Isolation and characterization of the sulfated glycosaminoglycans of the vitreous body

167

Biochimica et Biophysica Acta, 498 ( 1 9 7 7 ) 1 6 7 - - 1 7 5 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 28254

ISOLATION AND C H A R A C T E R I Z A T I O N OF THE S U L F A T E D GLYCOSAMINOGLYCANS OF THE VITREOUS BODY

WILLIAM S. A L L E N , E L I Z A B E T H C. O T T E R B E I N and A H M A D H. W A R D I *

Biochemical Research Department, Warren State Hospital, Warren, Pa, 16365 (U.S.A.) (Received N o v e m b e r 22nd, 1976)

Summary Ester sulfate containing glycosaminoglycans comprising approx. 3% of the total glycosaminoglycan content, have been isolated from protease-digested bovine vitreous b o d y by stepwise fractionation on AG-1X2(C1-) and gel filtration on Bio-Gel P-300. Two heparan sulfate and two chondroitin-4-sulfate fractions were isolated in nearly pure form. The heparan sulfate fractions were undersulfated and contained the same relative proportions of N- and O-sulfate (1 : 2), although the total sulfate content differed by approx. 100%. No chondroitin-6-sulfate was present in the isolates, based on evidence obtained from chondroitin ABC lyase experiments.

Introduction Several studies of the vitreous b o d y glycosaminoglycan constituents have mentioned the detection of small amounts of sulfate (trace to 0.24%) in addition to an almost equivalent amount of galactosamine in the hyaluronic acid isolates [ 1--4]. These isolations were carried out employing a variety of methods and the source of the sulfate was either not investigated, or was attributed to inorganic sulfate. In the course of studies on the pentose-rich glycoprotein of the vitreous b o d y in our laboratory [5], we have consistantly noted the presence of small amounts of sulfated glycosaminoglycans in the more polar fractions during AG1-X2 (C1-) fractionation of purified protease digested vitreous h u m o r isolates. The present investigation was undertaken to characterize and identify the sulfated glycosaminoglycans of bovine vitreous body. * Deceased January 29, 1977 Abbreviations: ADi-OSh, 2-acetamido-2-deoxy-3-O-(~-D-gluco-4-enepyranosyluronic acid)-D-glucose; ADi-OS, 2-acetamido-2-deoxy-3-O-(fl-D-gluco-4-enepyranosyl-uronicacid)-D-Galactose; ADi4S, 2-acetamido-2-deoxy-3-O-(~-D-gluco-4-enepyranosyluronicacid)-4-sulpho-D-galactose; ADi-6S, 2-acetamido-2-deoxy-3-O-(~-D-gluco-4-enepyranosyluronicacid)-6-O-sulpho-D-galactose; GlcN, glucosamine; GAIN, galactosamine.

168 Methods and Results

Analytical methods Paper chromatographic system: A, 1-butanol/ethanol/water (10 : 1 : 2, by vol.); B, ethyl acetate/pyridine/water (7.5 : 5 : 2, by vol.); C, ethyl acetate/ acetic acid/water (3 : 1 : 3, by vol.); D, 1-butanol/acetic acid/water (2 : 3 : 1, by vol). Amino acids, amino sugars, ester sulfate and uronic acid were determined as described previously [6]. N-Sulfate was determined by the indole-HC1 method [7]. Galactose and xylose were determined in acid hydrolysates by descending paper chromatography in solvent system A. The areas corresponding to the monosaccharides were eluted from the paper with 0.01 M HC1 and the reducing values measured according to the method of Spiro [8]. Cellulose acetate electrophoresis of the isolated fractions was performed in the system pyridine/acetic acid/water (1 : 9 : 115, by vol.) buffer at pH 3.5. The strips were stained according to the method of Seno et al. [9] with 5% alcian blue. The fractions were also subjected to extensive digestion with chondroitin ABC lyase [ 10] (Miles Laboratories, Kankakee Ill.). The digested fractions were then quantitated on cellulose acetate strips using a densitometer to measure the amounts of residual heparan sulfate. Infrared spectra were obtained in KBr pellets on a Perkin-Elmer Model 137B Spectrophotometer. Optical rotations were measured in 2 dm tubes on a 0.01 ° Zeiss Circle Polarimeter.

