On the adsorption of hyaluronan and ICAM-1 to modified hydrophobic resins

ht.

J. Biorhrm.

(‘r/l

i

Pergamon PII: S1357-2725(97)00058-7

On the Adsorption of Hyaluronan Modified Hydrophobic Resins

Bid. Vol. 29. No. IO. pp. 1179 1189, 199: 1997 Elsevier Science Ltd. All rights reserved Printed in Great Bntacn 1357-2725197 %17.(K) t 0.00

and ICAM-

to

PETER A. G. MCCOURT,* STEFAN GUSTAFSON Department qf Medical and Physiological Chemistry, University> oJ‘ Uppsala, Biomedical CL’IIIW. Box 575. S-751 23, Uppsala, Sweden Hyaluronan is a negatively charged glycosaminoglycan that occurs in connective tissue and has a wide range of mechanical and cell biological functions. The purpose of this study was to utilize affinity chromatography resinsfor purification of detergent (Tritou X-100) solubilized hyaiuronan binding proteins from liver, the major organ of hyaluronan clearance from the blood. However, during these studies we made the unexpected finding that hyaluronan binds to Sepharose substituted with a hexamethylene chain, a commonly used spacer arm in affinity chromatography resins,capped with either a terminal primary amine or a terminal acetoamido group. Hyaluronan did not bind the hydrophobic resins hexyl- or octyl-Sepharose under the same conditions. It was also found that rat liver intercellular adhesion molecule-l binds to resins containing the hexamethylene spacer arm, an interaction which could be inhibited with free hyaluronan oligosaccharides. Finally, we have determined that resins with ethylene spacer arms show no affinity for hyaluronan and can therefore be used to immobilize hyaluronan for chromatography of hyaluronan binding proteins. By using this resin we have purified two proteins of approximately 200 and 400 kDa from rat liver endothelial cells.In summary, this study demonstrates the efficacy of certain “capped-hydrophobic” resinsfor binding hyaluronan; these resins may provide a novel means for the study and/or purification of this glycosaminoglycan. This study further demonstrates the importance of the careful design of appropriate affinity columns for the specific purification of hyaluronan binding proteins. 0 1997 Elsevier Science Ltd Keywords: Hyaluronan 1-Amino-6-acetoamidohexane Sepharose molecule-l Hyaluronan binding Ligand affinity chromatography ht. J. Biochem. Cdl Biol. (1997) 29,

Intercellular adhesion

1179-1189

INTRODUCTION

*To whom all correspondence should be addressed. .4hhrrriotion.s: HA, hyaluronan; HABP. HA binding protein; LEC, liver endothelial cell(s); HARLEC, HA receptor on liver endothelial cells; ICAM-I, intercellular adhesion molecule-l ; LAC, ligand affinity chromatography; EDC, I-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EDA, ethylenediamine; DAH, 1.6-diaminohexane; control-EDAand control-DAHSepharose, I-amino-2-acetoamidoethaneand l-amino6-acetoamidohexane Sepharose 4B; HA-EDAand HA-DAH-Sepharose. HA-ethylenediamineand HA1.6-diaminohexane-Sepharose 4B; P-HA-DAHand P-control-DAH-Sepharose, Pharmacia-HAand -control-DAH Sepharose 4B; AH-Sepharose 4B, aminohexane-Sepharose 4B; AE-Sepharose, aminoethane-Sepharose 4B; EpAH-Sepharose, epoxylinked aminohexane-Sepharose 4B; control-EpDAHSepharose, epoxy-linked 1-amino-6-acetoamidohexane Sepharose 4B: HMBA, N,N’-hexamethylene-bisacetamide; PBS. phosphate-buffered saline; PBS-TB, PBS containing 0.1% (v/v) Triton X-100 and 10 mM benzamidine: PI, protease inhibitors. Received 6 January 1997; accepted 6 May 1997.

Hyaluronan (hyaluronic acid; HA) is a member of the family of glycosaminoglycans that occur in connective tissue, and has a wide range of functions, including space filling, lubrication and providing a hydrated matrix through which cells can migrate (Laurent and Fraser, 1992; Toole, 1990; Toole et al., 1984). Its simple chemical structure of repeating disaccharide units of N-acetyl-D-glucosamine and D-glucuronic acid linked by /I(]-4) and [j( 1-3) glycosidic bonds, respectively, belies the range of unique viscoelastic and physiological properties that this anionic polysaccharide has. Such qualities are probably conferred both by its high molecular weight and by its secondary and tertiary structures, which include two-fold helices and extensive branched networks (Scott et al., 1991), and possibly sheets and tubular 1179

1180

Peter

A. G. McCourt

structures (Mikelsaar and Scott, 1994). The formation of these secondary and tertiary structures is probably facilitated by both hydrophobic and hydrophilic interactions between HA monomers (Mikelsaar and Scott, 1994).

