From the Department of Medical Biochemistry and
Microbiology, Uppsala University, Biomedical Center, S-75 123 Uppsala,
Sweden and ¶ Repligen Corporation,
Needham, Massachusetts 02194
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ABSTRACT |
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Interleukin-8, a member of the CXC
chemokine family, has been shown to bind to glycosaminoglycans. It has
been suggested that heparan sulfate on cell surfaces could provide
specific ligand sites on endothelial cells to retain the highly
diffusible inflammatory chemokine for presentation to leukocytes. By
using selectively modified heparin and heparan sulfate fragments in a
nitrocellulose filter trapping system, we have analyzed sequence
requirements for interleukin-8 binding to heparin/heparan sulfate. We
demonstrate that the affinity of a monomeric interleukin-8 molecule for
heparin/heparan sulfate is too weak to allow binding at physiological
ionic strength, whereas the dimeric form of the protein mediates
binding to two sulfated domains of heparan sulfate. These domains, each
an N-sulfated block of ~6 monosaccharide units, are
contained within an ~22-24-mer sequence and are separated by a
region of 14 monosaccharide residues that may be fully
N-acetylated. Binding to interleukin-8 correlates with the
occurrence of the di-O-sulfated disaccharide unit
-IdceA(2-OSO3)-GlcNSO3(6-OSO3)-. We
suggest that the heparan sulfate sequence binds in horseshoe fashion
over two antiparallel-oriented helical regions on the dimeric
protein.
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INTRODUCTION |
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Glycosaminoglycans (GAGs),1 i.e. the linear, sulfate-substituted carbohydrate constituents of proteoglycans, are ubiquitous components of cell surfaces. They are generally believed to exert their biological functions by interacting with proteins. Heparin and heparan sulfate (HS) are sulfated GAGs of alternating hexuronic acid and glucosamine residues that have been shown to bind a variety of enzymes, cytokines/growth factors, and extracellular matrix molecules (for review, see Ref. 1). Such interactions may be highly specific, as illustrated by heparin/HS and antithrombin. A defined pentasaccharide sequence in the GAG chain binds antithrombin and thus strongly promotes its function as an inhibitor of the serine proteases acting within the coagulation cascade (2). Other proteins, such as histones, bind GAGs due to their overall basic character, without any apparent need for a specific saccharide sequence. Several attempts have been made to identify minimal consensus sequences in proteins required to bind heparin or HS (3, 4). Clusters of basic amino acid residues may provide binding sites that are either located within a short linear peptide stretch or composed of residues that occur topologically close together, but on different peptide loops (for review, see Ref. 5).
Sulfate groups on the GAG chains have been identified as important
determinants of protein-binding sites. They are introduced during the
process of polymer modification, through which the initial
(GlcUA1,4-GlcNAc
1,4)n polysaccharide chain is transformed
into the final product (6). While each reaction, catalyzed by a
distinct enzyme, generates the substrate for subsequent reactions,
these are generally incomplete, which leads to progressive structural
heterogeneity. Due to modulated activity of the GlcNAc N-deacetylase/N-sulfotransferase, i.e.
the enzyme that initiates polymer modification in HS and heparin
biosynthesis, variable proportions of GlcNAc units escape
N-deacetylation/N-sulfation. These residual
N-acetylated units are few and isolated in heparin and are
more abundant and typically arranged in consecutive sequence in HS. The
N-sulfate groups are prerequisite to substrate recognition by the GlcUA 5-epimerase and the O-sulfotransferases that
catalyze the subsequent modification reactions. Consequently, heparin
shows a high proportion of IdceA-containing, O-sulfated
disaccharide units and a relatively homogeneous overall sulfation
pattern, whereas HS is composed of alternating sulfated and nonsulfated sequences of variable length (7, 8). A typical HS chain will thus
contain essentially unmodified regions of up to 8-10 consecutive
GlcUA-GlcNAc disaccharide units. Clearly, the occurrence of such
regions will profoundly influence the mode of interaction with
proteins.
Multidomain interaction between HS and proteins has so far received
little attention. Such interaction would apply in particular to
oligomeric proteins composed of monomers carrying single
"heparin-binding sites." To study such a system, we have chosen the
heparin-binding cytokine interleukin-8 (IL-8). IL-8 is a
proinflammatory cytokine of the CXC chemokine family that
forms noncovalently linked dimers with a characteristic appearance (9).
