101

Biochimica et Biophysica Acta, 399 (1975) 101--112 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27691

r i lE CHARACTERISATION AND FUNCTION OF THE POLYSACCHARIDASES OF HUMAN SYNOVIAL FLUID IN RHEUMATOID AND OSTEOARTHRITIS

R.W. STEPHENS, P. GHOSH and T.K.F. TAYLOR

Raymond Purves Research Laboratories, (University of Sydney) Royal North Shore Hospital, St. Leonards, N.S. W 2065 (Australia)

(Received December 23rd, 1974)

Summary

A potential enzymic mechanism for the degradation of glycosaminoglycans was characterised using enzymes found in rheumatoid synovial fluid from the knee joint. This mechanism involves a true hyaluronidase together with the concerted action of fi-glucuronidase and fi-N-acetylhexosaminidase. The contri- but ion of the exopolysaccharidases to hyaluronate degradation was demon- strated by the use of specific inhibitors, while the distinct identi ty of a true hyaluronidase was shown by ammonium sulphate and agarose gel column frac- tionations. Only the hyaluronidase fraction was capable of degrading high mo- lecular weight hyaluronate.

The exopolysaccharidase activities were shown to be markedly elevated in rheumatoid as compared to osteoarthritic synovial fluid and also normal serum. On the other hand, hyaluronidase was similarly active in rheumatoid and osteo- arthritic synovial fluids; both these levels were lower than that of normal human serum. Hyaluronidase in synovial fluid may thus be derived by diffusion from serum, since it is of relatively low molecular weight (60 000).

The pH requirements of this enzyme system and the strong inhibition of hyaluronidase by synovial fluid make it unlikely that the mechanism operates extracellularly. It is proposed that as a lysosomal mechanism, however, it is an important contributing factor in the chronic erosion process characteristic of rheumatoid arthritis.

Int roduct ion

The mechanisms by which the polysaccharide components of articulm cartilage and synovial fluid are catabolized in health and disease are largely un- known.

Earlier work on the catabolism of the macromolecular polysaccharides oI

102

diarthrodial joints has mainly centred on the hyaluronic acid-protein complex of synovial fluid. Ragan and Meyer [1] observed the decreased viscosity of rheumatoid synovial fluid, and this led to the postulate that an endopolysac- charidase, similar to the hyaluronidases of bovine testicular extracts [2] and human serum [3] , was present. Such a hyaluronidase was further supported by Bollet et al. [4] , bu t later contested by Caygill and Ali [5].

The exopolysaccharidases, fl-glucuronidase and fl-N-acetylglucosaminidase, have been noted to be increased in rheumatoid fluids [6,7] , but their possible role in the catabolism of high molecular weight hyaluronate or chondroitin sulphate has not been shown experimentally.

Polysaccharidases may be implicated in cartilage erosion in rheumatoid [8] and osteoarthritis [9] , Recently Muir and co-workers [10] have proposed that a proport ion of cartilage proteoglycans exist as aggregates bound to hyaluro- nate chains to which a protein-link component is also bound. Depolymerisation of chondroit in sulphate and hyaluronate by enzymes capable of penetrating the matrix could thus lead to loss of tissue resilience, and the production of carti- lage lesions characteristic of rheumatoid arthritis.

This paper reports the characterisation of a true hyaluronidase activity in osteoarthritic and rheumatoid synovial fluid, and an evaluation of the role of this activity and exopolysaccharidases in bringing about destruction of high molecular weight hyaluronate and chondroitin sulphate.

Materials and methods

Fresh synovial fluid was obtained from knee joints classified as rheuma- toid or osteoarthritic according to Ropes et al. [11] . Cells were removed by centrifugation at 4000 X g for 10 min and the supernatant stored frozen. Loss of enzyme activity due to storage was found to be insignificant.

Enzyme su bstrates and inhibitors p-Nitrophenyl-2-acetamido-2-deoxyglucopyranoside and p-nitrophenyl-

2-acetamido-2-deoxygalactopyranoside for fl-N-acetylhexosaminidase assays, and phenolphthalein glucuronide for fl-glucuronidase assays were obtained from Sigma Chemicals, St. Louis, Mo. Human umbilical cord sodium hyaluronate type IIIS and shark and whale cartilage chondroit in sulphates (mixed isomers) type IIIS were also purchased from Sigma for hyaluronidase assays. Saccharo- 1,4-1actone was obtained from Calbiochem (Aust.), Sydney. Beaded agarose gel of exclusion limit 500 000 mol. wt was purchased from Bio-Rad Laboratories, Richmond, Calif.