Our laboratory has an interest in HA binding proteins (HABPs) that exist on the surface of liver endothelial cells (LECs) to endocytose free HA from the bloodstream (Smedsrod et al., 1990). In earlier studies, ligand affinity chromatography (LAC) using HA attached to some matrix has been used to identify HABPs (Forsberg and Gustafson, 1991; Gustafson and Forsberg, 1991; Mason et al., 1989; Sattar et al., 1992; Tengblad, 1979). LAC is a means of purifying a protein by immobilizing its natural ligand to a column matrix. The ligand can be coupled to the matrix directly, or via a spacer arm, typically hexamethylene, to set it away from the matrix to minimize steric effects. The introduction of the hexamethylene chain can, however, increase the risk of non-specific hydrophobic binding of unrelated biomolecules to the affinity matrix. We have previously applied LAC to identify intercellular adhesion molecule- 1 (ICAM- 1) (Dustin et al., 1986; Springer, 1990) as a putative LEC HABP (McCourt et al., 1994) using HA attached via a hexamethylene linker to a Sepharose matrix. Further studies into the nature of the suggested affinity of ICAMfor HA were subsequently initiated. It was during these studies that we made the surprising finding that both HA and lCAM-1 had an affinity for the capped hexamethylene linker, irrespective of whether HA was coupled to it or not. Furthermore, HA oligosaccharides could inhibit the interaction of ICAM- with this linker. However, using HA attached to Sepharose with a shorter ethylene diamine linker, we have identified two LEC surface proteins which can be specifically eluted with HA from this resin. Furthermore, neither HA nor ICAM-I bound this matrix. Taken together, these results demonstrate the potential utility of modified hexamethylene linkers for the purification and study of HA, and the important role such linkers play in the identification of HABPs using LAC. MATERIALS

AND

METHODS

Chemicals

Collagenase

(Grade

V),

hyaluronidase

and Stefm

Gustafson

(bovine testes type I), leupeptin, pepstatin A, PMSF, benzamidine, N,N’-hexamethylene bisacetamide (HMBA), poly-L-glutamic acid, ethylene diamine and Triton X-100 (molecular biology grade) were obtained from Sigma Chemical Co. (St Louis, MO, USA). Aprotinin was obtained from Bayer (Leverkusen, Germany). ‘25I was obtained from Nordion Inc. (Canada). Broad-range pre-stained molecular weight standards were obtained from Bio-Rad Laboratories (Hercules, USA). l-Ethyl-3-(3dimethylaminopropyl)-carbodiimide (EW was obtained from Fluka Chemika-BioChemika (Buchs, Switzerland). Cyanogen bromideactivated Sepharose 4B, EpAH Sepharose 4B and phenyl Sepharose were obtained from Pharmacia Biotech (Uppsala, Sweden). AH Sepharose 4B was a gift from Prof. Jan-Christer Jansson, Pharmacia BioProcess Technology, Uppsala, Sweden. HA-DAH-Sepharose, produced using Pharmacia’s AH Sepharose 4B as starting material for the coupling of HA (Tengblad, 1979) was a gift from the late Dr Anders Tengblad, Pharmacia, Uppsala, Sweden, and is referred to in the text as P-HA-DAH-Sepharose. The HA Radiometric assay was purchased from Pharmacia Diagnostics (Uppsala, Sweden). Hyaluronan (approximately 900 kDa) was a gift from Ove Wik, Pharmacia, Uppsala, Sweden and Healon (approximately 3.5 x 10’ kDa hyaluronan) was a gift from Pharmacia, Uppsala, Sweden. Heparin was a gift from Dr Marco Maccarana, Department of Medical and Physiological Chemistry, Uppsala, Sweden.

Preparation of oligosaccharides for coupling to Sepharose and for elution of bound proteins

Oligosaccharides were prepared as described previously (Forsberg and Gustafson, 199 1). Briefly, HA was digested with bovine testes hyaluronidase and the resulting oligosaccharides chromatographed on a calibrated S-300 size exclusion column. HA fractions corresponding to 25-40 kDa were pooled and dialysed against ammonium bicarbonate before being lyophilized to dryness. The resulting lyophilisate was dissolved either in water for coupling to aminoethyl- or aminohexyl-Sepharose or in phosphate buffered saline (PBS) (pH 7.4) (137 mM NaCl, 2.7 mM KCl, 6.5 mM Na,HPO, and 1.5 mM KH,PO,) for binding/ elution studies.

Binding

of hyaluronan

Preparation I$ HA-ethylenediamine 4B and 1-amino-2-acetoamidoethane 4B

and ICAM-I

Sepharose Sepharose

HA-ethylenediamine (HA-EDA) Sepharose 4B (Fig. lb) was prepared as follows. Aminoethyl (AE) Sepharose (Fig. la) was prepared according to Cuatrecasas and Anfinsen (197 1). Washed cyanogen bromide activated-Sepharose 4B was suspended in an equal volume of water containing 2 mm01 ethylene diamine (pH 10.0) per millilitre of Sepharose. The gel slurry was mixed end-overend at 4“C for 16 hr and then washed with two cycles each of 1 M NaCl, 100 mM NaHCO,, pH 10.0 followed by 1 M NaCl, pH 4.5 (70 ml buffer per millilitre Sepharose). The aminoethyl-Sepharose was washed further with 0.5 M NaCl, pH 4.5 and finally with water, pH 4.5 and then mixed with an equal volume of water at pH 4.5 containing 9.5 mg/ml hyaluronan (mean M, = 36 000). EDC was then added to a final concentration of 2.3 mM (corresponding to a HA disaccharide:EDC ratio of 1O:l) and the slurry mixed end-over-end at room temperature for 24 hr. The pH was maintained

(4

11x1

at 4.5-5.8 during this time by the addition of 0.1 M hydrochloric acid. The resin was washed as before and then mixed with an equal volume of 3.9 M NaCH,COO, pH 4.5, before the addition of EDC to a final concentration of 100 mM, and the slurry mixed end-over-end at room temperature for 24 hr in order to block the remaining unsubstituted amino groups on the gel. The resin was washed as before and stored in PBS containing 0.02% (w/v) NaN i as a 50% (v/v) slurry. The control resin 1-amino-2-acetoamidoethane (control-EDA) Sepharose 4B (Fig. 1c) was prepared in parallel, but with the elimination of the HA oligosaccharidej2.3 mM EDC coupling step.

