the chain conformation, it is conceivable that the aggregata- bility of the chains could also be affected by this modification. The results of the present study also ...
24.
THE JOURNALOF BIOLOCICUCHEMISTRY
Val. 256. No. Isaue of December 25, pp. 13039-13043,1981 Printed in L I S A .
Self-association of Heparan Sulfate DEMONSTRATION OF BINDING BY AFFINITY CHROMATOGRAPHY OF FREE CHAINS ON HEPARAN SULFATE-SUBSTITUTED AGAROSE GELS* (Received for publication, May 13, 1981)
Lars-&e FYanssonS, Birgitta Havsmark, and John K. Sheehang From the Department of Physiological Chemistry 2, University of Lund, S-220 07 Lund, Sweden
We have developed an affinity chromatography procedure which measures binding of heparan sulfate species to agarose gels substituted with different heparan sulfates. Three major subfractions of bovine lung heparan sulfate (HS2, HS3,and HS4) which differ in sulfate content and hexuronate composition have been used. Association-prone variants of these species (HS2-A, HS3-A, and HS4-A) were prepared by gel chromatography. Free heparan sulfate chains were applied to columns of various heparan sulfate-agaroses which were eluted with a linear guanidine gradient andbinding wasassessed by measuring the hexuronate content of the effluent. Associating heparan sulfate of a particular subfraction was chiefly bound to gels that were substituted with chains of the same kind, ie. HS2-A to HS2-A-agarose, HS3-A and HS4-A to HS3-A-agarose, and HS4-A to HS4-A-agarose. N-Desulfation and N-acetylation of HS2-A markedly reduced binding to HS2-A-agarose and periodate oxidation of glucuronate in HS3-A completely abolished binding to HS3-A-agarose. Partially oxidizedHS2-A was separated into bound and unbound material by affinity chromatbgraphy on HS2-A-agarose. Gel chromatography of these fractions indicated that unbound chains were significantly smaller than bound ones. It is concluded that association between heparan sulfate chains may be quite specific and that the strength of binding is dependent on co-operative interactions between a number of contact zones. The latter may correspond to the N-sulfated and iduronate- and glucuronate-containing segments.
Heparan sulfate comprises a family of heparin-related glycans that, like heparin, are based ona backbone of alternating glucosamine (a-D)and uronic acid (a-LOr P-D) residues joined via 1 44 linkages (1).In general, heparan sulfates are devoid of anticoagulant activity and they are less sulfated and more glucuronic acid-rich than heparin (1, 2). While heparin is largely located intracellularly (l),heparan sulfate seems to be a ubiquitous cell-surface component (3-6). On the cell surface, the polysaccharides are found assubpendant chains toa * This work was supported by grants from the Swedish Medical Research Council (567, 5731), “Greta och Johan K o c h Stitelser,” “Gustaf V:s 80-irs fond,” “Alfred Osterlunds Stiftelse,” “Riksforbundet mot reumatism,” and the Medical Faculty, University of Lund. The preceding articles in this series are Refs. 9 and 10. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “udvertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom requests for reprints should be addressed. 8 Recipient of a Visiting Scientist Fellowship from the Swedish Medical and NaturalScience Research Councils.
