22), demonstrated that the major genetic type of collagen in adult human skin is type I, while about 10% of the collagen consists of type III. In addition, a small but significant fraction of the total skin collagen was demonstrated to consist of al(I)-trimer molecules. DISCUSSION

and cY2-chains in an approximate ratio of 2:l (Figs. 5A and B). Cyanogen bromide peptide mapping and amino acid analyses (not shown) of the isolated a-chains confirmed the presence of al(I) and (~l(I11). These experiments indicated, therefore, that the 1.5 M NaCl precipitate contained both type I and type III collagen. On the basis of the total hydroxyproline content of each NaCl precipitation fraction and the a-chain composition, as estimated by CM-cellulose chromatography, it was possible to calculate the relative proportions of type I, type III, and al(I)-trimer

The synthesis of collagen molecules consisting of three al(I)-chains was initially demonstrated in chondrocyte cultures grown in the presence of 5-bromo-2’-deoxyuridine or cultivated under conditions allowing the cells to dedifferentiate to their mesenchyma1precursor form (8, 11). Under such conditions the cells, which initially synthesized only type II collagen, switched their collagen production predominantly to type I. In addition the cultured cells synthesized collagen molecules which were shown to consist of three identical al(I)-chains. Subsequently, it was demonstrated that

TYPEI-TRIMERCOLLAGENINHUMANSKIN

fibroblasts derived from diseased human gingiva or organ cultures of rat incisor odontoblasts synthesized al(I)-trimer collagen (9, 13). Furthermore, cultured cells obtained from a mouse teratocarcinoma were shown to synthesize [crl(I)], (10). The first demonstration that type I-trimer collagen can occur in vivo was obtained through analysis of collagen in a bone- and cartilage-forming mouse tumor induced by transforming salivary epithelial cells with polyoma virus (12). The results demonstrated that, in addition to type I collagen with a chain composition of [al(I)],aB, collagen with a chain composition of [al( was isolated. Recently, it has been suggested that [al(I)], collagen may be present in lathyritic chick embryo tendons and calvaria (23). In this study, an increased crll(r2 ratio was noted, as estimated by SDS-polyacrylamide disc gel electrophoresis, in material isolated by differential salt precipitation. Also, the amino acid composition of the isolated collagen resembled the composition of al(I)-chains. However, the genetic type of the isolated polypeptides was not examined further, and it is unclear whether the isolated molecules represented type I-trimer collagen. In the present study a significant fraction of the collagen in adult normal human skin was shown to consist of al(I)-trimer molecules. The [cyl(I)], collagen molecules, which were partially separated from type I and type III collagens by differential salt precipitation under nondenaturing conditions, were isolated by gel filtration and ion-exchange chromatography; these steps separate al(I)-chains from polypeptides of type IV and A-B collagens. The isolated a-chains were shown to be genetically related to al-chains of type I collagen by electrophoretic mobility on polyacrylamide gels in SDS, chromatographic behavior on CM-cellulose, and on the basis of cyanogen bromide peptide mapping. It should be noted, however, that a complete identity of the al-chains in type I-trimer molecules and in type I collagen cannot be established without more detailed analyses of the primary structure. Since the collagen molecules isolated from the human skin were initially solubilized by

377

pepsinization, an argument could be raised that the proteolysis might preferentially digest cy2-chains in normal type I collagen molecules. Such a possibility appears, however, very unlikely, since in case the a2-chain in an [al(I)],a2 molecule had been completely digested the remaining two al-chains would not stay in a conformation resistant to pepsin proteolysis. On the other hand, if pepsin produced only a limited number of cleavages in the cy2-chainwithout destroying the triple-helical conformation the subsequent differential salt precipitation, under nondenaturing conditions, would not separate minimally cleaved type I collagen molecules from intact ones. Examination of the al(I)-chains in type I-trimer molecules by amino acid analyses demonstrated that these chains have an amino acid composition very similar to those of cul-chains of type I collagen. An exception was the increased content of hydroxylysine which was approximately twice as high as that observed in the al(I)-chains isolated from type I collagen. However, the relative content of lysyl residues was correspondingly decreased, so that the sum of hydroxylysine plus lysine in the a-chains of type I collagen and al(I)-trimer collagen was the same. In the same analyses, the content of 3-hydroxyproline in [al(I)]:l was found to be increased, while the relative contents of 4-hydroxyproline and the remaining amino acids appeared to be unchanged. Similar observations have been made previously in studies on type I-trimer collagen from other sources (10, 23). The hydroxylysine in collagen is synthesized by a co-translational or post-translational hydroxylation of certain lysyl residues in the newly synthesized a-chains, and the hydroxylation reaction is terminated when the pro-a-chains of procollagen fold into the triple-helical conformation (24-27). In ordinary type I collagen the hydroxylation of the lysyl residues which appear in the hydroxylatable Y-position of the repeating X-Y-Gly sequence of collagen polypeptides is incomplete, and the content of hydroxylysyl residues in type I collagen can be markedly increased by incubating denatured a-chains with purified lysyl hydroxylase (28). On the basis of these