Preparation qfHA- 1,6 diaminohexane Sepharose 4B and l-amino-6-acetoamidohe.uane Sepharoso 48

HA- 1,6 diaminohexane (HA-DAH) Sepharose 4B (Fig. Id) and 1-amino-6-acetoamidohexane (control-DAH) Sepharose 4B (Fig. le) were prepared in parallel with the

NH *0-iL;-(cH2)2-~~2

AE-Sepharose

VH O-C-;-(CH&-;-C-M

(4

to resins

Y

HA-EDA-Sepharose

control-EDA-Sepharose

03

0

(9)

B

fH -C-r;l-(CH,)6-~-c-CH,

R

HA-DAH-Sepharose

control-DAH-Sepharose

OH @t-0-CH*-AH-N-(cH,)~-NH, H OH @--o-CH*-AH-;-(cH&-;-C-CH,

EpAH-Sepharose

9

9 Q H3C-C-;-(CH2)6-N-C-CH3 H

(h)

0 Fig.

PH -C-t$(CH&-;--C-M

= Sepharose

I. Diagrammatic

control-EpDAH-Sepharose

HMBA

HA = Hyaluronan representation

of the various

Sepharose

resins

and

HMBA.

1182

Peter

A. G. McCourt

previous two resins, but with the substitution of the longer linker 1,6-diaminohexane for ethylene diamine in the first step. Two further 1-amino-6-acetoamidohexane Sepharose 4B resins were prepared using either AH Sepharose 4B, a resin produced by Pharmacia BioProcess Technology from the coupling of 1,6-diaminohexane to CNBr-activated Sepharose 4B, or EpAH Sepharose 4B (Fig. 10, a resin produced by Pharmacia Biotech from the attachment of the same linker to Sepharose 4B via an epoxy coupling method. These control resins were prepared as was the control-DAH-Sepharose with the elimination of the 1,6-diaminohexane coupling step and are referred to in the text as P-control-DAH-Sepharose and control-EpDAH-Sepharose (Fig. lg), respectively. Preparation of hydrophobic resins of various chain length Ethylamine, n-propylamine and n-butylamine in water, pH 10, and n-pentylamine, n-hexylamine, n-octylamine, n-decylamine and n-dodecylamine in 50% (v/v) dioxane, pH 10, were coupled to CNBr-activated Sepharose 4B at 4 mmol/ml resin, under the same conditions as for the ethylene diamine coupling.

and

Stefan

Gustafson

PBS containing 0.1% (v/v) Triton X-100. HA oligosaccharides (0.95 mg in 0.1 ml H,O) were chromatographed on the columns at 2 ml/hr. Fractions of 0.5 ml were collected and assayed for the presence of HA using the HA Radiometric assay kit from Pharmacia Diagnostics according to the manufacturer’s instructions. All steps were performed at room temperature. Isolation of rat liver ICAM- 1 Rat liver ICAMwas isolated as described previously (McCourt et al.. 1994). Briefly, Triton X-100 solubilized protein from (routinely) 20 homogenized rat livers was sequentially chromatographed on Wheat germ agglutinin-Sepharose, Reactive Yellow 86agarose, Reactive Blue 4-agarose, Concanavalin A-Sepharose, P-HA-DAH-Sepharose and finally again on Concanavalin A-Sepharose as a concentration step. Isolation qf rat liver endothelial cells Single cell suspensions were prepared from the livers of male Sprague-Dawley rats by collagenase perfusion according to obrink (1982). LECs were isolated following Percoll gradient centrifugation and selective adherence according to Smedsrod and Pertoft (1985) and either solubilized directly in PBS containing 1% (v/v) Triton X-100 and protease inhibitors (PI) (10 mM benzamidine, 50 KIE/ml Aprotinin, 0.2 mM PMSF, 1 mg/ml leupeptin and 1 mg/ml pepstatin A) with or without 0.5 mM EDTA, or cultured overnight at 37°C in RPM1 1640 medium on fibronectin coated plates for surface labelling experiments (see below).

Assay of resins for the presence qf coupled HA Aliquots of 100 ~1 of 50% (v/v) slurries of the various Sepharose resins were each equilibrated in 0.15 M NaCl, 0.1 M NaCH,COO, pH 5.0 and then incubated with 40 units of bovine testes hyaluronidase in 550 ~1 of the same buffer, end-over-end for 2 hr at 37°C. The resins were allowed to stand overnight at 4°C before being remixed and centrifuged. Their respective supernatants (500 ~1) were then assayed for released HA oligosaccharide/glucuronic acid content (Bitter and Muir, 1962). The HA-EDAand the HA-DAH-Sepharose were substituted with 0.22 and 0.11 mg HA/ml wet gel, respectively. The P-HA-DAH-Sepharose was substituted with 0.7 mg HA/ml wet gel. There was no detectable HA on any of the control resins.

Surjke labelling qf rat liver endothelial cells Overnight cultures of approximately 10 x lo6 LECs on 60 cm2 plates were surface labelled with “‘1 using the lactoperoxidase method (Hubbard and Cohn, 1972) as described previously (Forsberg and Gustafson, 1991). The cells were washed six times with PBS and then solubilized in PBS/l % (v/v) Triton X- 100 (1 ml) containing PI with or without 0.5 mM EDTA.