Isolation of the sulfated glycosaminoglycan fractions Three batches of frozen bovine eyes each (mixed butcher run, mixed age and sex, Pel-Freeze Biologicals, Rogers, AR) were thawed, cleaned and washed with normal saline. The aqueous h u m o r was removed by piercing the upper and outer quadrants at the scelerocorneal junction with a No. 18 needle and rapid withdrawal with a syringe. The globes were bisected with an equitorial cut and the vitreous humor removed and strained through gauze. Care was taken to avoid contamination with blood and other occular tissues. The crude solids were precipitated with acetone and collected by filtration. The acetone powder was digested with papain-trypsin and the glycosaminoglycans precipitated with ethanol and partially purified as previously described [5,11]. The vitreous h u m o r glycosaminoglycans were dissolved in water, and the solutions treated with AG50W-X8 (H *) to remove basic impurities and the solutions filtered. The filtrates were adjusted to pH 7.5 with dilute NaOH. The glycosaminoglycan mixtures, containing approx. 4% ester sulfate, were then fractionated on 4 X 54 cm columns of AG1-X2 (Cl-, 200--400 mesh) using stepwise gradients of NaC1 through 0.5 M [5]. The columns were then eluted with 2.5 M NaC1 to recover the sulfated glycosaminoglycans. The eluates were dialysed against distilled water at 4°C and the retentates concentrated and lyophilized. The fractionations through 0.50 M NaC1, separated the pentose-rich glycopeptides [5] and approx. 98% of the hyaluronate. The combined 2.5 M NaC1 fractions yielded 0.35 g (approx. 4%) of a mixture of hyaluronate and sulfated glycosaminoglycans from 9.82 g of the protease digested material. The 2.5 M fraction was then refractionated on a 1.9 X 60 cm column of AG1-X2 (CI-, 200--400 mesh),

169

beginning the elution with water followed by the stepwise sequence 0.50, 0.65, 0.80, 1.00, 1.25, 1.75, 2.00 and 2.50 M NaC1. Elution at each NaC1 concentration was continued until negative tests were obtained for hexuronic acid (orcinol). The individual fractions were dialysed and lyophilized. The fractions eluted at 0.50, 1.00 and 2.5 M NaC1 contained only trace amounts of glycosaminoglycan and were discarded. The remaining fractions were gel-filtered on Bio-Gel P-300 (100--200 mesh, 2.9 X 150 cm columns) using 0.25 M NaC1 (containing 0.02% NAN3) as the elutrient. 3.0-ml fractions were collected and the tubes pooled into major fractions based on the uronic acid content [12] and the fractions dialysed and lyophilized. The 0.65 M NaC1 (I) and 0.80 M NaC1 (II) fractions gave single orcinol positive peaks on gel-filtration. The elution patterns on Bio-Gel P-300 for the remaining fractions (III--V) are shown in Fig. 1. Three peaks were detected in the 1.25 M NaC1 fraction (IIIA, IIIB, IIIC) and two peaks were noted in both the 1.75 M NaC1 fraction (IVA, IVB) and the 2.0 M NaC1 fraction (VA, VB). The leading peak of the 1.25 M NaC1 fraction (IIIA) from Bio-Gel P-300 emerged at approx. 100 ml later than the leading peaks of the other fractions indicating the presence of lower molecular weight compounds. Since all of the fractions were somewhat retained, the molecular weights in the fully hydrated form are presumed to be less than 3 • l 0 s.

Electrophoresis studies The electrophoretic analysis on cellulose acetate strips of the nine sulfate containing fractions is reproduced in Fig. 2. Fractions I and II showed very weak metachromatic bands at the origin and intense bands similar in mobility to pure hyaluronic acid. These bands consisted of two overlapping peaks when scanned on a densitometer. This suggests that these fractions consist of a mix-

.,, P--.