protein core as proteoglycans. There areonly two examples of thoroughly studied proteoheparan sulfates, those o f rat hepatocyte plasma membranes ( 7 ) and of hepatoma cells (8). Both are similar in size and gross chemical composition. They are comparatively small as they contain only three to four side chains. (In cartilage proteoglycans, well over 100 chains are typically found.) Binding of proteoheparan sulfate to the hepatocyte surface appears toinvolve both carbohydrate-protein and protein-phospholipid interactions (7).’ We have recently shown, using gel chromatography and light-scattering methods, that certain heparan sulfate chains are able to self-associate (9). This property is correlated with the presence of alternating ormixedGlcA-GlcNAc,GlcAGlcNS03, and IdoA-GIcNSOa repeating units2 (9, 10). In the present study, we have developed an affinity chromatography procedure which measures binding of different heparan sulfate species to agarose gels substituted with different chains. Various modifications of heparan sulfate have been carried out to assess the nature of this carbohydrate-carbohydrate interaction. EXPERIMENTALPROCEDURES
Muteriak-Heparin by-products from bovine lung (Glaxo) were purified according to the scheme outlined by Roden et al. (11). They were first treated with alkaline copper sulfate to remove dermatan sulfate and then fractionated by ethanol precipitation (as Ca” salts) to remove chondroitin sulfate which remains in thesupernatant solution a t 36%(v/v) ethanol. The heparan sulfate pool (36%ethanol precipitate) was subfractionated according to charge density by stepwise precipitation with cetylpyridinium chloride in the presence of decreasing concentrations of NaCl (see also Refs. 2, 9, IO). The following fractions were obtained: HS1 (0.2 to 0.4 M NaCl), HS2 (0.4 to 0.6), HS3 (0.6 to OB), HS4 (0.8 to LO), HS5 (1.0 to l.Z), and HS6 (complexes not soluble in 1.2 M NaCI). The subfractions HS2-4 were separated into more or less association-prone variants (9) bygel chromatography on a column (30 X 2000 m m ) of Sephacryl S-300 (Pharmacia) which was eluted with 0.5 M sodium acetate, pH 7.0, at a rate of 20 d / h . The effluent was analyzed by the orcinol method (see below), and fractions with K,, I0.4 (A)and 5 0.6 (C) were pooled and recovered by ethanol precipitation, followed by drying with absolute ethanol and diethyl ether. These fractions are essentially comparable to those described earlier (9,lO). For analyses, see Table 1. Other sources of materials were as follows: hyaluronate, dermatan sulfate, and heparin from the Chicago collection (Dr. M. B.Mathews, University of Chicago); CNBr and adipic acid dihydrazide, Fluka; Naf3H]BH4(100mCi/mg), New England Nuclear; Insta-gel, Packard; microgranular DEAE-cellulose (DE-%?),Whatman; solutions (7 M) of guanidine HCI practical grade (Sigma) were treated with activated charcoal under stirringovernight. After filtration through filter paper,
’ M. Hook, personal communication. The abbreviations used are: HexN, hexosamine; GlcN, GlcNAc, and GlcNS03, N-unsubstituted, N-acetylated, and N-sulfated glucosamine, respectively; HexA, hexuronic acid; GIcA, D-glucuronicacid IdoA, L-iduronic acid; -OSOS, ester sulfate; He&-GlcN, repeating disaccharide with reducing terminal glucosamine.
13039
Self-association Sulfate of Heparan
13040 TABLE I
Chemical analysesof heparan sulfate subfractions Yield"
Sample"
N-S07"/
O-SOdh/
R
9
HS2-A HS2-C 15 HS3-A HS3-C HS4-A HS4-C
HS5
20 15
Uronic acid composition
IdoA
$$;
%
a
a
5
75 70 65
40.25
0.20 20.40 0.30 100.35 0.35 0.60 13 0.40 20 40.85 0.60 10 0.72
0.80 0.40 0.90
20 605 605 505
15 35 20 35 45
GlcA
60 ~
Heparan sulfate was purified from bovine lung heparin by-products, fractionated according to charge density (fractions 1 to 5) as described under "Experimental Procedures." Each fraction was subsequentlyseparatedinto aggregating (A)and nonaggregating/less association-prone chains (C) by gel chromatography under associative conditions (9). The yields are expressed as the percentage of total heparan sulfate in the by-product. Total sulfate (O/N-SOJ) was determined after hydrolysis in 6 M HCI a t 100 "C for 8 h and N-sulfate (N-SO,) after hydrolysis in 0.04 M HCI a t 100 "C for 1.5 h (O-SOa = O/N-SO, minus N-SOa).In all fractions glucosamine constituted >95% of total hexosamine. Total uronic acid was determinedboth by the carbazole and orcinol methods. In bothcases, D-glucuronolactone was used as standard. Quantification of L-iduronic acid (total) and D-glucuronic acid was based on carbazole/orcinol ratios (2). Sulfated L-iduronic acid was determined after periodate oxidation a t p H7.0 and 37 "C for 24 h. Residual carbazole-positive material was considered to represent sulfated L-iduronic acid. T h e values are expressed as thepercentages of total uronic acid.