Chromatography of HA on various HA control resins Glass columns of 7.5 mm diameter packed with 4 ml each of hexyl-, octyl-, HA-EDA-, control-EDA-, control-DAH-, HA-DAH-, P-control-DAH-, EpAH- and trol-EpDAH-Sepharose, and equilibrated

Assay ,for binding of‘ ICAMto Sepharose matrices Aliquots of 20 ~1 (for binding studies of isolated rat liver ICAM-1) or 40 ~1 (for rat LEC extract binding studies) of 50% (v/v) slurries of various Sepharose resins were each equilibrated in PBS containing 0.1% (v/v) Triton X-100 and

and were AE-, Pconwith

Binding of hyaluronan

10 mM benzamidine (PBS-TB), centrifuged briefly and all supernatant decanted. ICAM[approximately 0.02 pg as determined from limiting dilutions of protein analysed with SDS-PAGE (Laemmli, 1970) and silver staining (Morrissey, 198 l)] in PBS-TB (10 ~1) or rat liver endothelial cell extracts (approximately 60 pg total protein, determined using the Pierce BCATM Protein Assay, with BSA as standard) in PBS-TB (200 ~1) were applied to each gel and mixed every 10 min for 1 hr at room temperature. The various agents [HA oligosaccharides (5 mg/ml), heparin (5 mg/ml), poly-L-glutamic acid (5 mgiml), HMBA (25 mg/ml), NaCl (0.75 and 1.5 M) and EDTA (0.5 and 5 mM)-final concentrations in PBS-TB indicated] were also added separately in some experiments to test their effect on the binding of ICAMto control-DAH-Sepharose and P-control-DAHSepharose. The supernatants were decanted and retained for analysis and the resins washed three times with PBS-TB (1 ml). The material that bound to the resins was released by mixing the resins with SDS-PAGE sample buffer containing 4% (w/v) SDS, 29% (w/v) sucrose, 0.008% (w/v) Bromophenol Blue and 0.08 M Tris-HCl, pH 8.8 (20 ~1) and boiling the mixture for 3 min. Bound and non-bound material was then analysed by SDS-PAGE and immunoblotting (Bjerrum and Schafer-Nielsen, 1986) with rabbit anti-HARLEC polyclonal antiserum (Forsberg and Gustafson, 1991) which recognizes rat ICAM- (McCourt et al., 1994) diluted 1:2000. EL&ion gf proteins from HA-EDA-, controlEDA-, HA-DAH- and control-DAH-Sepharose M’ith HA oligosaccharides Columns (17 mm diameter) were packed with 3 ml each of HA-DAH-, control-DAH-, HAEDA- and control-EDA-Sepharose and equilibrated with PBS containing 0.1% (v/v) Triton X-100 plus PI with or without 0.5 mM EDTA. To determine which cell surface proteins bound to the above resins, Triton X-100 extracts (corresponding to approximately 3 x 10” cultured LEC) of ‘?-surface labelled LEC were diluted to 0.1% (v/v) Triton X-100 with PBS (containing PI with or without 0.5 mM EDTA) (3.0 ml) and applied to the columns at 0.6 ml every 5 min. The columns were washed with equilibration buffer (15 ml) and then a 500 ~1 aliquot of 10 mg/ml HA oligosaccharides was applied. The columns were washed thereafter with equilibration buffer (500 ~1 every 10 min)

and ICAM250

11x3

to resins -.-

,

50 i

0

0

5

to

1‘I

II

Fig. 2. Chromatography of HA on control-EDA-. AE-, P-control-DA%Sepharose. HA P-HA-DAHand (0.95 mg) in Hz0 (100 ~1) was applied to 4 ml columns of control-EDA-, AE-, P-HA-DAHand P-control-DAHSepharose. Fractions (0.5 ml) were collected and andlyed for the presence of HA. (m: q , l , o) indicate HA (pg/fraction) eluted with PBS/O.I?/o Triton X-100 from control-EDA-. AE-. P-HA-DAHand P-control-DAH Sepharose, respectively.

and fractions collected. All chromatopraphic steps were performed at 6’C. Fractions containing most radioactivity were analysed by SDS-PAGE and autoradiography or immunoblotting with the rabbit anti-HARLEC polyclonal antibody. RESULTS

Binding oj’ HA to various resins To determine which matrices had some affinity for HA, HA oligosaccharides were chromatographed on various resins, in the presence of 0.1% Triton X-100. Of the HA applied (0.95 mg) to the resins, 100% was recovered from the hexyl- and octyl-Sepharose and from HA- and control-EDA-Sepharose (after elution with four column volumes of buffer), 61% was recovered from AE-Sepharose while only 23,22, 16 and 7% of that applied was recovered from the control-DAH-, P-HADAH-. control-EpDAH and P-control-DAHSepharose, respectively. Only 16% of the applied HA was similarly recovered from EpAH-Sepharose. Shown in Fig. 2 are chromatograms of HA eluted from control-EDA-. AE-, P-HA-DAH- and P-control-DAH-Sepharose. Trailing of HA was noted with all resins,

