Increased protocollagen proline hydroxylase activity in synovial tissue in rheumatoid arthritis

741

CLINICA CHIMICA ACTA

INCREASED IN SYNOVIAL

JOUNI

UITTO,

PROTOCOLLAGEN TISSUE

SEPPO

PROLINE

IN RHEUMATOID

LINDY,

PENTTI

Department of Medical Chemistry, University tology, University Central Hospital, Helsinki, Hospital, Heinola (Finland) (Received

HYDROXYLASE

ACTIVITY

ARTHRITIS

ROKKANEN

AND

KAUKO

VAIN10

of Helsinki, Department of Orthopaedics and Traumaand Department of Surgery, Rheumatism Foundation

June 30. 1970)

SUMMARY

Protocollagen proline hydroxylase activity was assayed in biopsy specimens of human synovial tissue. The enzyme required ascorbate, a-ketoglutarate, ferrous iron and molecular oxygen for its activity. The activity, determined from synovial tissue of 18 patients with rheumatoid arthritis, was increased as compared with values obtained

from control

patients.

INTRODUCTION

Protocollagen proline hydroxylase participates in the biosynthesis of collagen by hydroxylating proline in protocollagen, the proline-rich and hydroxyprolinedeficient polypeptide precursor of collagen. The hydroxylation of proline to collagen hydroxyproline by partially purified protocollagen proline hydroxylase has been shown to require ascorbate, a-ketoglutarate, ferrous iron and molecular oxygen (see for review ref. I). This enzyme activity has been demonstrated in various animal and lately its activity has been shown to be present in human skiney7, tissuesz-6 liver* and serums. Recent studies have suggested that the biosynthesis of collagen may be increased in patients with various connective tissue disorderslo(see for review, ref. 14). In the present study, the activity of protocollagen proline hydroxylase was determined in synovial tissue from patients with rheumatoid arthritis, and the values were compared with those of normal synovial tissue. In addition, the characteristics of protocollagen proline hydroxylase were investigated by determining its cofactor requirements. MATERIAL

AND METHODS

Biopsy specimens of synovial tissue were taken in connection with synovectomy from 18 patients suffering from definite rheumatoid arthritis. Control samples Clin. Chim. Acta, 30

(1970)

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UITTO et

742

al.

from patients having no rheumatic disease were taken in connection with traumatological and orthopaedic operations on the knee. Tissue specimens were minced with scissors for IO min in cold (o”) 0.1 M KC1 and 0.02 M Tris-HCl buffer, pH 7.8 at 24”, I ml per IOOmg of tissue, and then homogenized five times for 5 set at o0 with an Ultra-Turrax homogenizer. The homogenates were centrifuged at 15000 xg for 30 min at 4’, and portions of the supernatants were incubated with [14C]proline-labelled protocollagen substrateI (50000 disint.~min), 50 mM Tris-HCl buffer (pH 7.8 at 24’), z mM ascorbate, 0.5 mM cr-ketoglutarate, 0.08 mM FeSO,, and 0.05 mg/ml catalase (Sigma), in a final volume of 4.0 ml (refs. 15, 16). After 60 min incubation at 37’, an equal volume of concentrated HCI was added and the samples were hydrolyzed for 6 h at 136”. Thereafter, the total radioactivity’? and hydroxy-[l*C] proline18 were determined in the hydrolyzates. Another aliquot of the 15000xg supernatant of the tissue homogenate was hydrolyzed for 6 h at 136” and then assayed for a-amino nitrogen19. The protein content was calculated by multiplying the values of a-amino nitrogen by a factor of 6.8. The activity of protocollagen proline hydroxylase was expressed as disint./min hydroxy-[l*C]proline synthesized in an aliquot of 50 ooo disint./min [l*C]proline-labelled protocollagen substrate per mg 15000 xg supematant protein. The results were evaluated statistically, using Student’s t-test, and the differences were regarded as significant at the level of p < 0.05. RESULTS

The activity of protocollagen proline hydroxylase in human synovial tissue was studied by incubating aliquots of the 15 ooo x g supematant of the tissue homogenates with biologically prepared ~l~C~proline-labelled protocollagen substrate.

v

0

Fig. I. Assay indicate the protocollagen genate as the

I

1.0

t

2.0

PROTEIN (mg) of protocollagen proline hydroxylase activity in human synovial tissue. The values synthesis of hydroxy-[%]proline with 50000 disint./min of [l*C]proline-labelled as substrate and varying amounts of the rsooo x g supernatant of the tissue homosource of the enzyme.