Crystallographic and NMR studies have provided insights into the shape
of the molecule, which consists of a flat array of a -pleated sheet
with the
-helices of the two monomers arranged as antiparallel rods
on top of this sheet (10-12). The heparin-binding sites have been
tentatively localized to the exposed positively charged residues on the
top of these helices (13, 14).
The results of this study define constraints critical to the interaction of a HS chain with the IL-8 dimer. A minimal sequence of 18-20 monosaccharide units is required to span the two saccharide-binding sites on the dimer. The corresponding peptide-binding regions may be separated by a nonsulfated intervening stretch composed of up to 7 GlcUA-GlcNAc disaccharide units.
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EXPERIMENTAL PROCEDURES |
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Materials-- Recombinant human IL-8 was expressed in Escherichia coli and purified as described previously (15), except that a final step of affinity chromatography on heparin-agarose was added. Twenty mg of lyophilized IL-8 were dissolved in 10 ml of phosphate-buffered saline and passed over a 2-ml column of heparin-agarose (Sigma, type I) equilibrated in the same buffer. The column was developed with a linear salt gradient ranging from 0.15 to 2 M NaCl in 20 mM phosphate buffer, pH 8.2. The protein eluted as a single major peak at around 0.6 M NaCl. The pooled fractions were dialyzed against four changes of 50 mM ammonium bicarbonate and lyophilized. Bovine lung heparin (The Upjohn Co.) was purified as described (16). Heparan sulfate from bovine kidney was a gift of Seikagaku Corp. (Tokyo, Japan). Size-defined fragments of E. coli K5 polysaccharide were a generous gift of Dr. K. Lidholt (17). Heparinase from Flavobacterium heparinum (EC 4.2.2.7), which is specific for IdceA(2-OSO3), was obtained from Sigma, and heparitinase from F. heparinum (heparitin-sulfate lyase, EC 4.2.2.8; a mixture of heparitinases I and II according to the information by the supplier), specific for N-acetylated sequences, was purchased from Seikagaku Corp. Bio-Gel P-10 (superfine) was from Bio-Rad. Sephadex G-15, Sephadex G-50, Superose 6, and Superose 12 (prepacked 1.5 × 30-cm columns) and a prototype of a Superdex 30 column (2 × 60 cm) for fast performance liquid chromatography were obtained from Pharmacia (Uppsala, Sweden). A Partisil-10 SAX anion-exchange column (4.6 × 250 mm) and Whatman No. 3MM filter paper were purchased from Whatman Ltd. (Maidstone, Kent, Great Britain). NaB3H4 (24-28 Ci/mmol) and [3H]acetic anhydride (500 mCi/mmol) were obtained from Amersham International (Buckinghamshire, United Kingdom). Hydrazine hydrate was purchased from Fluka (Buchs, Switzerland). All other reagents were the best grade available.
Radiolabeling of Glycosaminoglycans-- Heparin was N-3H-acetylated at free amino groups as described (18) to a specific activity of 20,000 dpm of 3H/µg of hexuronic acid (~0.2 × 106 dpm/nmol of heparin). Bovine kidney HS (0.5 mg) was partially N-deacetylated by hydrazinolysis for 30 min at 96 °C in 1 ml of hydrazine hydrate (~30% water) and 1% hydrazine sulfate (19). The product was N-3H-acetylated with 2.5 mCi of [3H]acetic anhydride essentially as described (18) and extensively dialyzed against 1 M sodium acetate, water, and 0.25 M HIO3 to eliminate any hydrazides found (20) and finally against water. A specific activity of ~0.4 × 106 dpm/µg of uronic acid was achieved (corresponding to an average of ~4.6 × 106 dpm/nmol of HS based on an estimated molecular mass of 11.5 kDa for the HS chains labeled).
Chemical Modification of Heparin-- Selective chemical desulfation of heparin was performed as described previously (21) (see Table I). A preferentially 6-O-desulfated sample obtained by treatment with dimethyl sulfoxide/methanol (9:1, v/v) for 2 h at 93 °C was used as starting material for chemical depolymerization as described below.