1184

Peter A. G. McCourt and Stefan Gustafson

with the exception of hexyl- and octyl-sepharose, which eluted as sharp peaks (not shown). Binding of ICAM-I to HA-DAH-, controlDAH-, HA-EDA- and control-EDA-Sepharose To determine the affinity of ICAMfor various resins, a simple tube/resin assay was developed. Both isolated ICAM-I and LEC extracts were used as starting material for binding assays. Immunoblots of bound protein eluted from the resins by boiling in SDS show that more ICAM- (Fig. 3a) binds to P-controlDAH-Sepharose (lane 6) than to P-HA-DAHSepharose (lane 5). The same pattern is also apparent with more ICAMbinding to control-DAH-Sepharose (Fig. 3a, lane 4) than HA-DAH-Sepharose (Fig. 3a, lane 3), although these resins bind less ICAM- than the previous two. There is no binding of the purified ICAMto either control-EDA-Sepharose (Fig. 3a, lane 2) or HA-EDA-Sepharose (Fig. 3a, lane I). The non-binding fractions in the supernatants were also analysed by immunoblotting. Figure 3a shows that most ICAM-I remains in the supernatants of control-EDA-Sepharose (lane

7) and HA-EDA-Sepharose (lane S), less remains (in descending order) in those of HA-DAH-Sepharose (lane 9), control-DAHSepharose (lane 10) and P-HA-DAH-Sepharose (lane 11) and virtually none remains in that of P-control-Sepharose (lane 12). The band in Fig. 3a lane 13 represents the amount of ICAM-I applied to the resins in this experiment. The effect of EDTA on the binding of whole liver ICAM- 1 to P-control-DAH Sepharose was also tested. Figure 3b shows that the amount of ICAMbinding in the presence of 5 mM EDTA (lane 2) is reduced relative to that in the absence of EDTA (lane 1). In experiments using LEC extracts it was found that similar amounts of LEC ICAM-I bound to both HA-DAH- and control-DAH-Sepharose but to neither HAEDA- nor control-EDA-Sepharose (results not shown). The amount of LEC extract ICAMbinding to HA-DAHand control-DAHSepharose relative to that applied was rather less than if purified whole liver ICAM-I was used (results not shown). It should be noted that when the non-bound and SDS-eluted fractions were decanted for immunoblotting, there

(w

(a) 1

2

3 4

5

6

7 ,8

9

10 1112

12

13

203 118

Fig. 3. (a) Immunoblots of whole liver ICAM-I eluted from various resins with SDS, probed with the anti-HARLEC antibody. Whole liver ICAM- in the absence of EDTA was incubated with the various indicated resins and the bound/non-bound material analysed with immunoblotting. Shown is bound/non-bound material from HA-EDA-Sepharose (lane I/lane 7) control-EDA-Sepharose (lane Z/lane 8), HA-DAH-Sepharose (lane 3/lane 9), control-DAH-Sepharose (lane illlane lo), P-HA-DAHSepharose (lane 5/lane 11) and P-control-DAH-Sepharose (lane 6/lane 12). The band in lane 13 represents the material applied to the resins in this experiment. (b) The effect of 5 mM EDTA on binding was tested. Shown is eluted whole liver ICAM-I, which was bound to P-control-DAH-Sepharose in the absence (lane 1) and presence (lane 2) of EDTA.

Binding of hyaluronan and ICAM-I

remained material in the liquid phase of the Sepharose gel which was not assayed. Thus, on the immunoblots, the sum of bound plus unbound bands appears to be less than the band representing the total ICAMapplied to the Sepharose resins. Binding of ICAMto other matrices To investigate if the binding of ICAMto control-DAH-Sepharose was of a hydrophobic nature, as suggested by the requirement of the extra four methylene groups in the DAH linker, we tested a range of hydrophobic affinity resins with increasing aliphatic chain length for their ability to bind ICAM-1. ICAM- purified from whole rat liver was incubated with ethyl-, propyl-, butyl-, pentyl-, phenyl- hexyl-, octyl-, decyl- and dodecyl-Sepharose, as well as control-DAH-Sepharose, and bound protein eluted with SDS and analysed with immunoblotting as before. There was no detectable ICAMbinding to the ethyl-, propyland phenyl-Sepharose resins, some binding to the butyl-, dodecyl-, decyl-, octyl- and pentylSepharose resins (increasing in that order) and more binding to the hexyl- and control-DAHSepharose resins (roughly equal) (data not shown). However, P-control-DAH-Sepharose bound more ICAMthan control-DAHSepharose (Fig. 3a, compare lanes 4 and 6). Non-substituted cross-linked Sepharose 4B and Tris-blocked cyanogen bromide Sepharose did not bind ICAM-I (data not shown). EJ&t of HA, heparin, poly-L-glutamic acid, HMBA and NACL on the binding of ICAM- to P-control-DAH-Sepharose To investigate which agents could inhibit the ICAM- / 1-amino-6-acetoamidohexane Sepharose 4B interaction, purified rat liver ICAM- 1 or LEC extracts, in the presence of various agents, were incubated with aliquots of P-controlDAH--Sepharose. Bound protein released by SDS was analysed as before. The band in Fig. 4 (lane 1) is the amount of ICAMbound to P-control-DAH-Sepharose in the absence of any additives. HA at a concentration of 5 mg/ml significantly inhibits the binding of ICAM(Fig. 4, lane 2) to P-control-DAHSepharose. Heparin (5 mg/ml) apparently had no effect on the binding of ICAMto P-control-DAH-Sepharose, though two extra immunoreactive bands at approximately 60 kDa were also evident (Fig. 4, lane 3). Poly-L-glutamic acid (5 mg/ml), while not

to resins

11x5

12345678

203 118 86

Fig. 4. Inhibition of ICAM-I binding to P-control- and control-DAH-Sepharose with various reagents. Whole liver [CAM-l in the absence of EDTA wds incubated in the presence of the indicated reagents with P-control-DAH-Sepharose, and the bound material eluted with SDS and analysed by immunoblotting with the anti-HARLEC antibody. Shown is the amount of ICAMbound to P-control-DAH-Sepharose (lane 1) in buffer alone, or in the presence of: 5 mg/ml HA (lane 2), 5 mg/ml heparin (lane 3). 5 mg/ml poly-L-glutamic acid (lane 4) 25 mg/ml HMBA (lane 5). 0.75 and I .5 M NaCl (lanes 6 and 7, respectively). The band in lane 8 represents the material applied to the resins.

