70 (1976) 61-69 Publishing Company,
in The Netherlands
M. ADAM, R. VITASEK,
Z. DEYL, G. FELSCH,
and Z. OLSOVSKA
Research Institute for Rheumatic Diseases, and Physiological Institute, Academy of Science, Prague (Czechoslovakia) and Medical Clinic, Friedrich-Schiller-University, Jena (G.D.R.)
Summary It has been proved that collagen from rheumatoid patients contains a distinctly higher proportion of insoluble fraction resistant to heat solubilization. In accordance with this fact is the finding that rheumatoid skin and nodules contain a higher proportion of collagen type III.
Introduction In recent year the interest of rheumatologists has been concerned with collagen, a protein present almost everywhere in the organism. Klemperer et al. [l] introduced the term “collagen diseases” during their study of lupus erythematodes generalisatus pathology, because they supposed that collagen is the part of the connective tissue which is primarily involved. The term “collagen diseases” was then broadened to include other rheumatic inflammatory diseases as well. Despite various approaches to clarify the biochemical background of these diseases, besides general agreement about the pathogenetic mechanism of these disorders, the knowledge is rather fragmentary. It has been stated by Uitto et al.  that the activity of the protocollagen-proline hydroxylase in rheumatoid synovial tissue is definitely increased and, in parallel, that the proportion of hydroxyproline in the nondialysable fraction of this tissue also increases. Therefore, they suggest increased collagen biosynthesis in rheumatoid synovial tissue. On the other hand, a more detailed biochemical knowledge of collagen structure formed under these conditions is rather scanty. However, an increased collagenolytic activity in rheumatoid synovial tissue was reported by Harris et al. . In combination with the results of Uitto et al.  this latter fact helps in the understanding of the increased levels of urinary hydroxyproline in these diseases [ 41. The present study was devoted to collecting data about the solubility of collagen obtained from skin and synovial tissue of rheumatoid patients and in its
second stage to the partial ch~a~~rization which is markedly increased in the disease.
of an insoluble
Material and methods Sk& ~~~~es for solubility studies were obtained either during surgical operations in controls or from the arm in patients with rheumatoid arthritis, for the more detailed studies they were obtained at necropsy. Rheumatoid nodules were localized in the elbow region and removed under local anesthesia. The diagnosis of rheumatoid arthritis was made according to the A.R.A. criteria,
Skin specimens or synovial tissue were freed from subaqueous tissue or fat and then extracted sequentially with NaCl/phosphate buffer (pH 7.4,10.5) containing 28.1 g NaCl, 1.96 g Na2HP04 . 12 Hz0 and 0.16 g KH2P04 per litre (neutral salt-soluble collagen, NSC), citrate buffer (pH 3.7) containing 10.9 g citric acid monohydrate, 100 ml 1.0 M NaOH and 50 ml 1.0 M HCl per litre (acid-soluble collagen, ASC). The residue was subjected to lyotropic solubilization by heating in 0.05 M acetic acid to 40°C for 30 min (insoluble collagen solubilized under denaturation conditions, GSC&) as described in detail elsewhere [ 5,6]. The amount of collagen in each fraction was calculated from the hydroxyproline content using correction factors with respect to different hydroxyproline content in both collagen types (7.46 for type I and 5.5 for type III). Rheumatoid nodules were freed from fat and collagen type III was extracted by the use of pepsin according to the method of Chung and Miller [‘?I in the modification of Fujii and Kiihn [Zl], We compared the result with the collagen type 111 content of normal lungs, bron~hophe~monia lungs, rat skin of different ages and polyureth~-sub~u~neous granuloma in rats. Preparation of thiosem$wrbazide-solubilized fraction (TSC) Skin specimens were extracted at first according to Rubin et al. . In addition to the acid-soluble fraction, thiosemic~bazide-solubil~zed, insoluble collagen was also subjected to further investigation during this study. Depolymerization of insoluble collagen was carried out according to Rosmus et al. [9 f : 10 g of insoluble collagen were washed with water and suspended in 20 ml acetate buffer (pH 5.6) containing 4 X 8 ml 0.2 M acetic acid and 45 X 2 ml 0.2 M sodium acetate per litre. After 12 h the supernatant was centrifuged and another portion of 20 ml acetate buffer was added together with 0.35 g of thiosemicarbazide. The reaction mixture was shaken for 24 h at room temperature and all low molecular weight compounds were removed by dialysis against tap water (72 h), The pH of the high molecular weight fraction was adjusted to 10.0 by the addition of 0.4 M potassium bicarbonate. The mixture was kept standing for 5 h at 65*C. The insoluble residue was removed by centrifugation at 5000 rev./min for 30 min. The supernatant was collected and dialysed against water to neutral pH and lyoph~ized. C~romutograp~y on Sepharose 4-B was carried out according to Zimmer-