[J-Glucuronidase assay Synovial fluid (0.2 ml) was incubated at 37°C for 2 h (unless otherwise

specified) with 50 pl of 10 mM glucuronide in a final volume of 1.0 ml made up with 0.1 M formate buffer pH 4.0. A glycine stopping-buffer (pH 10.7, 3.0 ml) was then added, the mixture centrifuged at 4000 X g for 10 min, and the supernatant absorption then measured at 540 nm in a Varian 6350 spectro- photometer . Production of phenolphthalein was linearly time-related for at least 8 h of incubation. Varying the amount of synovial fluid in the assay

103

DILUTION OF ~-GLUCURONIDASE ACTIVITY

IN RHEUMATOID SYNOVIAL FLUIDS

0.15-

0-10-

A

0"05-

Fluid 1

Fluid 2

i 50 150 250

pl SF

Fig. 1. Synov ia l f luid ( S F ) f r o m t w o r h e u m a t o i d pat i ent s was di luted wi th assay b u f f e r to a c o n s t a n t f inal v o l u m e o f 1 .0 ml. Pheno lphtha le in p r o d u c t i o n was m e a s u r e d in a t w o h o u r i n c u b a t i o n w i t h p h e n o l p h t h a - lein g l u cu r o n i d e .

between 10 and 200 pl showed f~.at activity fell off at higher concentrations of synovial fluid (Fig. 1), suggesting the presence of an endogenous inhibitor (cf. ref 12). When saccharo-l,4-1actone was used to completely and specifically inhibit ~-glucuronidase, this was added as an aqueous solution (40 pl, 2 mM) to give a final concentrat ion of 80 pM.

{J-N-acetylglucosaminidase and fl-N-acetylgalactosaminidase assays Synovial fluid (0.2 ml) was incubated at 37°C for 30 min with 50 pl of 10

mM (37 °C) p-nitrophenylglycoside in a final volume of 1.0 ml made up with 0.1 M formate pH 4.0. The reaction was stopped and the mixture centrifuged as above. The absorption was read at 400 nm. Production of p-nitrophenol was linearly time related for at least 2 h, and was also linearly related to the amount of synovial fluid in the range I0 - -200 pl. When acetate was used as an inhibitor this replaced the formate buffer.

Hyaluronate degrading activity Synovial fluid (0.1 ml) was incubated with 200 pg of human umbilical

cord hyaluronate at 37°C for 20 h in a final volume of 0.5 ml 0.1 M formate buffer pH 4.0. N-acetylhexosamine reducing groups were then determined by the method of Bonnet and Cantey [3] , using 0.10 ml saturated potassium tetraborate solution. Production of reducing groups was approximately linear for the first 5 h (Fig. 2) but was non-linearly related to the amount of synovial fluid, falling off rapidly with high concentrations.

Results

Conditions for synovial fluid hyaluronate degradation When synovial fluid was incubated with formate buffer pH 4.0 without

104

RELEASE OF N-ACETYL GLUCOSAMINE IN

RHEUMATOID SYNOVIAL FLUID INCUBATION

NO ADDED HYALURONATE 5-

~ j N - A C E T Y L 3-

GLUCOSAMINE

RELEASED

1-

I I I

2 4 6 HOURS INCUBATION

Fig. 2. R h e u m a t o i d synov ia l f l u id (0.1 m l ) was incubated w i t h 0.1 M fo rma te pH 4.0 (0 .4 roD. N-acety l - h e x o s a m i n e r e d u c i n g ends were m e a s u r e d a f t e r the t ime in tervals ind ica ted .

the addition of hyaluronate, a time-dependent release of N-acetylhexosamine reducing groups could be detected by formation of a coloured product with p-dimethylaminobenzaldehyde (Fig. 2). This product was identifiable by its visible spectrum, which was similar to that published by Reissig et al. [ 13 ].

Column chromatography on Sephadex G100 confirmed that high molecu- lar weight hyaluronate in the synovial fluid was depolymerised to fragments of low molecular weight.

The addition of umbilical cord hyaluronate to this system resulted in a concentration-dependent increase in the release of N-acetylhexosamine (Fig. 3).