causing any significant reduction in whole liver ICAMbinding, did result in a distorted ICAM- band migrating at a slightly higher IV, (Fig. 4, lane 4). HMBA, even at a concentration of 25 mg/ml, had no effect on the binding of ICAMto P-control-DAH-Sepharose (Fig. 4, lane 5). NaCl at concentrations of 0.75 and 1.5 M (Fig. 4, lanes 6 and 7, respectively) reduced the binding to some extent, but not to the same degree as HA (Fig. 4, lane 2). The band in Fig. 4 lane 8 represents the amount of ICAMapplied in this experiment. El&ion of ICAMand other protein1 f&m control-DAH-Sepharose and other resin.\ To demonstrate that ICAM-I and other proteins could be eluted from control-DAH-Sepharose, mini columns of the same resin, as well as HA-DAH-, control-EDAand HAEDA-Sepharose, were used to affinity purify material from Triton X-100 extracts of “‘1 surface labelled LEC in the presence and absence of 0.5 mM EDTA. Immunoblotting demonstrated that HA elutes comparable amounts of ICAMfrom HA-DAH and control-DAH-Sepharose in the absence of

1186

Peter

A. G. McCourt

EDTA (data not shown) but autoradiography of the same material revealed that many other protein bands, besides those in the 80-90 kDa region, were eluted by HA also from the same resins (Fig. 5a, lanes 3 and 4, respectively). Similarly, in the presence of EDTA several bands were eluted from both HA-DAH and control-DAH-Sepharose (Fig. Sb, lanes 3 and 4, respectively). No detectable amount of ICAMwas released by HA from HA-EDAand control-EDA-Sepharose in the absence of EDTA as analysed by immunoblotting (data not shown). However, proteins which appeared to bind more specifically to HA were observed in the eluate from HA-EDA-Sepharose. From this resin HA eluted 400, 200 and 84 kDa bands in the absence of EDTA (Fig. 5a, lane l), and similar bands in the presence of 0.5 mM EDTA, though the 84 kDa band was markedly reduced in intensity (Fig. 5b, lane 1) and was not apparent in all experiments. From controlEDA-Sepharose HA eluted 220 and 84 kDa bands in the absence of EDTA (Fig. 5a, lane 2), though the 220 kDa band was not apparent in all experiments, and only small amounts of the 84 kDa component in the presence of EDTA (Fig. 5b, lane 2). It should be noted that the estimation of the molecular weight of the

and Stefan

Gustafson

400 kDa band is approximate, as we did not have standards larger than 207 kDa. DISCUSSION

HA, a polyanionic polysaccharide, is typically considered a hydrophilic molecule. However, another feature of HA is the existence of large hydrophobic patches (of eight abutting CH groups) extending over three sugar units, repeated on alternate sides of the molecule throughout its length (Scott, 1989). In aqueous solution there is also extensive H-bonding within the HA molecule, via H,O bridges, between the acetoamido groups on N-acetylglucosamine residues and the carboxyl groups on glucuronic acid residues. The internal H-bonding causes considerable local stiffness in the chain (Laurent, 1970; Scott, 1989) and, together with interactions between the hydrophobic patches, also allows HA to form networks with itself and possibly other components of connective tissue (Scott, 1989; Scott et al., 1991). In this study we have tested a range of substituted Sepharose resins for their ability to bind HA in the presence of Triton X-100 (see Fig. 2 and results). We have found that HA has

w

Fig. 5. SDS-PAGE/autoradiographic analysis of material eluted from various resins with HA. Extracts of “‘1 surface labelled LEC in the (a) absence and (b) presence of 0.5 mM EDTA were applied to the indicated resins in columns, and the HA eluted material analysed by SDS-PAGE and autoradiography. Shown is material eluted from HA-EDA-Sepharose (a lane 1 and b lane I), control-EDA-Sepharose (a lane 2 and b lane 2), HA-DAH-Sepharose (a lane 3 and b lane 3) and control-DAH-Sepharose (a lane 4 and b lane 4).