Chemical Depolymerization of Polysaccharides-- Limited deaminative cleavage of native and modified heparin was performed as follows to create size-defined fragments. To cleave chains at N-sulfated glucosamine units, 10 mg of native or 2-O-desulfated heparin (which still contains most of the N-sulfates) were dissolved in water adjusted to pH 1.5 with H2SO4 (final volume, 1 ml). Following the addition of NaNO2 (140 µg in 10 µl), the solutions were kept on ice for 3 h. The reaction was stopped by adjusting the pH to 9 with Na2CO3, and the cleavage products were reduced with 2.5 mCi of NaB3H4 overnight at room temperature, followed by 10 mg of unlabeled NaBH4 for 2 h. The resultant labeled oligosaccharides were desalted on a column (1.2 × 66 cm) of Sephadex G-15 in 0.2 M NH4HCO3 and separated by gel chromatography on a column (1 × 146 cm) of Bio-Gel P-10 in 0.5 M NH4HCO3. Preferentially 6-O-desulfated heparin was cleaved at some of the N-unsubstituted amino groups of glucosamine residues that had been created by the preceding desulfation step. A 10-mg sample was dissolved in water, and the solution was adjusted to pH 4.0 with CH3COOH and rendered 0.1 M in NaNO2 (final volume, 1 ml). The sample was incubated on ice for 30 min. The reaction was terminated, and the sample was 3H-labeled as described above. All O-desulfated heparin fragments were re-N-sulfated as described (21) before final separation by gel chromatography on Bio-Gel P-10. The specific activities of the 3H-labeled modified heparin fragments ranged between 0.25 and 0.5 × 106 dpm/nmol of oligosaccharide.
Enzymatic Depolymerization of Polysaccharides-- Size-defined HS fragments were produced enzymatically. Ten µg of N-3H-acetylated bovine kidney HS (specific activity, 0.4 × 106 dpm/µg of uronic acid, ~4.6 × 106 dpm/nmol of HS) were digested with 0.04 units of heparinase in 200 µl of 5 mM sodium Pi, pH 7.0, 150 mM NaCl, and 0.1 mg/ml bovine serum albumin for 1 h at 37 °C. The digest was separated on a Bio-Gel P-10 column as described for the heparin fragments. HS fragments of approximate 20-24-mer size (by reference to heparin oligosaccharide standards) were isolated and used in binding experiments with IL-8.
In a protection approach, the N-3H-acetylated HS (~1 µg) was incubated with 0.1-1.0 milliunit of heparitinase (a mixture of heparitinases I and II specific for N-acetylated glucosamine units) in the presence or absence of 10 µg of IL-8 in 200 µl of 50 mM Hepes buffer, pH 7.0, and 1 mM CaCl2. Digestion was maintained for 60 min at 43 °C and was then stopped by heating at 96 °C for 5 min. The digests were separated on a Superose 12 column in 50 mM Tris, pH 7.5, and 1 M NaCl, and effluent fractions were analyzed by scintillation counting and pooled as indicated.Binding Assays-- Radiolabeled GAGs or size-defined fragments were incubated with protein in 10 mM phosphate buffer, pH 7.4, containing different amounts of salt (see figure legends) as described (22). Protein along with any bound oligosaccharides was trapped on a nitrocellulose filter (25-mm diameter unless otherwise stated). The filter was washed with the incubation buffer, protein-bound oligosaccharides were dissociated from the membrane in 2 M NaCl, and the radioactivity was measured or the sample was desalted for further analysis. In preparative experiments, 1 nmol each of the indicated 3H-labeled modified heparin 18-mers was incubated with 10 nmol of protein in a final volume of 1 ml of phosphate-buffered saline. The bound fractions were isolated by filter absorption over two 45-mm filters and recovered as described above. The amounts of bound material were calculated based on recovered 3H and the specific radioactivity of the respective preparations.
Analysis of GAGs-- GAGs were quantified by colorimetric determination of hexuronic acid using the meta-hydroxydiphenyl method (23) with GlcUA as a standard. A factor of 3 was arbitrarily employed to convert values to saccharide mass.