SUBSTRATE DEPENDENCE OF SYNOVIAL FLUID HYALURONIOASE

25-

~g N-ACETYL 15-

GLUCO6AMINE

RELEASED

10-

5-

I I I

~HYALURON~ ACID ADDED /~J ASSAY ~IXTURE

Fig. 3. H u m a n umbi l i ca l cord h y a l u r o n a t e was a d d e d to an i n c u b a t i o n of r h e u m a t o i d synovia l fluid in 0.1 M f o r m a t e b u f f e r p H 4.0. N - a c e t y l h e x o s a m i n e r educ ing ends were m e a s u r e d a f t e r 20 h. No te activity with e n d o g e n o u s h y a l u r o n a t e a lone.

105

DILUTION OF SYNOVtAL FLUIO H Y A L ~ ACTIVITY

22-

FORM~E

18-

. . . . . . . ~ . - 4 .r - . . ACETATE

p(:J N'ACETYL 14- /" " ,

GLUCO6AMBIE / , " " - . "~

RELEASED / • • ~ FORMATE

§ - / " / A FORMATE+NICI

i i i 1@0 I N 2 0 0

i A S'VNOVIAL FLUID

Fig . 4. D e g r a d a t i o n o f h y a l u z 6 n a t e b y v a r y i n g a m o u n t s o f s y n o v i a l f l u i d w a s m e a s u r e d i n i n c u b a t i o n s w i t h = "-, 0 .1 M f o r m a t e p H 4 . 0 ; ~ . . . . . . . e , 0 .1 M a c e t a t e p H 4 . 0 ; • -e , f o r m a t e + 1 0 m M M g 2+ a n d • . . . . . . 4 , f o r m a t e + 0 . 3 M N a C I .

This high molecular weight hyaluronate thus acted as a substrate complement- ing endogenous synovial fluid hyaluronate.

When the amount of synovial fluid in the assay was varied, the activity was non-linearly related, falling off rapidly at higher concentrations (Fig. 4). This is consistent with the presence of an endogenous inhibitor as has been described earlier in serum [14 ] . Mg 2+ is known to increase the effectiveness of this inhibitor [15 ] , and this was observed in the case of synovial fluid {Fig. 4). Other effectors o f the activity were acetate and simple increase in ionic strength by 0.3 M NaC1.

pH DEPENOENCE OF SYNOVIAL FLUID HYALURONIDASE

0.20 -

A 5 8 5 nm

0 . 1 0 -

/ i • . . . . . . m,

A - - A $O mM CITRATE

• . . . . . • O.1OM ACETATE 4" 0,15 M NaCI

i i i i

2 4 8 pH

F i g . 5. D e g r a d a t i o n o f h y a l u r o n a t e b y r h e u m a t o i d s y n o v i a l f l u i d a t v a r i o u s p H v a l u e s in • • , 50 m M citrate and s- ..... •, 0.10 M acetate + 0.15 M NaCI.

106

The pH opt imum for hyaluronate degradation in the assay was around pH 3.5, the activity being higher in citrate than in acetate (Fig. 5, cf. ref. 16). The pH requirement was thus typical of lysosomal acid hydrolases.

Assays of pathological fluids The hyaluronate-degrading activity was assayed in 32 rheumatoid and 16

osteoarthritic synovial fluids. For comparative purposes, using the same assay, the average serum activity for six healthy people was 20.2 + 1.7 pg N-acetyl- glucosamine.

Surprisingly, no significant difference could be found in the activity levels of these two groups, (Fig. 6). However, in the light of the dilution effect noted above, the levels measured may not reflect the true functional activity in vivo.

When exopolysaccharidases were measured, however, marked increases in the levels of both glucuronidase and fl-N-acetylglucosaminidase were found in rheumatoid fluids compared to osteoarthritic fluids (Figs 7 and 8). The osteo- arthritic fluid activities were comparable with those in normal serum.

Specific inhibitor studies The possible role of increased levels of ~-glucuronidase and fl-N-acetyl-

glucosaminidase in the seemingly constant presence of hyaluronidase was fur- ther investigated by the use of specific inhibitors.

Previous work has shown the specific and potent effect of saccharo-

HYALURONIDASE ACTIVITY OF ARTHRITIC SYNOVIAL FLUIDS

FREQUENCY

_

0

OSTEO ARTHRITIS

RHEUMATOID ARTHRITIS

0-1"9 2-,3"9 4-5"9 6-7-9 8-9-9 10-11'~ 'I2-13"9 '14-1:5~ '16-1,~9

V9 N-ACETYLGLUCOSAMINE RELEASED

Fig. 6. The hyaluronate-degrading activities of 32 rheumatoid and 16 osteoarthritic synoviai fluids were measured; the act iv i t ies were divided in to the s u b - g r o u p s s h o w n and p l o t t e d against the n u m b e r ot sampl e s in each s u b g r o u p .