Binding of hyaluronan

some affinity for resins containing a hexamethylene chain with a terminal amine (Fig. If) or a terminal acetoamido group (Fig. le and g), less affinity for a resin containing an ethylene chain with a terminal amine (Fig. la), and no affinity at all for a resin with an ethylene chain and a terminal acetoamido group (Fig. lc). This showed that there was a requirement for a chain longer than two methyl groups for HA binding to the acetoamido resins, suggesting a hydrophobic interaction. However, as there was no HA binding to either hexyl- or octyl-Sepharose, the binding was not simply of a hydrophobic nature (see also Uchiyama et al., 1985 and discussion below), but also required the presence of a terminal acetoamido group. Thus, the six methyl groups and the terminal acetoamido group may act in concert to bind the hydrophobic patches and carboxyl groups on HA, similarly to the binding of detergent to dermatan sulphate recently suggested by Scott pt LII. (1995) (see Scheme 2 of that paper). It has also been proposed by Scott (1992) that such co-operative binding could be the basis of the aggregation of HA and other glycosaminoglycans. Furthermore, as the interaction described in this report occurs in the presence of Triton X- 100, a derivative of isooctane which would bind to both HA and the resin, it could be stronger in the absence of such detergents. It is therefore possible that the l-amino-&acetoamidohexane group on resins depicted in Fig. le and g represents a novel HA binding moiety, which may also provide an alternative means for the purification or concentration of HA. This ligand may also prove to be a simple and useful tool for the study of HA interactions that maintain shape in extracellular matrix. Further studies to find the optimal aliphatic chain length (in this moiety) for HA binding are warranted. The isourea substituent, which results from the reaction of the cyanate ester on CNBr activated-Sepharose with amino compounds, is positively charged at neutral pH and may further act to stabilize the binding of the negatively charged HA to Control-DAH-Sepharose (Fig. le), though this is not an essential requirement for HA binding. The resins containing a terminal amine at the end of an aliphatic chain, namely AE-Sepharose (Fig. la) and EpAH-Sepharose (Fig. If), also bound HA. In the case of AE-Sepharose, this is most likely an ionic interaction between the positively charged (at neutral pH) primary amine and the negatively charged HA. However, the extra four

and ICAM-I

to resins

11x7

methyl groups on EpAH-Sepharose seemed to enhance greatly the binding of HA. It has previously been reported that glycosaminoglycans can be chromatographed on hydrophobic resins such as phenyl- and octyl-Sepharose in the presence of 0.01 M hydrochloric acid and high concentrations (4.0-2.0 M) of ammonium sulphate (Nagasawa and Ogama, 1983; Uchiyama et al., 198s). These high salt concentrations were necessary for the binding of polysaccharides to the resins, which were then eluted by decreasing salt gradients. As the behaviour resembled a hydrophobic interaction between polysaccharides and resins the process was originally considered to be hydrophobic interaction chromatography (Nagasawa and Ogama, 1983). However, subsequent analysis showed that the retention of the polysaccharides on the columns was related to their solubility in ammonium sulphate (Uchiyama rt al., 1985), and the authors concluded that the fractionation depended on the ability of the polysaccharides to precipitate on the gel rather than hydrophobic interactions. We have also tested some of the above resins for their ability to bind partially purified proteins from rat liver and LEC. These studies were necessary in the light of our previous findings that a 90-100 kDa protein bound to and could be eluted (with free HA) from HA-DAH-Sepharose (Forsberg and Gustafson, 1991; McCourt et al., 1994). After the identification of this protein as rat ICAM(McCourt et al., 1994), studies were initiated to identify potential HA binding sites in fragments of ICAM-1. It was then found that considerable amounts of ICAM-I bound to both HA-DAH and control-DAH resins (Fig. 3). This led to a further investigation of the nature of the affinity chromatographic process. In the present study we show firstly that HA actually has an affinity for P-HA- and P-control-DAH-Sepharose (Fig. 2) and other similar resins and secondly that several LEC surface proteins, including ICAM-1. can be eluted with HA oligosaccharides from both HAand control-DAH-Sepharose (Fig. S and data not shown). Thus, the elution of these proteins with HA may not be a specific affinity phenomenon, but instead a displacement phenomenon whereby HA competes with these proteins for the 1-amino-6-acetoamidohexane moiety on control-DAH-Sepharose (Fig. Id), which should also exist on the P-HA- and

1188

Peter

A. G. McCourt

HA-DAH-Sepharose as a result of the blocking of unsubstituted amino groups on the resin with acetate. It is interesting that of three tested polyanions, namely HA, heparin and poly-Lglutamic acid, only HA could inhibit ICAM-I binding to P-control-Sepharose (Fig. 4) suggesting that neither heparin nor poly-Lglutamic acid has as high affinity for this resin as does HA. It would be of interest to investigate whether other polysaccharides, such as dermatan sulphate, chondroitin sulphate or keratan sulphate, have any affinity for this resin. We have also attempted to identify the moiety to which ICAM-I binds on control-DAHSepharose (1 -amino-6-acetoamidohexane Sepharose 4B). We have shown that P-controlDAH-Sepharose made using Pharmacia’s AHSepharose 4B binds the most ICAM-I of all resins tested. That the control-DAH-Sepharose, made with 1,6-diaminohexane and CNBractivated Sepharose, did not bind ICAMas effectively is probably because we did not have optimal conditions for the coupling of the linker that the manufacturer would have been likely to use. In studies with other resins made using CNBr-activated Sepharose, it was found that hexyl-Sepharose and control-DAH-Sepharose bound comparable amounts of ICAM(not shown), though control-DAH-Sepharose bound rather less than P-control-DAH-Sepharose (Fig. 3). That the octyl-, decyl- and dodecyl-Sepharose resins bound less ICAMmay be because of reduced coupling of these ligands owing to their lower solubility, even in the presence of 50% (v/v) dioxane. The importance of the six carbons on the control resin with the terminal amide group suggested that the antineoplastic agent HMBA (Marks et al., 1994) (Fig. If) might have been a ligand for ICAM-1. However, the finding that even quite high concentrations of HMBA (25 mg/ml) could not hinder ICAM- binding to the control Sepharose (Fig. 4) indicates that this is not the case. The interaction of ICAM- with the same resin was inhibited somewhat with high concentrations of NaCl, though not to the same degree as with HA (Fig. 4), which suggests that the binding is not purely of a hydrophobic nature. Thus, hexane attached by the cyanogen bromide linkage to Sepharose via a secondary amine may represent a new ligand for rat ICAM-I and may provide a novel means of purifying ICAM- 1. Since the DAH linker proved to be a source of non-specific binding, the HA-DAH-Sepha-