/

400

,v~

't . ~ - - c ~

i

500

,\ 600

•

4

/, 3OO

40O

5O0

300

400

50O

ml EFFLUENT VOLUME

Fig. 1. E l u t i o n p r o f i l e s o n B i o - G e l P - 3 0 0 gel f i l t r a t i o n o f f r a c t i o n s I I I - V o b t a i n e d f r o m r e f r a c t i o n a t i o n of t h e t o t a l s u l f a t e d f r a c t i o n s o n A G 1 - X 2 (CI-). E l u t i o n w a s w i t h 0 . 2 5 M N a C I ( c o n t a i n i n g 0 . 0 2 % N A N 3 ) f r o m a 2.9 X 1 5 0 c m c o l u m n ( v o i d v o l u m e a p p r o x . 2 5 0 m l , b l u e d e x t r a n ) . A b s o r b a n c e a t 6 7 0 r i m , o r c i n o l .

170

I IT T1TA mB TITC 1VA 1VB ~A ~B S

Fig. 2. Diagrammatic r e p r e s e n t a t i o n o f the migration o f the sulfate c o n t a i n i n g fractions I-VB in cellulose acetate electrophoresis. S, standard strip; Hyaluronate, heparan sulfate (bovine lung), chondroitin-4-sulfate, f r o m left to right respectively. C o n d i t i o n s as described in Analytical Methods.

ture of HA and undersulfated glycosaminoglycans. Fractions IIIA and IIIB showed two bands having mobilities between hyaluronic acid and heparan sulfate, with the most intense band being the slower moving. Fractions IIIC and IVA showed three bands; two of these bands were located between hyaluronic acid and heparan sulfate with the third band having a slightly slower mobility than authentic chondroitin-4-sulfate. Fraction IVB gave two bands, with the less intense band moving just ahead and hyaluronic acid and the more intense band coinciding with authentic chondroitin-4-sulfate. Fractions VA and VB showed very weak bands at the origin and intense bands with the same migration as authentic chondroitin-4-sulfate.

Infrared studies The infrared spectra of fractions I and II revealed a weak S-O group absorption at approx. 1220 cm -~. The remaining fraction exhibited definitive S-O stretching vibrations at 1220--1240 cm -1 with increasing absorption through fraction VB. In addition, the following characteristic infrared profiles were noted: fractions IIIA and IIIB exhibited C-O-S stretching vibrations at 822 cm -~, suggestive of sulfation at C-6 of hexosamine (equitorial orientation) and a C-N-S stretching band at 894 cm -1 (N-sulfate); fractions IIIC and IVA also showed C-N-S absorption at 894 cm -~ as well as C-O-S stretching bands at both 820 cm -~ and 855 cm -1. The additional C-O-S vibration at 855 cm -~ is indicative of sulfation at C-4 of the hexosamine moiety (axial orientation); fraction IVB showed a C-O-S stretching vibration at 850 cm-'; fractions VA and VB revealed C-O-S stretching bands at 855 cm -~ and the complete infrared profiles were identical to authentic chondroitin-4-sulfate. Although these studies in themselves are not necessarily diagnostic of the position and/or configuration of a sugar ester sulfate [13,14], they are useful as supportive evidence for conformational assignments.