107

' 8.

,.=

4.

- GLUCURONIDASE ACTIVITY OF

ARTHRITIC SYNOVIAL FLUIDS

~ - OsteoarthntDc

[ ] RheumatoKI

~ -N - ACET YLGLUCOSAMINIDASE

ACTIVITY OF ARTHRITIC SYNOVIAL FLUIDS

16.

12.

=es.

[ ] Osteoarthritic I m Rheumatoid

4 .

2 4 6 8 16 32 48 64 80 nmoles PP / mm / ml SF nmoles NP / rain i ml SF

Fig. 7. The f l -g lueuron idase ac t iv i t i e s o f 4 8 r h e u m a t o i d a n d 2 6 o s t e o a r t h r i t i c s y n o v i a l f lu ids we re m e a - su red ; t h e ac t iv i t i es were d iv ided i n t o the s u b - g r o u p s s h o w n a n d p l o t t e d aga in s t t he n u m b e r o f s a m p l e s in e a c h s u b - g r o u p . PP, p h e n o l p h t h a l e i n ; SF , s y n o v i a l f luid.

Fig. 8. The f l - N - a c e t y l g l u c o s a m i n i d a s e ac t iv i t i e s o f 29 r h e u m a t o i d a n d 2 0 o s t e o a r t h r i t i c s y n o v i a l f lu ids were m e a s u r e d ; t h e ac t iv i t i es we re d iv ided i n t o the s u b - g r o u p s s h o w n a n d p l o t t e d a g a i n s t t he n u m b e r o f s a m p l e s in e a c h s u b - g r o u p . NP, p - n i t r o p h e n o l .

1,4-1actone on ~-glucuronidase activity [17] . With synovial fluid fi-glucuroni- dase assays it was shown to give very significant inhibition at only 1 pM con- centrat ion (Fig. 9). At 80 pM saccharo-l,4-1actone inhibition was complete.

Acetate has previously been shown to inhibit human placental fi-N-acetyl-

INHIBITION OF SYNOVIAL FLUID I]-GLUCURONIDASE

BY SACCHARO-1,4 -LACTONE

0.15-

CONTROL

0.10 " i

.2540 n rn

0.0~- -- IpM SL

f S . . . . . . ~ . . . .

i 1 1 2 4 8

HOURS INCUBATION

F;~ _~. =; ,e ,u lpthaleLn produced b y the ac t ion o f r h e u m a t o i d synov ia l f l u i d ~-glucuronidase was measured over the t ime course shown; w i t h ( A - - . ---. - -4 ) and w i t h o u t (m i ) the add i t i on o f 20 /~1 o f 50/~M saccharo-l,4-1actone (SL) to the standard assay.

108

ACETATE INHIBITION OF COLUMN PURIFIED

13-N - ACETYLGLUCOSAIIINIDASE

~'M NITROPHENOL

~ ' - ~ FORMATE / ~ /

• ACETATE /

/ /

/

/ /

q,

/ /

/

/ . /

/ /

o . , / /

r i i 50 100 t50

pl FRACTION

Fig. 10 . Inh ib i t ion o f f l -N-ace ty lg lucosaminidase act iv i ty by 0 .1 M ace ta te . The e n z y m e used was partial ly puri f ied b y f rac t i ona t i o n o f r h e u m a t o i d synovia l fluid o n an agarose gel c o l u m n o f e x c l u s i o n l imi t 5 0 0 0 0 0 m o l wt . Act iv i ty w i t h f o r m a t e o - - . . . . o ; w i t h ace ta te = ~ .

glucosaminidase [18]. With synovial fluid assays, 0.1 M acetate moderately inhibited this activity (Fig. 10).

When either of the specific inhibitors was added to assays of hyaluronate degrading activity, this produced partial inhibition (Table I). Increasing the concentration of saccharo-l,4-1actone beyond that required for total inhibition of ~-glucuronidase did not further inhibit hyaluronate degradation. Also, if acetate was used together with saccharo-l,4-1actone, inhibition was not signifi- cantly higher than with saccharo-l,4-1actone alone.

It can be seen from Table I that when both exopolysaccharidases were strongly inhibited, the major cause of hyaluronate degradation by synovial fluid was unaffected.