and

Stefan

Gustafson

rose, under the conditions used, was deemed unsuitable for the purification of the HA receptor on LEC. Furthermore, given the size of the coupled HA (mean M, = 36 000) the longer DAH linker does not confer any advantages in reducing steric hindrance from the matrix. LeBaron et ul. (1992) coupled HA to aminoethyl-Sepharose for the study of HA binding regions on versican. This prompted us to test a similar HA resin, coupled via the same ethylene diamine based linker (HA-EDASepharose) (Fig. lb), for the purification of the HA receptor on LEC. During the course of these studies we did indeed find that HA-EDASepharose was effective in the purification of two proteins of (approximately) 400 and 200 kDa that specifically bound to and were eluted with HA from this resin (Fig. 5). These same proteins were not eluted from the equivalent control resin. We also found that ICAMdid not bind this resin or the equivalent control resin (Fig. 3). Yannariello-Brown et al. (1992) have identified two large polypeptides (166 and 175 kDa) on the surface of rat LEC, with an affinity for HA coupled to a photoaffinity cross linking reagent. They proposed that the two polypeptides may exist as a heterodimer of approximately 340 kDa and have also found ‘*‘I-HA binding activity at 400 kDa in detergent solubilized LEC membranes fractionated with gel filtration (Yannariello-Brown and Weigel, 1992). This polypeptide pattern has some similarities to the two protein species of (approximately) 400 and 200 kDa isolated from HA-EDA-Sepharose in this study. Work is now under way to isolate sufficient amounts of these proteins for sequencing and antibody production. Acknowledgements-We are grateful to MS Kajsa Lilja for expert technical assistance, and to MS Anne-Marie Gustafson and Dr Jukka Melkko for excellent rat liver perfusions. We thank Dr Bianca Tomasini-Johansson, Dr Nick Bonham, Dr Robert Moulder and Prof. Torvard Laurent for their invaluable advice and discussions during this study. We thank Dr Staffan Johansson and Prof. Laurent for their critical review of this manuscript. This work was supported by Agnes and Mac Rudbergs fond, Erland Wesslers fond, Konung Gustaf V:s 804rsfond and the Swedish Medical Research Council.

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Hubbard A. L. and Cohn 2. A. (1972) The enzymatic codination of the red cell membrane. J. Cell Biol. 55, 390-~4os. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nurure 227, 680-685. Laurent T. C. (1970) Structure of hyaluronic acid. In C‘honisrry and Molcwi~~r Mrrtri.u (Edited by Balazs

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Mikelsaar R.-H. and Scott J. E. (1994) Molecular modelling of secondary and tertiary structures of hyaluronan, compared with electron microscopy and NMR data. Possible sheets and tubular structures in aqueous solution. G~~,~,~~[,~~~?j~,~~7~~, J. 1 I, 65-71. Morrissey .I. H. (1981) Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. And. Biochem. 117, 307-310. ktgdsawa K. and Ogama A. (1983) Process for separation of carbohydrates. United States Patent no. 4,421,650. obrink B. (1982) Hepatocyte-collagen adhesion. Mel/z. I:ri:~wro/. 82, 513-529.

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Springer T. A. (1990) Adhesion receptor\ of the immune system. Nr~rurc~ 346, 425 -4.34. Tengblad A. (1979) Affinity chromatography on unmobi1iLed hyaluronate and its application to the isoiatlon of hyaluronate binding proteins from cartilage. R~rrc~iri,~~. Biophw. /iCtfJ 578, 281 289. Toole B. P. (1990) Proteoglyclans and hyalurolum in morphogenesis and differentiation. In (‘(>I1 h&~x:i, af eulrrrc&ulor nrurriu (Edited by Hay E. D.). pp. 705 339. Plenum Press, New York. Toole B. P.. Goldberg R. L., Chi-Rosso G.. Underhlll <‘. B. and Orkin R. W. (1984) Hyaluronate-cell interaction.\. In Thr Role of E.~trcml/ultrr Mrrtrix in Deot~lopmcwt (Edited by Trelstad R. C.), pp. 43.-66. Alan R. Liss. Neh \I’ork. Uchiyama H.. Okouchi K. and Nagasawa K. (1985) Chromatography of glycosaminoglycans on hydrophobic gel. Correlation between chromatographic beha\lour 01 glycosaminogiycans on phenyl Sepharose CL-IB and their solubility in the presence of high concentrations of ammonium sulfate. C‘trrhoh~tfrare Re.s. 140, 139 349. Yannariello-Brown J., Frost s. J. and Weigel P. H (1992) Identification of the Ca’ --independent cndocytic hyaluronan receptor m rat liver sinusoidal endothelial cells using a photoatlinity cross-linking reagent. ./. Bid. Chem. 267, 2045 I -20456. Yannariello-Brown J. and Weigel P. H. (1992) Detergent solubiliration of the endocytotic (.‘a’ -independent hyaluronan receptor from rat liver endothelial cells and separation from a Ca’ -dependent hyaluronan-binding activity. Biodwmisrr~~ 31, 576--5X4.