C&n. Claim. Acta, 30 (1970) 741-744

743

PROTOCOLLAGEN PROLINE HYDROXYLASE IN RHEUMATOID ARTHRITIS

When varied amounts of the 15 ooo xg supernatant were added to the incubation system, it was found that, up to 0.5 mg, the amount of hydroxy-[14C]proline synthesized was dependent on the amount of supernatant protein added. However, if more protein was used for enzyme determination, the synthesis of radioactive hydroxyproline deviated markedly from linearity (Fig. I.) Therefore, subsequent determinations of protocollagen proline hydroxylase activity in human synovial tissue were carried out with 0.2 to 0.5 mg of the 15000 xg supematant protein. TABLE

I

COFACTORREQUIREMENTS

OF

PROTOCOLLAGEN

PROLINE

HYDROXYLASE

FROM

HUMAN

SYNOVIAL

TISSUE

Incubation system

Hydroxy-[W]proline formed (disint. /min)

Per cent of complete system

Complete Minus ascorbate Minus g-ketoglutarate Minus ferrous iron Minus all cofactors Minus oxygen

1427

IO0

20

I

65 203 34

5 ‘4 2

120

a

300 pg of the I 5 ooo x g supernatant protein of the synovial tissue homogenate was incubated [iK]proline-labelled protocollagen substrate (50000 disint./min) as described in MATERIAL METHODS, and the amount of hydroxy-[Klproline formed was assayed.

with AND

Protocollagen proline hydroxylase purified from animal sources has been shown to require ascorbate, cr-ketoglutarate, ferrous iron and molecular oxygen for its activity. The enzyme in human synovial tissue required the same cofactors, and omission of any of these substances resulted in very low activities (Table I). TABLE

II

PROTOCOLLAGEN

PROLINE

Group Controls Rheumatoid

arthritis

HYDROXYLASE

ACTIVITY

IN

RHEUMATOID

No. of patients

Protocollagen proline hydroxylase activity*

IO

1502 f 37’ 3127 zt 2571

18

proline

hydroxylase

activity

TISSUE

P

<0.05

* The values are expressed as disint. /min hydroxy- [WJproline [i%]proline labelled protocollagen substrate per mg 15000 xg homogenate (mean & S.D.). Protocollagen

SYNOVIAL

synthesised per 50000 disint. /min supernatant protein of the tissue

was determined

in synovial

tissue

18 patients suffering from rheumatoid arthritis. The activity of this enzyme was increased in rheumatoid synovial tissue about z-fold as compared to the values obtained from control subjects (Table II). from

DISCUSSION

Using radioactive isotope incorporation methods, some authors have demonstrated that the biosynthesis of collagen is increased in the skin of some patients with rheumatoid arthritislOand other disorders affecting connective tissue 11-13+. In addition, the activity of protocollagen proline hydroxylase has been shown to be high in the skin of patients with so-called collagen diseases as compared with that of healthy contro1s6~7.In the present study, protocollagen proline hydroxylase activity was C&Z.Chim. Acta,

30 (1970)

741-744

744

UITTO et

al.