378

JOUNI

observations it is possible that the increased hydroxylysine content of type I-trimer collagen may be a result of slower folding of three al(I)-chains into the triple-helical conformation. Since the association of three pro-a-chains through their noncollagenous extension peptides is a prerequisite for rapid intracellular helix formation [see (29)], such a slow folding of the polypeptides might reflect imperfect or delayed recognition and association of the extension peptides when three identical pro-al(I)-chains are involved in the synthesis of a collagen molecule. The slower folding of the molecule into the triple helix would likewise explain the increased content of 3-hydroxyproline, since this amino acid is also introduced into the collagen polypeptides by post-translational hydroxylation and its synthesis is limited by triple-helix formation (30). On the other hand, the relatively normal content of 4-hydroxyproline in the [~yl(I)]~ molecule can be explained by the fact that the hydroxylation of susceptible prolyl residues even in normal type I collagen al(I)-chains is nearly maximal (29), and therefore, slower folding of the polypeptides would not appreciably increase the 4-hydroxyproline content of the chains. Even though genetically distinct types of collagen have been isolated in various connective tissues, their physiologic significance is currently unknown (2). Several lines of evidence suggest, however, that certain chemical features of the collagen molecule may play a significant role in the stability of collagen fibers formed in the extracellular space. For example, the presence of hydroxylysine in the molecule is of special importance in stabilizing the intermolecular collagen crosslinks (31-33). On this basis it is possible that the small but significant amount of al(I)-trimer collagen, which demonstrates an increased hydroxylysine content, might play a role in the stability and tensile strength of normal human skin and other connective tissues. Defects in the biochemistry of this particular type of collagen may then be associated with connective tissue diseases, as has been demonstrated in the case of other genetically distinct collagens (3).

UITTO ACKNOWLEDGMENTS The author gratefully acknowledges the expert technical assistance of Ruth Allan and Kathy Polak. Kenneth Henderson and Greg Grant helped in performing the amino acid analyses.

REFERENCES 1. UITTO, J. (1978) Cl&. Res. 26, 301A. 2. MILLER, E. J. (1976) Mol. Gel/. Biochem. 13, 165-192. 3. UITTO, J., AND LICHTENSTEIN, J. R. (1976) J. Invest. Dermatol. 66, 59-79. 4. EPSTEIN, E. H., JR. (1974) J. Biol. Chem. 249, 3225-3231. 5. BURGESON, R. E., ELADLI, F. A., KAITILA, I., AND HOLLISTER, D. W. (1976) Proc. Nat. Acad. Sci. USA 73, 2579-2583. 6. BORNSTEIN, P.. AND ASH, J. F. (1977) Proc. Nat. Acad. Sci. USA 74, 2480-2484. 7. RHODES, R. K., AND MILLER, E. J. (1978) Biochemistry 17, 3442-3448. 8. MAYNE, R., VAIL, M. S., AND MILLER, E. J. (1975) Proc. Nat. Acad. Sci. USA 72, 4511-4515. 9. NARAYANAN, A. S., AND PAGE, R. C. (1976) J. Biol. Chem. 251, 5464-5471. 10. LITTLE, C. D., CHURCH, R. L.. MILLER, R. A., AND RUDDLE, F. K. (1977) Cell 10, 287-295. 11. BENYA, P. D., PADILLA, S. R., AND NIMNI, M. E. (1977) Biochemistry 16, 865-872. 12. MORO, L., AND SMITH, B. D. (1977) Arch. Biochem. Biophys. 182, 33-41. 13. MUNKSGAARD, E. C., RHODES, M., MAYNE, R., AND BUTLER, W. T. (1978) Eur. J. Biochem. 82, 609-617. 14. PIEZ, K. A. (1968) Anal. Biochem. 26, 305-312. 15. PIEZ, K. A., EIGNER, E. A., AND LEWIS, M. S. (1963) Biochemistry 2, 58-66. 16. UITTO, J., LICHTENSTEIN, J. R., AND BAUER, E. A. (1976) Biochemistry 15, 4935-4942. 17. EPSTEIN, E. H., JR., SCOTT, R. D., MILLER, E. J., AND PIEZ, K. A. (1971) J. Biol. Chem. 246, 1718-1724. 18. KING, J., AND LAEMMLI, U. K. (1971)J. Mol. Biol. 62, 465-477. 19. STUDIER, F. W. (1973) J. Mol. Biol. 79, 237-248. 20. KIVIRIKKO, K. I., LAITINEN, O., AND PROCKOP, D. J. (1967) Anal. Biochem. 19, 249-255. 21. CHUNG, E., AND MILLER, E. J. (1974) Science 183, 1200-1201. 22. EPSTEIN, E. H., JR., AND MUNDERLOH, N. H. (1978) J. Biol. Chem. 253, 1336-1337. 23. JIMENEZ, S. A., BASHEY, R. I., BENDITT, M., AND YANKOWSKI, R. (1977) Bioehem. Biophys. Res. Commun. 78, 1354-1361.

TYPE

I-TRIMER

COLLAGEN

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