mann et al. [lo]. Column temperature was maintained at 25°C and Sepharose gel was equilibrated with 1.0 M CaClz containing 0.1 M sodium acetate and 0.02% sodium azide.‘The same buffer was used as the mobile phase. Column size was 2.5 cm X 60 cm. Collagen samples were dissolved in the starting buffer and heated for 30 min at 40°C; about 25 mg of collagen in lo-15 ml of starting buffer were applied to the column. The flow rate was 6 ml/h and 3-ml fractions were evaluated by absorbance measurement at 230 nm. CM-ceklose chromatography followed the original method of Piez et al. [ll]. A column 2.5 cm X 15 cm, thermostated at 4O”C, with a flow rate of 80 ml/h was used. The effluent was monitored on a Pye-Unicam spectrophotometer of 230 nm using a micro flow-through cuvette. DEAE-cellulose chromatography of fraction I from the Sepharose column was done in order to remove proteoglycans from polymerized collagen. The actual separation followed the technique of Miller 1121, Po~yacrylamide gel electrophoresis was done using &&nine buffer (pH 4.3) P31. Other analytical procedures Amino acid analysis was done on a laboratory
assembled amino acid analyser, the main parts of which were from Technicon. ~yd~xyproZine was assayed according to Stegem~n . The presence of neutral sugars was determined by using the orcin reagent of FranCois et al. u51. Results Investigation of the collagen solubility in skin samples from rheumatoid patients compared to controls revealed a higher resistance of patholo~c~ly affected collagen towards heat solubilization (Fig. 1). A similar phenomenon was
Fig. 1. &Ability
of skin collagen in patients with rheumatoid arthritis.
COLLAGEN HUMAN NSC,
insoluble residue after heating.
.._... NO. of cases
Percentage of total collagen ________-_____I____-. NSC
tmgil g wet tissue) _______.__I____
.~. _ -._~~_-~~~.~~.-__..-.
.- _-_ --
Rheumatoid synovlal tissue
___ .. .
observed in rheumatoid synovial tissue as indicated by the results in Table I and Fig. 2. Similarly, as observed in other pathological situations, the most distinct differences were observed in the insoluble fraction, susceptible to heat solubiiization, from this residue. The dispersion of results is rather high, which is probably due to age differences of the granulation tissues investigated. These introductory findings stimulated a further effort to elucidate the nature of the MEUTRAL
fSOIUIIiLIZfD w 40°C 3omm
Fig. 2. Solubility of collagen from rheumatoid synovial tissue.
120 180 240
EFFLUENT Fig. 3. Chromatographic profiles of ASC and TSC from skin on Sepharose 4-B columns. For technical details see Methods. (a) ASC, controls;(b) TSC, controls; (c) ASC, rheumatoid arthritis: (d) TSC. rheumatoid arthritis. Note the increased occurrence of the high molecular weight fraction in arthritic patients.
differences between collagen from rheumatoid arthritis and controls. We characterized collagen from rheumatoid patients and controls by chromatographic separation on a Sepharose 4-B column (Fig. 3). Practically no difference was observed in the amounts of fractions II and III in both the investi-
FIWZTIOW I. FR0N-I N!&y pdvwm
Fig. 4. Polyacrylamide
gel tracings of fractions separated
160 180 200 280 300
Fig. 5. Chromatographic separation of high molecular weight fraction I from a Sepharose 4-B column on DEAE-ceIlulose. The faster-moving fraction represents coltagen. The other peak consists of proteinpolysaccharide complexes.
gated collagens. According to polyacrylamide gel electrophoresis, fraction II was composed mostly of p-fraction and higher polymers besides a minor proportion of a-fraction (Fig. 4). Fraction III was formed mostly of single 01chains. Rheumatoid collagen contained, however, in comparison with controls, an evidently increased amount of high molecular weight fraction I in both ASC and IS&,.