T A B L E I

E F F E C T OF I N H I B I T O R S

Inhib i t ion was m e a s u r e d o n h ya lu r on id a te degradat ion , /~ -N-acety lg lucosaminidase act iv i ty and ~-glucu- rorddase act iv i ty o f r h e u m a t o i d synovia l f luid. The e x o p o l y s a c c h a r i d a s e s w e r e m e a s u r e d b y the use o f spec i f i c g l ycos i de substrates .

~ -N-acety l - ~-g lucuronidase Hyalt tronidase g lucosamin idase degradat ion

1 0 m M MgCI 2 0 0 4 8 0 .3 M NaCI 0 0 96 8 0 / ~ M s a c c h a r o - l , 4 - 1 a c t o n e 0 1 0 0 28 0 .1 M a c e t a t e 6 8 0 1 5 A c e t a t e + s a c c h a r o - l , 4 - 1 a c t o n e - - 1 0 0 31

109

AGAROSE A0-Sm PROFILE OF SYNOVIAL FLUID HYALURONIDASE

1"0- e...=..= PROTEIN ----- HYALURONIDASE

~,% e.,,.=.= • p -GLUCURONIDASE .--=" ~. 1 p'N-ACETYL

0"8- ~ ~ GLUCOSAMINIDASE / \ ~=~p-N-ACETYL

~ GALACTOSAMINIDASE

0-6.

A / i 0-4.

t .=- "o,,,,,.,,,,,*,,,,,,,,,,~...,.,,,,., ~ ~

0.2. ~ %%

FRACTION

Fig. 11. Rheumatoid synovial f luid was ~ a c t i o ~ t e d wi th ammonium sulphate at pH 4.0. The precipitate after 50% saturation was resuspended and applied to a~ agarose gel column of exclusion l im i t 500 000. The eluting buf fer was 0.1 M formate pH 4.0. ]~xopolysacchaxidases were assayed w i th specific glycoside substrates.

Fractionation of synovial fluid Ammonium sulphate fractionation of synovial fluid (5 ml) at 4°C in ci-

trate buffer (5 ml) pH 4.0 showed that fi-glucuronidase, fi-N-acetylglucosamini- dase and hyaluronate-degrading activity could be precipitated by 50% satura- tion. This resuspended precipitate (3 ml) was applied to an agarose gel column (1.6 × 60 cm) of exclusion limit 500 000 mol wt and previously equilibrated with 0.10 M formate buffer pH 4.0. All fractions were assayed for ~-glucuroni- dase, ~-N-acetylglucosaminidase, ~-N-acetylgalactosaminidase and hyaluronate- degrading activity (Fig. 11).

It was then clearly shown that fractions containing ~-glucuronidase and hexosaminidase activities were not active in degrading high molecular weight hyaluronate.

However, hyaluronate-degrading activity was associated with a more re- tarded peak (Fig. 11) which had an estimated mol. wt of 60 000. This enzyme also degraded chondroitin sulphate, though at about one-fifth the rate with hyaluronate. It was thus considered to be a true hyaluronidase, and was not inhibited by acetate saccharo-l,4-1actone or Mg 2÷, but was totally inhibited by increasing the ionic strength of the assay to 0.3 M NaC1.

Discussion

The results obtained provide evidence for a potential mechanism of gly- cosaminoglycan catabolism which utilizes a true hyaluronidase together with the concerted action of two exopolysaccharidases; ~-glucuronidase and ~-N- acetylhexosaminidase.

110

The contribution of exopolysaccharidases to hyaluronate depolymerisa- tion in this mechanism was demonstrated by the use of specific inhibitors, while the distinct identity of a true hyaluronidase capable of depolymerising high molecular weight hyaluronate and chondroitin sulphate was shown by ammonium sulphate fractionation and agarose gel permeation chromatography.

Incomplete depolymerisation of hyaluronate to an oligosaccharide mix- ture is a slow reaction, even under optimal in vitro conditions [19]. However, if exopolysaccharidases are present which can degrade these oligosaccharides, a low hyaluronidase activity may be amplified by these additional actions to give more rapid depolymerisation of hyaluronate to monosaccharides and N-acetyl- hyalobiuronic acid (Fig. 12). If oligosaccharides produced by the action of hyaluronidase can subsequently reduce its activity by a feed-back mechanism [28] then destruction of these oligosaccharides by exopolysaccharidases would remove this inhibition.