of mannitol, and oxyglycans were recovered as described above. Reduction of oxyglycans obtained after partial oxidation was accomplished by the addition of 2 mg of Nar3H]BH4 (33 mCi/mmol)/ mg of substrate in aqueous solution (5 mg/ml). Excess borohydride was decomposed with glacial acetic acid and [3H]heparan sulfate was recovered after extensive dialysis and freeze-drying. N-Desulfation of heparan sulfate was carried out by the solvolytic procedure of Nagasawa et al.(13). N-Acetylation was performed with acetic anhydride (14). Affinity Chromatography-Binding studies were performed a t room temperature on columns (6 X 50 mm) containing agarose gels substituted with various heparan sulfate species. The columns were equilibratedwith 0.15 M NaCI, and samples (100 pg to 1 mg) of heparan sulfates, dissolved in 4 M or 6 M guanidine HCI (100 pl) if not stated otherwise, were allowed to drain into thegel. A liquid head of 0.15 M NaCl (approximately 1 ml) was placed above the gel and the outlet of the column was kept closed for 12 to 15 h with three changes of the liquid head above the gel. Then the column was eluted with a linear gradient of 0.15 M NaCl to 1.5 M guanidine HCI (total volume, 100 m l ) a t a rate of 3 ml/h. The shape of the gradient was checked by conductivity measurements. The effluent was collected in 1-ml fractionsand analyzedfor uronate (orcinol) or 3H radioactivity. The recovery was 80 to 90%. RESULTS
Characterization of Heparan Sulfates-Bovine lung heparan sulfateencompasses awide variety of species with sulfate contents ranging from 0.44 to 1.23 mol/mol of hexosamine and with iduronic acid contents ranging from 25% to 50% of total uronic acid (2,9).An ion exchange chromatogram of the total heparan sulfatefrom bovine lung isshown in Fig. 1. The subfractions HSl to HS5 were obtained by stepwise precipitation with cetylpyridinium chlorideat themolarities of NaCl indicated under "Experimental Procedures." These subfractions were individually chromatographed on the samecolumn
the A2m of the clarified solution was 0.05 (water blank); other chemicals were of analytical grade. Preparation of Affinity Matrices-Adipic acid dihydrazide-substituted agarose was prepared by mixing 10 g of dihydrazide with 100 ml of CNBr-activated Sepharose 4B (Pharmacia) in the presence of 130 HA HS1 2 3 4 5 DS Hep ml of 0.1 M NaHCO,, pH 9. After shaking a t 4 "C overnight, the gel was washed with 10 volumes of distilled water and suspended in 0.1 M sodium acetate, pH 5.0. Oxyheparan sulfates were obtained after brief periodate oxidation (5 min) of polysaccharide fractions (2 mg/ml) in 0.02 M NaI04, 0.05 1 .o M sodium formate, pH 3.0, a t 4 "C (-5% oxidation of GlcA residues, see Refs. 2, 12). Reactions were stopped by addition of a 10-fold molar excess of D-mannitol. The partially oxidized chains, which were rev) covered after dialysis and freeze-drying, were dissolved in 10 ml of 0 v) acetate buffer (5 to 10 mg/ml) and mixed with 10 ml of adipic acid dihydrazide-agarose. After shaking at 4 "C overnight, the gels were washed with 100 ml of 0.5 M NaCl and 100 ml of distilled water. These 0.5 washings were combined and dialyzed, and unbound material was recovered by freeze-drying. The gels with bound material (the aldehydes of oxyglycans forming an unstable aldimine with the spacer group of the gel) were then suspendedin 10 ml of 0.1 M sodium acetate, pH 5.0, and 25 mg of NaBH4 was added during stirring in four portions a t approximately 15-min intervals. After the aldimine bonds had been stabilized by reduction, the gels were washed with I I 1 -h/ distilled water and suspended in the appropriate buffer (see below). 