$2 IO- a N
ca,$OI, a (1)
06 0” f
250 300 350
Fig. this toid was
6. CM-cellulose chromatography of the fast fraction from DEAE-cellulose separation (Fig. 5) after particular fraction has been subjected to pepsin treatment. Skin samples: (a) controls: (b) rheumaarthritis. Note the increased proportion of the intermediate peak between al(I) and ~yz(I) which tentatively designated as [arl (III)] 3.
AMINO ACID COMPOSITION OF SKIN 4-B CHROMATOGRAPHY (See Fig. 3) Amino
Hydroxyproline Aspartic acid Threonine Swine* Glutamic acid Proline Glycine Alanine VaIine Me thionine * * Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Lysine Histidine* Arginine Cyst&w* * Extrapolated ** No correction
of residues per 1000 residues per collagen fraction ~~
108 43 20 38 18 89 338 110 23 3 12 30 3 18 18 25 8 35 0
108 44 20 36 75 87 332 100 23 3 12 30 3 16 8 46 9 46 0
101 50 20 36 76 81 330 100 24 3 12 29 3 16 10 43 10 49 0
to zero time of hydrolysis. for oxidation was introduced.
While fraction III of ASC and of TSC, as shown by the amino acid analysis, represents practically pure collagen (Table II), the high molecular weight fraction I was very rich in carbohydrates; rheumatoid TSC fraction I contained 6.0 percent of neutral sugars (calculated as glucose) whereas control TSC,fraction I only contained 0.4 percent of sugars. Therefore, we subjected the rheumatoid TSC fraction I to DEAE-cellulose chromatography which separated collagen and protein-polysaccharides complexes (Fig. 5). The collagenous fraction was afterwards digested by pepsin as described for collagen type III isolation and subjected to CM-cellulose chromatography when it revealed a high peak beTABLE
III IN VARIOUS
Ratio type I Skin, rat. 6 months old Skin, hamster, 8 months old Skin, rat, 11 months old Lung. human, 61 years old Lung bronchopheumonia, 71 years old Sponge granuloma Rheumatoid nodules
1.75 2.03 2.91 2.67 0.63 0.29 0.71
TO ITS PRESENCE
tween peaks of the ei- and e2-fractions (Fig. 6). According to Epstein 1161, this peak is formed by [cY~(III)]~. In comparison with controls the proportion of this peak was distinctly increased in rheumatoid skin, This latter result agrees well with the direct fractionation of collagen from rheumatoid nodules, in which an increase of collagen type III was also established (Table III). Discussion In general it can be concluded that the solubility of collagen from rheumatoid patients is altered. The proportion of insoluble collagen withstanding solubilization by heating is distinctly higher in samples originating from rheumatoid tissues. This tendency is noticeable both in synovial tissue and in skin. From this point of view it was worth further investigation to elucidate the basic chemical properties of rheumatoid collagen. Because of the paucity of synovial tissue or nodule material this part of our investigation was carried out on skin samples. The results obtained by Sepharose 4-B chromatography indicated the presence of a considerable amount of a high molecular weight fraction in rheumatoid collagen which is in good agreement with the overall balance studies. The low molecular weight fraction of rheumatoid collagen exhibits an amino acid composition which is in reasonable agreement with the controls. In view of the recent investigations of Chung and Miller  and Trellstad et al. [ 171, it is necessary to bear in mind the fact that our TSC samples were obviously a mixture of at least two types of collagen, namely type I and III. The increased proportion of high molecular weight fraction I in rheumatoid collagen might suggest a shift between collagen type I and III in favour of the latter. Collagen [a,(III)] 3 molecules in comparison with cartilaginous [al(D)] 3 collagen molecules have a more ubiquitous distribution. Miller et al. [ 181 found collagen type III in dermis, major