To ensure complete N-substitution following chemical N-sulfation, saccharide samples were tested for N-unsubstituted glucosamine residues by treatment with nitrous acid at pH 4 (24), followed by gel chromatography on a column (1 × 150 cm) of Sephadex G-50 in 1 M NaCl. Size separation of selectively desulfated heparin preparations on a Superose 6 column gave an average molecular mass for all preparations of 10-11 kDa. Thus, no appreciable cleavage had occurred during chemical processing. The composition of native or modified heparin chains or oligosaccharides was determined after complete deamination at pH 1.5 for N-sulfated chains or at pH 4 following hydrazinolysis of N-acetylated chains (4 h at 96 °C) (25). The resultant disaccharides were reduced with NaB3H4, recovered by gel chromatography on Sephadex G-15 (1 × 200 cm), and separated by anion-exchange high performance liquid chromatography on a Partisil-10 SAX column. The proportions of nonsulfated disaccharides were determined by high-voltage paper electrophoresis on Whatman No. 3MM paper in pyridine acetate buffer, pH 5.3. ![]() |
RESULTS |
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Minimal Size of Heparin Oligosaccharide Binding to IL-8--
The
smallest oligosaccharide fragment able to bind to IL-8 was identified
by in-solution binding studies using radiolabeled size-defined heparin
fragments. Under physiological buffer conditions, the smallest fragment
with appreciable affinity for IL-8 was a heparin 18-mer (Fig.
1A). The interaction between
heparin and IL-8 was previously shown to be of relatively low affinity,
with a Kd of ~6 × 106
M (26), and we therefore considered the possibility of a
charge-dependent interaction involving a shorter saccharide
sequence that had escaped detection due to the ionic conditions of the
assay. Indeed, washing of the nitrocellulose filter at lower ionic
strength (10 mM phosphate buffer without added NaCl)
revealed a remarkably distinct binding profile with a heparin 8-mer as
the smallest binding species (Fig. 1A). In accord with this
finding, small heparin oligosaccharides were able to compete with
full-sized radiolabeled heparin for binding to IL-8 at physiological
ionic strength. The smallest oligosaccharide that inhibited the binding
of labeled heparin for 50% or more was a 6-mer (Fig.
1B).
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Sulfate Dependence of Heparin Binding to IL-8-- The importance of different sulfate groups in the binding of heparin-related saccharides to IL-8 was evaluated using a competition assay with variably modified heparin chains. Full-length radiolabeled heparin was allowed to interact with IL-8 in the presence of unlabeled native or selectively modified heparin (Fig. 2 and Table I) . Native bovine lung heparin showed the highest inhibitor capacity, above that of any of the modified heparins. A drop in competition effectiveness by a factor of ~10 was seen with two of the selectively monodesulfated species, i.e. the selectively N-desulfated and the 2-O-desulfated preparations, whereas the preferentially 6-O-desulfated preparation was slightly less efficient (it should be noted that chemical 6-O-desulfation is accompanied by partial 2-O-desulfation). The more completely 2,6-O-desulfated sample was a hundredfold less efficient than native heparin. The remaining preparations, i.e. the N-/2-O-, N-/6-O-, and completely N-/2-O-/6-O-desulfated samples, were all ineffective as competitors. These results indicate that N-, 2-O-, and 6-O-sulfate groups all contribute appreciably to IL-8 binding.
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Affinity Fractionation of Partially Desulfated Heparin Oligosaccharides-- To further define the role of O-sulfate groups, 3H-labeled 18-mers derived from selectively O-desulfated heparins (see "Materials") were affinity-fractionated, and the disaccharide composition of IL-8-bound fractions was compared with that of the corresponding unfractionated material. Application of a preferentially 6-O-desulfated 18-mer to this procedure resulted in retention of 6% of the material on the nitrocellulose filter, complexed with IL-8, whereas the 2-O-desulfated fraction yielded 15% of IL-8-bound material.