This type of three-enzyme mechanism was first proposed by Linker et al. [ 20] for the digestion of hyaluronate by crude bovine testicular extracts. These workers concluded from a study of oligosaccharides and monosaccharides

ENZYMIC DEGRADATIO~N OF HYALURONATE

HYALURONIDASE I

H H

+

~ ° GLUCURONIOASE

H H

~p - N -/K~ T Y LGLUCOSAMINIOASE

H H

A G = N ACETYLGLUCOSAMINE

GAin GLUCURONJC ACID

Fig. 12. Scheme for the mechan i sm of total degradation of hya luronate by rheumato id synovia l f lu id High m o l e c u l a r weight substrate is first cleaved by hya lu ron idase , and the o l igosacchar ides p r o d u c e d ar~ fu r t h e r a t t a c k e d by exopolysaechar idases .

111

formed in digests that hyaluronidase action was followed by exopolysaccha- ridase breakdown of oligosaccharides to monosaccharides.

Aronson and De Duve [21] have shown that lysosomal enzymes from rat liver digest hyaluronate by the same mechanism. The occurrence of all three enzymes above was demonstrated by these workers to occur in the lysosomes, and their individual contr ibut ion to hyaluronate depolymerisation was exam- ined by using saccharo-l,4-1actone and dermatan sulphate as specific inhibitors.

Chondroitin sulphate as well as hyaluronate may be degraded by this mechanism, as shown by Buddecke et al. [22,23] working with bovine arterial wall extracts. This is especially relevant to cartilage erosion. Further, human serum can also catabolize hyaluronate by this three-enzyme mechanism [24] .

From these and other studies, there is an emerging picture of a mecha- nism, which is common to a variety of tissues, for the complete degradation of hyaluronate and chondroit in sulphates. From the present work with the en- zymes found in rheumatoid synovial fluid, it appears likely that this mechanism of macromolecular degradation is also a feature of the inflammatory tissues of rheumatoid joints.

D

The principal difference found between rheumatoid and osteoarthritic fluids was the greatly increased exopolysaccharidase activities in the former. These high rheumatoid synovial fluid levels reflect the highly active lysosomal mechanisms of phagocytic cells in the fluid in vivo [29] . The cell type involved in product ion of the enzyme appears to be the activated rheumatoid lining cell rather than the leukocyte. No correlation was found between white cell counts of fluids and their exopolysaccharidase activities, (cf. refs 6,7,25 and 26). In osteoarthritis, the chondrocyte may be a source of localised cartilage de- struction [27] and an increase in lysos0mal enzymes in synovial fluid would not then be expected.

In contrast to the findings with exopolysaccharidases, the hyaluronidase activity of fluids from the two pathological groups were found to be similar. Further, the levels were always less than that for normal serum. Hyaluronidase in synovial fluid does not, therefore, appear to be a product of inflammatory cells within the joint. In view of its low molecular weight (estimated by column chromatography to be 60 00.0), it is likely that in fact the enzyme may be derived from a remote source via diffusion from serum. Serum hyaluronidase is indeed very similar [3] in properties to the synovial fluid enzyme described above, and is known to be inhibited by a substance universally present in mammalian blood [14] .

The strong inhibition of hyaluronidase in assays with high concentrations of synovial fluid (Fig. 3) is similar to that observed with serum [3] , and sug- gests the presence of the same endogenous inhibitor. This result, coupled with the requirement for acidic conditions makes it unlikely that the three-enzyme system elaborated above is active in synovial fluid in vivo. Rather, the lysoso- mal environment within phagocytic cells would appear as the most favourable one for glycosaminoglycan catabolism by the mechanism outlined.

The occurrence of aggregates of cartilage proteoglycans with hyaluronic acid has recently been established in vivo, and its experimental dissolution by a bacterial hyaluronidase demonstrated [30] . This result clearly shows that hy- aluronic acid within the aggregate may indeed be reached and acted upon by an

1 1 2

enzyme, despite any steric hindrance by the attached proteoglycans. Further, unlike the bacterial enzyme used in this demonstration, it must be noted that the human synovial fluid hyaluronidase studied above is also capable of degrad- ing chondroitin sulphate. The proposed mechanism above therefore makes pos- sible the dissolution of the aggregates and also the further breakdown of pro- teoglycan units by the combined action of hyaluronidase, fi-N-acetylhexos- aminidase and ~-glucuronidase.

Acknowledgements

The support of the National Health and Medical Research Council is grate- fully acknowledged. Synovial fluids were kindly provided by Dr J. Webb of the Sutton Rheumatology Laboratory, Royal North Shore hospital. The authors acknowledge the technical assistance of Miss J. Benson.

References

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