Molecular size and xylose content of urinary glycosaminoglycans

13

BIOCHIS,IICAET BIOPI-IYSICAACTA BBA 26692

MOLECULAR SIZE AND X Y L O S E CONTENT OF U R I N A R Y GLY COSAMINOGLYCANS

~,_ WASTESON AND E W E S S L E R

Instztt~te of l~/fedwal Chemzstry, Umvers,ty of Uppsala, Uppsala (Sweden) (Received June I4th, 1971)

SUMMARY

I The molecular weights of the non-ultrafilterable glycosanllnoglycans isolated from normal human urine were determined b y gel chromatography, and their neutralsugar contents were analysed b y g a s - h q m d chromatography 2 The molecular weights (~rw) of the polysaccharldes were as follows" chondroltm sulphate fractions, Irom 8ooo to 13 ooo; dermatan sulphate, 16ooo, heparan sulphate fractions, from 8ooo to approx 3o ooo, the non-sulphated glycosammoglycans (hyaluromc acid and chondroitin), 8ooo The ratio of Mw to M~ did not exceed I 5 in any of the fractions The calculated Stokes' radii of the chondroitin sulphate fractions were 24-36 ~, whereas the Stokes'radn of the largest heparan sulphate fractions were about 6o A 3 The chondroitm sulphate% the dermatan sulphate, and the heparan sulphates contained shghtly less than one residue of xylose per chain, independently of size. This finding suggests that no slgmficant endopolysaccharIdase activity had been exerlLed on the excreted glycosamlnoglycans 4 The non-sulphated urinary glycosammoglycans, which consisted of hyaluronic acid ("/3) and chondroltln (1/3), were found to contain about one xylose residue and about two residues of galactose per chain

INTRODUCTION

The u r m a r y glycosammoglycans consist of a mixture of all species present in connective tissue Not until recently has it been possible to resolve this mixture completely into chemically chstlnct fractions I Molecular weight determinations on unfractmnated material have indicated a polychsperse distribution with an average molecular weight of about IO ooo 3-5, considerably lower than that of the tissue glycosamxnoglycans This discrepancy m a y be due to degradation of polysacchande chains and/or to a selective filtration barrier in the glomerull In the present investigation this problem has been approached b y subjecting each separate fraction of the urinary glycosaminoglycans to molecular weight distribution analysis An a t t e m p t has been Bwchzm Bwphys Acta, 252 (1971) 13-17

I~

~

WAb±'ESON E WEbbLFI¢

made to evaluate the significance of the glomerular filtration barrier FinalE- tht possible role of endopolysaccharldases has been mvestl
Fractions of chondroltm sulphate, dermatan sulphate, heparan sulphate, and non-sulphated glycosammoglycuronans, respectively, were isolated irom pooled urine of healthy men aged about 2o The procedures employed, lnvo!vmg ultrafittratlon, proteolytm digestion, ran-exchange chromatograph?-, and electrophoresls have been described ~ Weight-average (M~) and number-average (M~) molecuiar weights of poly~acchande fractions were determined by analytlca! gel chromatography m o 2 M N a G as described b y WASTESONT,s (For theoretical aspects on gel chromatography, see e g LAURE~T et a! 9 ) According to this method, samples are chromatographed on a colul~qn of Sephadex G-2oo, previously cahbrated with chondromn 4-sulphate fractions ot known molecular weight I t should be pointed out that the appllcatmn of this method to other polysacchandes depends on the plausible assumption thae their exclusion from the Sephadex gel is similar to that of chondro~tln sulphate Stokes' radn of the polysacchande fractions were calculated from the K ~ values of the maxima m the gel chromatograms ~° Hexosamme was determined by the method of ELSON AND 5IoxGaX ~ as described previously x Xylose and galactose were determined as their aldl+ol acetates b y g a s - h q m d chromatography *-" as described by LINDAI~L~3, with the exception ~hat the po!ysacchandes were hydrolysed for z h only RESULTS AND DISCUSSION -

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m

The molecular weights (M~ and Mn) of the polysaccharlde fractions are given in Table I As can be seen from Table I, the differences betweer. ?PT;. and 5-f~ were compaiatlvely small with the ratio of ~tTiwto 7~r~ not exceeding I 5 Furthermore, the peaks observed in most of the gel chromatography experiments were generally narrow The low degree of &sperslty indicated by these observations was not unexpected since the 1on-exchange chromatography, used to prepare these tractions, is known t<~ separate glycosammoglycans both according to charge and molecular welghtn, x~ The molecular weight of the urinary chondroltm sulphate (8ooo-I3 ooo) wa~. considerably lower than that reported for tissue chondromn sulphate (I 3 ooo-3o ooo) (for re?s, see e g re? !o) This discrepancy might be due to the action ot giycosidases on the tissue chondroltln sulphate and/or selective excretion of low molecular weight material Degradatmn ot tissue polysacchandes might be excerted b y endopolysaccharidases or by the concerted actmn ot hexosamlmdases and glycuromdases with exo-enzyme properties If the chondromn sulphate in urine had been subjected to extensive endopolysacchandase actlwty, the number of polysacchande fragments should have increased, and therefore the amounts of the sugars constituting the prorein-carbohydrate linkage region in each polysacchande fragment should have been Bwch~m Bwphys Acta, 252 (197 I) i 3 - 1 7