These bound fragments were dissociated from nitrocellulose-adsorbed protein by sodium chloride and desalted. In analytical assays, aliquots of the bound fractions were compared with the respective unfractionated starting material for binding to IL-8. Both preparations, independent of the type of modification, showed increased binding to IL-8 as compared with the unfractionated starting material. Under similar interaction conditions, 1.8 pmol of unmodified heparin, 0.8 pmol of 2-O-desulfated, and 0.3 pmol of 6-O-desulfated heparin were bound to IL-8. From the recovered, once IL-8-bound pools, 1.4 pmol of the selectively 2-O-desulfated and 0.9 pmol of the corresponding, preferentially 6-O-desulfated preparations were sequestered on the filter in a second binding experiment. The preselected 2-O-desulfated sample thus approached the binding ability of unmodified heparin. Compositional analysis of the IL-8-bound fractions showed only marginal differences in the overall degree of sulfation and distribution of O-sulfate groups compared with the corresponding unfractionated materials (Fig. 3 and Table II). The bound material derived from a 2-O-desulfated 18-mer was thus abundantly 6-O-sulfated, but low in 2-O-sulfate groups, whereas that recovered from a 6-O-desulfated 18-mer showed the reverse proportions. However, both fractions yielded significantly increased amounts of the minor 2,6-di-O-sulfated disaccharide representing -IdceA(2-OSO3)-GlcNSO3(6-OSO3)- structures in the intact polysaccharide; this increase was particularly conspicuous for the 6-O-desulfated material. The amounts of the di-O-sulfated disaccharide unit approached 1 mol/mol of either type of IL-8-bound 18-mer (i.e. 11% of the total disaccharide units), and it therefore seems reasonable to conclude that this structure is of importance to the interaction between IL-8 and heparin or HS. While most of the additional 2-O- and 6-O-sulfate groups, located on mono-O-sulfated disaccharide units, are most likely redundant for IL-8 binding, we cannot exclude that selected residues contribute to the interaction.
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Domain Structure of IL-8-binding Heparan Sulfate-- The interaction studies using native or modified heparin oligomers demonstrated that the IL-8 dimer may be spanned by exclusively N-sulfated, extended saccharide sequences. However, since the physiological GAG ligand for IL-8 at the cell surface is presumably HS rather than heparin, we proceeded to study HS-derived IL-8-binding oligosaccharides, with the explicit aim of assessing to what extent such species may contain N-acetylated domains. N-[3H]Acetyl-labeled kidney HS was first degraded by limited digestion with heparinase to cleave glucosaminidic linkages between GlcNSO3(±6-OSO3) and IdceA(2-OSO3) units, thus generating HS oligomers with internal (labeled) N-acetylated sequences flanked on both sides by N-sulfated, presumably IL-8-binding regions (Fig. 4A). After size separation, a fraction corresponding to a heparin ~20-24-mer was selected for binding to IL-8 using the nitrocellulose trapping system. Following incubation with the protein, the bound (~10% of added 3H under conditions of low-salt wash) and unbound fractions were recovered and treated exhaustively with nitrous acid at pH 1.5. The resulting labeled, exclusively N-3H-acetylated fragments were separated by gel chromatography (Fig. 5). The IL-8-unbound fraction yielded a spectrum of variously sized oligomers, with a predominant component larger than a heparin 12-mer or a K5 16-mer (see legend to Fig. 5). This component was largely lacking in the deamination products from the IL-8-bound fraction. Instead, these products showed a larger proportion of smaller labeled oligosaccharides, which ranged in size from heparin 4- to 12-mers. This result indicates that an IL-8-binding region in a HS chain may contain up to 6-7 contiguous N-acetylated disaccharide units, which separate two smaller N-sulfated binding sites, each composed of 4-6 monosaccharide units. Judging from the distribution of smaller N-[3H]acetyl-labeled oligosaccharides, N-sulfate groups may occur also in the intervening sequence bridging the two terminal sulfated regions (in accord with the results of experiments involving labeled heparin oligomers).
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DISCUSSION |
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A number of cytokines and chemokines, including
granulocyte/macrophage colony-stimulating factor (27), interleukin-3
(28), IL-8 (9), macrophage inflammatory protein-1 (29), and platelet factor-4 (22, 30), have been shown to bind to proteoglycans/GAGs. While
the functional implications of these interactions are not fully
understood, it has been proposed that the proteoglycans may act as
storage sites for the small highly diffusible molecules or aid in their
presentation to receptors (28, 31-34). A model study involving various
chemokines and heparin suggested that GAG chains might provide
specificity to chemokine action also in vivo (26), yet
little information has been available regarding the structural
properties of these chains as required to mediate protein binding.