increased in synovial tissue taken from patients with rheumatoid arthritis, as compared with control values. Further, the enzyme in human synovial tissue was found to be similar to that extracted from animal sources, in that it had the same cofactor requirements. Protocollagen proline hydroxylase performs the hydroxylation of peptide-bound proline to collagen hydroxyproline, and its activity has been shown to be high in tissues where active synthesis of collagen occurs, e.g. in embryonic21*az and granulomatous22+23tissues. In addition, in granuloma tissue its activity has been shown to parallel the rate of collagen formation 23. Therefore, increased protocollagen proline hydroxylase activity in rheumatoid synovial tissue may be a reflection of increased synthesis of hydroxyproline. Thus, the results seem to suggest that increased collagen formation in the synovial tissue may form an essential part of the disease process in rheumatoid arthritis. ACKNOWLEDGEMENTS

This study was supported by a grant from the National Research Council for Medical Sciences, Finland. REFERENCES I J. ROSENBLOOM AND D. J. PROCKOP, in J. E. DUNPHY AND W. VAN WINKLE (Eds.), Repair and Regeneration: The Scienti$c Basis ofSurgical Practice, McGraw-Hill Book Co Inc., New York, 1968, p&3. 117-135. z J. J. HUTTON AND S. UDENFRIEND, Proc. Natl. Acad. Sci. U.S., 56 (1966) 198. 3 R. 0. LANGNER AND G. C. FULLER, B~o~hem. Biophys. Res. Commun., 36 (1969) 559. 4 J. HALME AND M. J~AsKEL~INEN~.B~oc~~~. J., 116 (1970) 367. 5 !;;ALME, J. UITTO, K. KAHANPAA, P. KARHUNEW AND S. LINDY, J. Lab. C&z. Med., 75 (1g70) P. PELTOKALLIO AND K. I. KIVIRIKKO, Stand. J. Clin. Lab. Invest., 23 (1969) 241. 7 J. UITTO, M. HANNUKSELA AND 0. RASMUSSEN, Alzn. C&n. Res.. z (1970) 235. 8 T. TAKEUCHI AND D. J. PROCKOP, Gastroenterology, 56 (1969) 744. 9 H. D. STEIN, H. R. KEISER AND A. SJOERDSMA. Lancet, i (1970) 106. rc U. GERLACH, Z. Ges. Ezptt. Med., 139 (1965) 542, XI 0. LAITINEN, J. UITTO, M. HANNUKSELA AND K. K. MUSTAKALLIO, Ann. Clin. Res., I (1969) 63. 12 H. R. KEISER AND A. SJOERDSMA, Clin. Chim. Acta, 23 (1969) 341. 13 J. UITTO, P. HELIN, 0. RASMUSSEN AND I. LORENZEN, Ann. Clin. Res., 2 (1970) 228. 14 J. UITTO, Collagen Biosynthesis in Human Skin in vitro. A Biochemical Study on Patients with Connective Tissue Disorders and on the Eflekct of D-Penicillamine, Academic Dissertation, Universitv of Helsinki, Helsinki. 1970. 15 K.-I. KIVIRXK~ AND D.. J.-PROCKOP, J. Biol. Chem., 242 (1967) 4007. 26 K. I. KIVIRIKKO AND D. J. PROCKOP, Proc. NatE. Acad. Sci. U.S., 57 (1967) 782. 17 D. J. PROCKOP AND P. S. EBERT, A%aE. Biochem., 6 (1~63) 263. 18 K. TUVA AND D. 1. PROCKOP, Anal. Biochem., IS (1~66) 77. 19 H. k. RUBIN~~I& AND D. J. PRYCE, J. Clin. P&&l., ii i1959) 80. 20 0. LAITINEN, J. UITTO, M. IIVANAINEN, M. HANNUKSELA AND K. I. KIVIRIKKO, C&z. Chim. Acta, 21 (1968) 321. 21 K. JUVA AND E. HEIKKINEN, Abstr. 5th Meeting Federation Europ. Biochem. Sot., Prague, 1968, p. 141. 22 E. MUSSINI, J. J. HUTTON AND S. UDENFRIED, Scienca, 157 (1~68) 927. 23 K. JUVA, Acta PhysioE. &and., Suppr. 308 (1968) 56. 6 J. UITTO, J. HALME, M. HANNUKSELA,

C&Z. Chim. Acta, 30 (1970) 741-744