30 40 50 60 70 80 In general, the amount of bound material was between 4 to 5 mg/ml of gel. EFFLUENT VOLUME (ml) Analytical Methods-The quantities of hexosamine, sulfate, and FIG. 1. Ion exchange chomatography of the total heparan hexuronate were measured with the methods listed previously (2, 9, sulfate pool. The total heparan sulfate pool from bovine lung was 10). Separations of glucosamine and galactosamine were performed on a Bio-Cal automatic amino acid analyzer using a pH 6.45 buffer. prepared asdescribed under "Experimental Procedures." The column The proportions of IdoA (with or without -0SO:d and GlcA were was DE-52 DEAE-cellulose (6 X 140 mm) equilibrated with 0.1 M obtained from the carbazole/orcinol ratios (2). Periodate oxidation sodium acetate, pH 5.0; amount applied, 5 mg. Elution was with a (pH 7; 37 "C; 24 h ) of heparan sulfate was conducted to estimate the linear gradient (0.1to 2.5 M sodium acetate, pH5.0; total volume, 100 amount of sulfated IdoA (2, 12). The extentof oxidation was quanti- ml) a t a rate of 3 d / h . The shape of the gradient was checked by conductivity measurements; the effluent was analyzed with an autofied by carbazole measurements. Radioactivity was assayed with a Packard 2650 liquid scintillation counter using Insta-gel (0.5 ml of mated version of the carbazole-borate method. The points of elution (peak positions) of hyaluronate (HA), dermatan sulfate (DS; IdoAsample mixed with 5 ml of liquid) as scintillator. rich), and heparin (Hep) as well as those of the heparan sulfate Modification Reactions-Periodateoxidation of glucuronate in subfractions HS1 to 5 are indicated in the graph. Although bovine (GlcA-GlcNAc). block regions of heparan sulfatewas carried out with 2 mg of polysaccharide/ml of 0.02 M NaIO,, 0.05 M sodium formate, lung heparan sulfate comprises material with the same elution posipH 3.0, at 4 "C either for 5 min (partial oxidation) or 24 h (complete tion as dermatansulfate, the galactosamine content of the preparation was 5 5 % of total hexosamine. oxidation) (see also Ref. 12). Reactionswere stopped by the addition
I
I I Ill I I
Sulfate Heparan Self-association of
13041
and had the elution positions indicated in the figure. Subfractions HS1 andHS2 accounted for a smaller portion of bovine lung heparan sulfate with a lower charge density, whereas HS3 and HS4 comprise the majority of the material. From the position of HS5 and onward, the amount of material was steadily decreasing. Subfractions HS2 to HS4 which contain self-associating variants (9) were separated into more (A fractions) or less (Cfractions) association-prone chains by gel filtration under associative conditions. Chemical analyses of these specimens are shown in Table I. In general, the C fractions contained relatively more IdoA-OS03 residues than did the A fractions (most association-prone).In this respect, the C fractionswere akin to HS5 which contains no aggregatable species (9). Affinity Chromatography of Heparan Sulfate VariantsEach of preparations HSP-A, HS3-A, and HS4-A was immobilized on agarose. Thus, gels containing heparan sulfate chains with glucuronic acid contents of 75%, 65%, and 60% of total uronic acid (Table I) were available as affinity matrices. Free heparan sulfate chains (associating as well as nonassociating), dissolved in a small volume of 4 or 6 M guanidine HC1, were applied to the gels which contained 0.15 M NaC1. The guanidine was allowed to diffuse away from the application zone into theliquid head above the gel or downward into the gel matrix. If this protocol was not adopted, no material was retarded on the column as estimated from orcinol analyses of the effluent (a gradient of 0.15 M NaCl to 1.5 M guanidine). As shown in Fig. 2, a portion of HS2-A ( a ) was retarded on HS2-A-agarose whereas the nonassociating HS2-C ( b ) ,the associating HS3-A (c), andHS4-A (not shown), and the nonassociating HS5 (not shown) were not bound when the gels were charged with 100-pg samples dissolved in 100 pl of 4 M guanidine HCl. Binding of HS2-A to HSZ-A-agarose was not seen when 1 mg/100 pl of 4 M guanidine HCI or 100 pg/100
p1of 6 M guanidine HC1 was applied. However, binding was observed when 100 pg or 1 mg of HS2-A in 100 p1 of 0.15 M NaCl was added. In the latter case, HS2-A was completely retained on the column (see also Fig. 6). When HS3-A-agarose was used as affinity matrix, the results shown in Fig. 3 were obtained. Fractions HS3-A ( b )and HS4-A ( d ) displayed the largest tendency to bind; in the former case, on elution with a guanidine gradient, bound material emerged at a slightly higher concentration. The
10
20
0.1
0
r-
(D