One aim of this study was to define the role of sulfate substituents in GAG binding to IL-8 as reflected through the interaction with fully sulfated, 3H-labeled heparin. The interpretation of such experiments is complicated by the potential redundancy of sulfate groups in saccharide sequences shown to be involved in binding. A striking example is the interaction of basic fibroblast growth factor with a fully sulfated heparin hexasaccharide region, in which only one out of two or three IdceA 2-O-sulfate groups and none of the GlcN 6-O-sulfate residues were found to be essential for binding (35, 36). One approach to the analysis of sulfate requirement is to apply selectively desulfated heparin derivatives in competition experiments with fully sulfated, radiolabeled heparin and the protein ligand. Preferential removal of N-, 2-O-, or 6-O-sulfate groups was thus found to appreciably impede the interaction with IL-8, suggesting that all types of sulfate substituents contribute to binding. This conclusion was verified in direct binding experiments utilizing partially O-desulfated, 3H-labeled heparin 18-mers. Preferential 2-O- and 6-O-desulfation both yielded products from which minor fractions could be sequestered by binding to IL-8. While the overall degree of sulfation of each IL-8-binding species did not differ significantly from that of the corresponding parent preparation, the two binding fractions differed markedly in composition from each other (Table II). Clearly, it must be assumed that neither all the IdceA 2-O-sulfate groups present in the IL-8-binding fraction derived from the preferentially 6-O-desulfated heparin 18-mer nor all the GlcN 6-O-sulfate groups in the corresponding species obtained following preferential 2-O-desulfation could be essential for the interaction. However, both fractions of binding components were appreciably enriched in the minor di-O-sulfated -IdceA(2-OSO3)-GlcNSO3(6-OSO3)- disaccharide unit; this finding applied in particular to the preferentially 6-O-desulfated material (Table II). We conclude that the 2-O- and 6-O-disulfated disaccharide structure promotes binding. On the other hand, each 18-mer contains two binding sites for IL-8 (see below), whereas neither of the two IL-8-binding fractions contained more than 1 mol of di-O-sulfated disaccharide unit/mol of 18-mer. Therefore, it must be assumed that some of the mono-O-sulfated disaccharides may substitute for a "missing" di-O-sulfated disaccharide unit. The precise and optimal arrangement of variously sulfated sugar units in the IL-8-binding site remains to be defined.
The interaction of heparin with IL-8 is relatively weak (26) and was
found in this study to require fragments 18-mers for measurable
binding under physiological ionic conditions (Fig. 1). However, under
conditions of reduced ionic strength, a distinct 8-mer lower size limit
for binding was detected using the nitrocellulose filter trapping
assay. Considering the cleavage specificity of the nitrous acid
reaction used to randomly depolymerize the heparin starting material,
the actual protein-binding site might be as small as a pentasaccharide
sequence within the recovered 8-mer (cf. the analogous
antithrombin-binding site in heparin (37)). Accordingly, heparin
oligosaccharides <10-mers could efficiently compete with full-sized
radiolabeled heparin for binding to IL-8 (Fig. 1). Taken together with
the known propensity of IL-8 to form dimers (12, 38), these findings
are readily interpreted in terms of a trimeric complex involving
an extended sequence that spans two small binding sites, one on each
IL-8 monomer. Indeed, dimerization of IL-8 upon binding to heparin was
recently demonstrated (39).
The physiological saccharide ligand for the IL-8 dimer presumably is not heparin, but rather HS proteoglycans at the surface of the vascular endothelium (32, 34). Witt and Lander (26), using affinity co-electrophoresis, noted that HS bound to IL-8 with about the same affinity as heparin having a much higher overall degree of sulfation. This finding was attributed to the orientation of the Glu-63 residues in the IL-8 homodimer, which would create a patch of negative charge between the two clusters of basic amino acid residues that form the individual "heparin-binding sites" on each IL-8 monomer. It was hypothesized that this arrangement would favor binding of polysaccharide chains containing sulfated regions interspersed by a nonsulfated sequence of appropriate length that would minimize charge repulsion due to the Glu units. Indeed, this study revealed an internal sequence of up to 7 consecutive N-acetylated disaccharide units in HS fragments capable of binding to IL-8.