SIZE A N D X Y L O S E C O N T E N T OF U R I N A R Y G L Y C O S A M I N O G L Y C A N S

TABLE

I5

I

MOLECULAR

WEIGHT

AND

GLYCOSAMINOGLYCURONANS

SIZE,

XYLOSE

OF NORMAL

AND

SULPHATE

HUMAN

CONTENT

OF NON-ULTRAFILTERABLE

URINE

T h e p r e p a r a t i o n a n d i d e n t i f i c a t i o n of t h e f r a c t i o n s h a v e b e e n d e s c r i b e d p r e v i o u s l y (see r e f I) T h e R o m a n figures indicate t h e order m w h i c h t h e fractions e m e r g e d f r o m t h e ion e x c h a n g e r d u r i n g e l u t i o n w i t h s a l t s o l u t i o n s of i n c r e a s i n g c o n c e n t r a t i o n s CS = c h o n d r o l t m s u l p h a t e , D S = d e r m a t a n s u l p h a t e , I-IS = h e p a r a n s u l p h a t e P e r c e n t of t o t a l u r i n a r y g l y c o s a m m o g l y c a n h e x o s a m m e a r e g i v e n in t h e s e c o n d c o l u m n ( f r o m r e f i) M w = w e i g h t - a v e r a g e m o l e c u l a r w e i g h t M n = n u m b e r - g v e r a g e m o l e c u l a r w e i g h t M o l e c u l a r raze IS g i v e n as t h e r a d i u s (~-) of t h e e q u i v a l e n t s p h e r e ( S t o k e s ' r a d i u s ) X y l o s e v a l u e s a r e e x p r e s s e d as r e s i d u e s p e r c h a i n S u l p h a t e v a l u e s ( f r o m re± I) a r e g i v e n as m o l a r r a t i o t o h e x o s a m m e

Fractzon I II III IV V VI VII VIII VIII III IV V VI VII VIII

CS CS CS CS CS ca ca DS HS ]KS HS ]KS ]KS ~IS

% 6 2 IO 5 12 16 9 2 o I I 2 4 4 o

o 9 I 3 2 9 2 2 7 5 9 9 2 4 7

Mw

Mn

8ooo 8ooo 9ooo 9000 8000 IOOOO ILOOO 13000 16000 8000 9 °0o 12000 17000 27000 3° 0 0 o

7ooo 7ooo 8ooo 8ooo 7000 8000 9000 90o0 12000 7ooo 7 °00 9000 13000 19 ° 0 0 19000

Stokes' r adms ( d )

Xylose

Sulphate

22 25 26 24 24 3° 36 31 41 23 23 3° 4° 55 64

1 2 I I o 8 o 9 o 6 o 6 o 7 -o 7 o 8 o 5 o 5 o 4 -o 6

o o o o I I I I I o o o o o o

o 2 5 8 2 3 3 3 2 5 5 6 7 9 9

considerably reduced However, the number of xylose residues per chain, calculaLed from the number-average molecular weight, was found to range between o 6 and I I (Table I) The molar ratios of galactose to xylose invariably fell below the theoretical value of 2 o (ref 6) as expected, since galactose is more slowly hydrolysed from the polymer than xylose la. Neutral sugars other than galactose and xylose were only found in trace amounts Since the values were not corrected for degradation dunng hydrolysis and are thus to be regarded as mmimum values, the results lndmated that all oi most of the chains of each fraction contained the linkage region It therefore seems reasonable to conclude that urinary chondroltln sulphate IS not to any major extent the product of endopolysacchandase degradation The molecular weight of the urinary heparan sulphate was 8ooo-3o ooo (Table I). The high molecular weight fractions had the same molecular weight as reported earlier for tissue heparan sulphate (24 ooo-29 ooo)18 The number of xylose residues per chain was tound to be o 4-o 8 (Table I) Had extensive endopolysaccharldase degradation occurred, the amount of xylose per chain would have been drastically reduced, as m Hurler's syndrome 16 Smce this is not the case, the low molecular weights observed are apparently not due to endopolysaccharidase degradation The origin of the urinary glycosamlnoglycans IS not established Intravenously rejected polysaccharldes were partly excreted In the urine 17 However, the molecular size was not determined. I t has been found that dextran molecules with Stokes' radn less than about 3oA are freely filterable, whereas dextran molecules larger than about 5oX are completely retained Ill the plasma18,19 For polyvlnylpyrrohdone, the upper size limit for free filtration has been found to be 24 A and the lower size hmlt for B~och,m Bzophys Acta,