171 TABLE I ANALYSIS OF SULFATE BODY *

CONTAINING

GLYCOSAMINOGLYCAN

Fraction

% of t o t a l **

Uronic acid ***

GlcN

GaIN

SO 4

I II IIIA IIIB IIIC IVA IVB VA VB

27.0 43.0 3.7 1.9 4.9 4.0 5.5 4.5 4.9

1.10 1.05 1.06 1.08 1.14 1.06 0.88 1.09 1.03

0.82 0.75 0.96 0.93 0.68 0.77 0.11

0.18 0.25 0.04 0.07 0.32 0.23 0.89 1.00 0.99

0.23 0.18 0.54 1.16 0.79 0.43 0.87 0.97 1.01

* ** *** T

0.65M t 0.80M 1.25M 1.25M 1.25M 1.75M 1.75M 2.00M 2.00M

0.01

FRACTIONS

N-SO 4

0.23 0.55 0.20 0.15

FROM VITREOUS

Amino acids

(a)~ degrees

0.10 0.13 0.25 0.19 0.17 0.19 0.23 0.21 0.17

--40 --33 +52 +61 +38 +27 --10 --23 --27

Percent dry w e i g h t a f t e r Bio-Gel P-300 filtration. M o l a r r a t i o s w i t h t o t a l h e x o s a m i n e as 1.00. M e a n o f t h r e e d e t e r m i n a t i o n s . C a r b a z o l e u s i n g g l u c u r o n o l a c t o n e as s t a n d a r d . NaC1 c o n c e n t r a t i o n u s e d d u r i n g final f r a c t i o n a t i o n o n A G 1 - X 2 (C1-).

Chemical composition Monosaccharide analysis of acid hydrolysates of the sulfate containing fractions by paper chromatography in solvent system B, revealed that all of the fractions contained uronic acid, hexosamine, galactose and xylose. Examination of the hydrolysates in solvent system C, showed that fractions IIIA-IVA contained both D-glucuronic and L-iduronic acid as the uronic acid constituents; D-glucuronic acid was the only uronic acid present in the remaining fractions. The composition of the fractions are shown in Table I, expressed as molar ratios to total hexosamine. In general the analyses show approx, equimolar ratios of uronic acid and hexosamine in all of the fractions with variations in the ester sulfate and amino acid ratios. In fractions I and II, glucosamine is the principal hexosamine present and the ester sulfate to total hexosamine ratio is approx. 0.2 which coincides with the galactosamine content. The electrophoresis data indicated a two-component mixture and the optical rotations are midway between hyaluronic acid and choindroitin-4-sulfate. Thus these fractions are composed of hyaluronate and probably a partially sulfated chondroitin sulfate hybrid. Fractions IIIA and IIIB contained N-sulfate in an approx. ratio of 1 : 2 to total sulfate but fraction IIIA had approx, one half of the total sulfate content of fraction IIIB. The principal hexosamine present was glucosamine, and from the overall chemical composition and optical rotations, both fractions appear to be undersulfated forms of heparan sulfate. Fractions VA and VB had O-sulfate to galactosamine ratios of approx. 1 : 1 and apparently consist of chondroitin-4-sulfate fractions of different molecular size. Table II summarizes the neutral sugar and serine contents of the various fractions expressed as molar ratios to serine. The molar ratios of galactose/xylose/ serine are nearly all of the order of 2 : 1 : 1 respectively with the exception of the hyaluronate-containing fractions I and II. In fraction I, the principal amino acid is glutamic acid. Serine is the principal amino acid present in the other fractions with glycine being the next most dominant. These results suggest that

172 T A B L E II NEUTRAL TIONS *

SUGAR

CONTENT

OF

THE

SULFATE-CONTAINING

Fraction

NaC1 (M)

Serine

Xylose

G a l a c t ose

I II IIIA IIIB IIIC IVA

0.65 0.80 1.25 1.25 1.25 1.75

2.09 2.88 5.15 4.63 7.62 3.75

2.14 2.80 5.05 4.79 7.70 3.41

2.82 5.15 9.19 9.39 13.45 6.65

IVB VA VB

1.75 2.00 2.00

4.67 3.31 3.42

3.93 3.26 2.96

7.94 6.68 5.81

GLYCOSAMINOGLYCAN

Serine/Xylose/Galactose

1.00 1.00 1.00 1.00 1.00 1.00

: : : : : :

1.02 0.97 0.98 1.06 1.01 0.91

: : : : : :

FRAC-

**

1.35 1.44 1.78 2.03 1.76 1.77

1.00 : 0.84 : 1.70 1.00 : 0.98 : 2.02 1.00 : 0.87 : 1.70

* E x p r e s s e d as p m o l p e r 1 0 0 ~ m o l o f h e x o s a m i n e . ** M o l a r r a t i o s w i t h s e r i n e as 1 . 0 0 .

the sulfated glycosaminoglycans of vitreous humor are similar to those isolated from other sources [15--17] in that the carbohydrate-peptide linkage region is comprised of the sequence uronosyl-galactosyl-galactosyl-xylosyl-serine.