vessels and leiomyoma and it is very probable that type III is present in various tissues together with type I. The ratio of type I to III varies with age. A preponderance of type III in fetal tissues and a relative paucity of this type of collagen in adult tissue suggest its importance in facilitating growth and remodelling of connective tissue. Bailey et al. 1191, on the other hand, claimed that a stable keto-cross link is formed in rheumatoid arthritis. Our own results obtained with CM-cellulose chromatography of the pepsin digest of TSC fraction I indicate that the proportion of collagen type III in rheumatoid skin is definitely increased. This finding is supported by the ratio between collagen types I and III in rheumatoid nodules, which was about 3 : 4, whereas in normal healthy tissue it was about 3 : 1. Thus, the preponderance of the insoluble collagen type III can account for the low solubility of rheumatoid arthritis collagen. Certainly it cannot be excluded that another type of collagen is present in inflamed tissues. Concomitantly in rheumatoid arthritis the level of active tissue collagenase is increased. According to Harris et al.  both types of collagen are broken down by tissue collagenase at the same rate but our results showed a greater resistance of type III to pepsin digestion. Currently it is hard to say what relation the increased collagen catabolism bears to the increased concentration of collagen type III. The role of collagen type III is still unknown. Results of Wood
 showed the importance of cross-linking for the fibril formation. It could be, therefore, anticipated that collagen type III containing disulfide bridges acts as an aggregation center in fibrogenesis. On the other hand, the higher degree of cross-linking causes a reduced swelling of the collagen structure, which indisputably alters the transport ability of connective tissue and which may affect further fibroblast metabolism. Replacement of one type of collagen by another involves the regulation and control of collagen synthesis in the sense that collagen formation proceeds from different genetic loci. The failure to regulate correctly collagen synthesis in various tissues could be responsible for different connective tissue disorders manifesting various clinical signs. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Klemperer, P.. Pollaek. A.D. and Bachr. G. (1941) Arch. Pathol. 32, 569 Uitto, J., Lindy, S.. Turto. H. and Vainio, K. (1972) J. Lab. Clin. Med. 19. 960 Harris Jr., E.D., Di Bona. D.R. and Krane, SM. (1969) J. Clin. Invest. 48, 2104 Kivirikko, K.I. (1970) Int. Rev. Connect. Tissue Res. 5. 93 Adam, M., Fietzek. P. and Kuhn. K. (1968) Eur. J. Biochem. 3, 411 Deyl, 2.. Juficova. M.. Rosmus, J. and Adam. M. (1971) Exp. Gerontol. 6, 383 Chung. E. and Miller, E.J. (1974) Science 183, 1200 Rubin, A.L.. Drake, M.P., Davison. P.Z.. Pfahl, D., Speakman. P.I. and Schmitt. F.O. (1965) chemistry 4. 181 Rosmus. J.. VanEfkova. 0. and Deyl, Z. (1972) KoZdstvi 22, 246 Zimmermann. B.K., Pikkarainen. J.. Fietzek. P.P. and Kuhn. K. (1970) Eur. J. Biochem. 16, 217 Pier. K.A.. Eigner, E.A. and Lewis, M.S. (1963) Biochemistry 2. 58 Miller, ES. (1971) Biochemistry lo,1652 Stark, M. and Jfiihn, K. (1968) Eur. J. Biochem. 6, 584 Stegemann, H. (1958) Z. Physioi. Chem. 311.41 Francois, C.. Marshal, R.D. and Neuberger. A. (1962) Biochem. J. 83.335 Epstein Jr., E.H. (1974) J. Biol. Chem. 249. 3225 Trellstad. R.L.. Rizzie. K.R. and Rubin. D.F. (1974) Biochem. Biophys. Res. Commun. 57. 717 Miller, E.S., Epstein Jr., E.H. and Pier, K.A. (1971) Biochem. Biophys. Res. Commun. 42,1024 Bailey, A.J., Bazin. S. and Delauney, A. (1973) Biochim. Biophys. Acta 328,383 Wood, G.C. (1964) Int. Rev. Connect. Tissue Res. 2, 1 Fujii. T. and Kuhn. K. (1975) Hoppe Seyler’s Z. Physiol. Chem. 356.1793