Combining structural data for the protein and polysaccharide moieties
defines the constraints of potential interaction models. Crystallographic and NMR studies of the IL-8 dimer point to a 2-fold
symmetrical arrangement of the monomers, with the two -helices in
antiparallel orientation (12, 40). The heparin-binding sites have been
tentatively localized to the positively charged amino acid residues
exposed on these ridges (13, 14). The dimensions of the IL-8 dimer have
been estimated to 40 × 42 × 32 Å, with the two
-helices
being 12-14 Å apart. Since the length of a heparin disaccharide unit
is estimated to ~8.5 Å (41-43), a fully extended HS 22-24-mer
would measure ~100 Å and thus would fit in horseshoe-like fashion
across the dimer. According to this model, the postulated internal
N-acetylated domain (
12-14-mer) would bridge the two
-helices, whereas the two adjacent sulfated regions (5-6-mer each)
would each run alongside one helix (Fig. 7). Notably, the symmetry of the protein
dimer would allow the HS chain to accommodate each of the two
interaction sites with similar polarity.
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The functional role of the intervening saccharide sequence remains to
be clearly defined. Variations within this structure could conceivably
modulate the protein/HS interaction. Conversely, a comparison of
different chemokines with similar structures revealed distinct
structural differences in the polypeptide domain facing the GAG ligand
that were potentially implicated in the discrimination between
different polysaccharide ligand subspecies (26).
N-Acetylated sequences bridging separated sulfated
protein-binding domains were found also in HS species binding to
dimeric interferon-, another cytokine not related to IL-8 (44), and
to tetrameric platelet factor-4 (30). The bridging domain in the case
of platelet factor-4 seems to be similar in size to the one identified
for IL-8 (30), although the domain is suggested to stretch over the
intersection between two dimers and not, as in the suggested model for
IL-8, over two monomers. In the case of interferon-
, the bridging
domain was considerably longer (15-16 disaccharide units) than that
associated with IL-8 binding and therefore was thought to extend in a
more loosely knit loop structure. Such extended N-acetylated
regions, while clearly present in the bovine kidney HS investigated,
were essentially absent from the IL-8-associated fragments recovered
following digestion with either heparinase or heparitinase (Figs. 5 and
6). The significance of this difference in domain structure as related
to the roles of HS in cytokine function is not understood. Conceivably,
the variability could be exploited to modulate the efficiency of
chemokine retention on different cell surfaces or to regulate the
selection between different cytokine targets.
It has been proposed that HS promotes IL-8-mediated attraction of neutrophils by providing a trap on the endothelium or in the extracellular matrix that sequesters the secreted chemokine and prevents its diffusion from an inflammatory site (32, 34) as well as its clearance from the blood stream through nonspecific chemokine receptors on erythrocytes (45). Moreover, HS and heparin had a positive effect on IL-8-mediated chemotactic activation of neutrophil migration in vitro (14), although haptotactic activation could not be excluded in this study. A secondary effect of added GAGs was inactivation of elastase released from the neutrophils. HS thus could promote chemoattraction by enhancing the local concentration of IL-8, either by retaining the chemokine on the HS chain or by protecting it from rapid degradation by the released protease (46, 47). It has been noted that whereas HS appears to preferentially bind the dimeric form of IL-8 under physiological conditions (39), monomeric IL-8 has been claimed to be fully active in eliciting a functional response (38, 48). The IL-8/HS interaction may therefore primarily serve to retain the cytokine at its site of production and secretion.
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ACKNOWLEDGEMENT |
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We thank Dr. Kerstin Lidholt for the gift of size-defined E. coli K5 oligosaccharides.
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FOOTNOTES |
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* This work was supported by Swedish Medical Research Council Grant 2309 and by Polysackaridforskning AB (Uppsala, Sweden).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Medical Biochemistry and Microbiology, Uppsala University, P. O. Box 575, S-75 123 Uppsala, Sweden. Tel.: 46-18-471-43-67; Fax: 46-18-471-42-09. E-mail: Dorothe.Spillmann{at}medkem.uu.se.
1 The abbreviations used are: GAGs, glycosaminoglycans; HS, heparan sulfate; IL-8, interleukin-8; aManR, 2,5-anhydromannitol.
2 The largest HNO2-resistant fragments co-elute with heparin 12-mer and K5 16-mer and therefore would correspond to ~14-16-mers in terms of an N-acetylated HS fragment. Since the terminal disaccharide unit must represent an N-sulfated GlcN residue in the intact HS, the N-acetylated stretch would be maximally 14 residues long.
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REFERENCES |
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