Digestion with chondroitinase ABC lyase Since chondroitinase ABC lyase does not digest heparan sulfate, while it does attack hyaluronate, chondroitin and chondoitin sulfates, the presence of these types of glycosaminoglycans can be assessed by the use of this type of enzyme

I

r

I

I

7 - - r

].2 •

0.8

~

- --o.

--o

0,2 IO

20

30

40 60 240

TIME(rain) F i g . 3. R a t e a n d were carried out 232 nm plotted 0.05 M KCI/HCI I; A ~, I f ; D VB.

extent of degradation of fractions I-IV with chondroitinase ABC lyase [10]. Incubations w i t h 0 . 0 0 8 u n i t o f e n z y m e p e r 0 . 1 / ~ m o l o f s u b s t r a t e as u r o n i c a c i d a n d a b s o r b a n c e a t against reaction time. The reaction was stopped at the indicated times by the addition of buffer. At 60 min, an additional 0.01 unit of enzyme was added. Substrates: o o, • m, I I I A , I I I B ; / /, I I I C ; • e, I V A ; • A IVB; X X, V A ;

173

preparation. The rate and extent of digestion of the sulfate containing fractions with chondroitinase ABC lyase is shown in Fig. 3 as measured by the increase in absorbance at 232 nm over a period of 240 rain. Fractions I and II are degraded at a constant rate through the 60-min reaction time (approx. 60% depolarization). Fractions IIIA and IIIB were resistant to digestion during this time. Fractions IIIC and IVA were degraded swiftly within the first 3 min, followed by a leveling off which reached a b o u t 30% depolarization after 60 min. During the first 5 min, fractions IVB, VA and VB were approx. 60% depolarized, after which the rate gradually leveled off, reaching complete depolarization after 60 min. The addition of a further 0.01 pmol of enzyme at 60 min resulted in minimal or no appreciable change in the absorbance of fractions IIIA-VB, whereas fractions I and II showed an increase in reaction rate which after 240 min had leveled off at approx. 100% depolarization. Quantitative identification of the liberated unsaturated disaccharides was carried o u t by digestion of the fractions with chondroitinase ABC lyase at pH 8.0 overnight along with suitable blanks containing no enzyme. The mixtures were then applied to Whatman No. 1 paper, and the dissaccharides separated by descending chromatography in solvent system D overnight [18]. The disaccharides were visualized with an ultraviolet lamp. The regions containing the disaccharides (and their corresponding blanks) were cut into strips and eluted with 0.01 HC1 at 50°C for 10 min. Their molar fractions were determined by ultraviolet absorbance at 232 nm with suitable corrections applied based on the molar extinction coefficients. The results are shown in Table III. The principle disaccharide of fractions I and II was :~Di-OSh along with minor components ADi-4S and ADi-OS. This indicates that fractions I and II contained small amounts (approx. 20%) of undersulfated chondroitin-4-sulfate or its hybrides with the remainder consisting of hyaluronic acid. Fractions IIIA and IIIB were resistant to digestion suggesting that the fractions consisted of heparan sulfates. Fractions IIIC and IVA yielded ADi-4S and ADi-OS as digestion products (approx. 38% and 23% respectively). Fraction IVB yielded 86 mol% of ADi-4S. Fractions VA and VB were completely digested, yielding 100% of ADi-4S, characteristic of chondroitin-4-sulfate. In order to further substantiate the absence of heparin sulfate in fractions I and II, cellulose acetate electrophoresis was performed on samples prior to and

TABLE III MOLAR FRACTIONS OF THE UNSATURATED WITH CHONDROITINASE ABC LYASE

DISACCHARIDES

OBTAINED

BY DIGESTION

M o l a r f r a c t i o n s d e t e r m i n e d by u l t r a v i o l e t a b s o r p t i o n at 2 3 2 n m a f t e r s e p a r a t i o n b y paper c h r o m a t o graphy and e l u t i o n w i t h 0 . 0 1 M H C I a t 5 0 ° C f o r 10 rain.