252 (1971) 1 3 - 1 7

I6

.~ WASTFSOR, E V~E%%LER

complete retention 6oA a0 Since the molecular weights of the chondrottm sulphake preparations were snnflar to those of urinary chondroltm sulphate obtained w, thou~ the use of proteolytlc enzymes ~'-~ the molecular size of urinary chondrottm qulphate (24-36A, Table I) is compatlble with the assumptmn that it originates from the blood w a glomerular filtration The reason why large heparan sulphate molecules (Stokes' radma 64/~ Table ~) occur in the urine ~s obscure t t IS possible that the urinary hepaian sulphate ~s not exclusively a product of glomerular filtration, but originates partly lrom the renal t~ssue The presence of heparan sulphate m rat and dog kidney has been reported 21 3~. Since it is still uncertain 1~*hyaiuromc acid is bound ~o protein it is worthy of note that the non-sulphated fraction, whmh consisted of s/a of hyaluromc acid and 1/~ of chondroltln ~ contained the neutral sugars (I 2 residues of x3dose and 2 2 residues of galactose per chain), characteristic ot the protem-polysaccharlde linkage reg~oo~ of the sutphated glycosamlnoglycuronans 6 Similar qualitative findings have been reported by CHAKRAPANI AND BACHHAWAT 2a I t Is seen m Table I that the !ow molecular weight fractions of heparan sulphate contained less sulphate than the h~gh molecular weight ones Since the proportmn c{ chains containing a xylose resMue was essentially the same in al! iractlons of heparan sulphate, this observation suggests that the polysacchande chum contains less sup phate in the nelghbourhood of the protem-carbohyd_~ate linkage regmn than in other portions of the chum Similar conclusions have been reported earlier for heparm ~ and heparan sulphate as The distribution of sulphate within the urinary chondrmtm sulphate chains is more difficult to eslmate since the relation between sulphate conkent and molecular weight is less apparent for the chondrmtm sulphate than for the heparan sulphate fractions (Table I) ACKNOWLEDGEMENTS

This work was supported by grants from the Swedish Medical Research Council (B7o-I3X-4-o6B), the Swedish Cancer Society (53-B69-o4X), Koaung Gustaf V 8o-gtrsfond, Svenska Saltskapet for Medlcmsk Forsknlng and the Medical Faculty of the University of Uppsala The excellent technical assistance ot )/Ilqs G Anderson and Miss B H m r t is gratefully acknowledged REFERENCES I 2 3 4 5 6 7 8 9 lO I1 12 13

WEssLE~,B~ochem J , 122 (1971 ) 373 DI FERRANTE AND C RICH, CI*~ Chum Acta, I (1956) 519 P ~ARADI, J A CIFONELLI AND A DORFMAN Beoch~m Bzophys Acla, 141 (1967) lO 3 CONSTANTOPOULOS,Nature, 22o (z968) 583 CONSTANTOPOULOS, A S DEKABAN AND W R CARROLL, Anal •zochem , 31 (1969) 59 RODkN, in E R o s s I AND E STOLL, B~ochemastry of Glycoprote~,zs and Related S*tbsta~zces, Part II, s K a r g e r AG, Basel, 1968 p 185 A WAST~SOm-, Baochzm Bzophys Acta, i 7 7 (1969) 152 3. WASrESON, J Chromatogr, 59 (1971) 87 T C LAURENT, B OBRINK, ~ H1gLLSING AND 2~k~:ASTESON, I~ TH GERRITSEI,,', ]Uoder~a S eparat, ou ~aIethods of Macromolecules and Partacles, Vol 2, J %Wiley, N e w Y ork, 1969, p I99 A \VAsTESON, Bzoehem J , i 2 2 (i971 ) 477 L A ELSO~- ANI) W T J MORGAN, Bzochem J , 27 (1933) 1824 P ALBERSH~ClM, D J NEVlNS, P D ENGLISH AND A ]~ARR,Carbohydr Res , 5 (1967) 34 ° U LINDAHL, B,0chem J , 116 (197 ° ) 27 E N D G G L

B~och~m ~ o p h y s Acta, 2.52 (1971) I 3 - 1 7

SIZE AND XYLOSE CONTENT OF URINARY GLYCOSAMINOGLYCANS i4 15 16 17 18 19 20 2I 22 23 24 25

T T J D G I~ B A C B U J

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C LAU~ENT AND A ANSETH, Exp Eye Res, I (I96I) 99 C LAURENT AND J • SCOTT, Nature, 202 (1964) 661 KNECHT, J A CIFONELLI AND A DORFMAN, J B,ol Chem, 242 (1967) 4652 KAPLAN AND K MEYER, J Clan Invest, 41 (1962) 743 ARTURSON AND G WALLENIUS, Scand J Cl*n Lab Invest, 16 (1964) 81 A GRANATH AND B E KVIST, jr Chromatogr, 28 (1967) 69 HULME AND J HARDWICKE, Cl*n Sc*, 34 (1968) 515 ALLALOUF, A BEe AND N SHARON, B~ochzm Bzophys Acta, 83 (1964) 278 W CASTORAND J A GREENE, J Clzn Invest, 47 (1968) 2125 CHAKRAPANI AND B I~ BACHHAWAT, Ind*an J B~ochem, 5 (1968) 9 LINDAHL, B,och,m B,ophys Acta, 13 ° (1966) 368 A CIFONELLI, Carbohydr Res, 8 (1968) 233

B,och~m B,ophys Acta, 252 (1971) 13-17