Disaccharide

Fractions . I

ADi-OSh * ADi-OS ADi-4S • Mol %.

0.80 0.04 0.16

II

nIA

IIIB

IIIC

IVA

IVB

VA

VB

0.72 0.07 0.19

0.04

0.04

0.08 0.30

0.04 0.19

0.08 0.86

1.02

0.98

174 following exhaustive digestion with chondroitin ABC lyase. No residual glycosaminoglycans were observed in these fractions following digestion for 24 h under the conditions used for the time-course study previously described. Discussion

Fractionation of the purified protease digested glycosaminoglycan isolates from bovine vitreous humor indicates that approx. 3% of the total glycosaminoglycans are sulfated to varying degrees. Two fractions of undersulfated heparin sulfate (IIIA, IIIB) and two fractions of fully sulfated chondroitin-4-sulfate (IVA, IVB) were isolated in nearly pure form based on physical and chemical characteristics. Both species differed somewhat in molecular size according to gel-filtration separation on Bio-Gel P-300. The heparan sulfate fractions contained the same proportions of N- and O-sulfate, although the lower molecular size fraction contained a b o u t twice as much total sulfate. Several minor sulfated components present in the other fractions appear to be mixed hybrides of heparan sulfate and sulfated chondroitin-4-sulfate (fractions IIIC, IVA, IVB). Undersulfated heparan sulfates have been found in a number of tissues; for example, normal human aorta [16], the liver and urine of patients with Hurler's syndrome [16], rat brain [17], normal human urine [19], bovine lung [20] and human umbilical cord [21]. To our knowledge, heparan sulfate has not been previously demonstrated to be a constituent of any of the occular tissues. On the other hand, chondroitin sulfates have been isolated from cornea [22,23], and a hyaluronidase sensitive half-sulfated chondroitin sulfate has been found in the retina [24] of cattle eyes. Our method of careful removal of the vitreous humor from the bovine eye would seem to preclude any chance of contamination with glycosaminoglycans from other occular sources. In addition, the presence of heparan sulfate in the vitreous humor and its absence from other occular tissues suggests that this type of sulfated glycosaminoglycan is native to the vitreous humor. Sulfated glycosaminoglycans have also been isolated from bovine aqueous humor in our laboratory (Allen, W.S. et al, unpublished) in approx, the same percentage amounts of total glycosaminoglycans. Finally, on the basis of evidence obtained from experiments using chondroitinase ABC lyase, it can be concluded that no chondroitin-6-sulfate is present in the vitreous humor. Acknowledgement We are indebted to Mr. C. Wolf for the photographic reproductions. References 1 2 3 4 5 6

M e y e r , K. a n d P a l m e r , J.W. ( 1 9 3 4 ) J. Biol. C h e m . 1 0 7 , 6 2 9 - - 6 3 4 L a u r e n t , T.C. ( 1 9 5 5 ) J. Biol. C h e m . 2 1 6 , 2 6 3 - - 2 7 1 Balazs, E.A., L a u r e n t , T.C: a n d L a u r e n t , U.B.C. ( 1 9 5 9 ) J. Biol. C h e m . 2 3 4 , 4 2 2 - - - 4 3 0 B e r m a n , E . R . ( 1 9 6 2 ) B i o c h i m . B i o p h y s . A c t a 58, 1 2 0 - - 1 2 2 Allen, W.S. a n d W a r d i , A . H . ( 1 9 7 3 ) B i o c h i r n . B i o p h y s . A c t a 2 9 7 , 1 - - 1 0 Allen, W.S., O t t e r b e i n , E.C., V a r m a , R . , V a r m a , R.S. a n d W a r d i , A . H . ( 1 9 7 6 ) J. N e u r o c h e m . 879--885 7 L a g u n o f f , D. a n d W a r r e n , G. ( 1 9 6 2 ) A r c h . B i o c h e m . B i o p h y s . 9 9 , 3 9 6 - - 4 0 0

26,

175

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

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