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CHEMOKINES
From the Paterson Institute for Cancer Research,
Manchester; the National Institute for Biological Standards and
Control, Potters Bar; and the Beatson Institute for Cancer Research,
Glasgow, United Kingdom.
The CC chemokine macrophage inflammatory protein 1 Stem cells may exist in niches in the bone marrow
environment where stromal cells, specific extracellular matrix
components, and bound cytokines regulate proliferation, commitment, and
terminal differentiation of undifferentiated hematopoietic progenitor
cells (HPCs).1,2 A key regulatory cytokine of HPCs is the
heparin-binding CC chemokine MIP1 In the absence of stroma, specifically sulfated heparan sulfate (HS)
glycosaminoglycans are required for maintenance of long-term culture-initiating cells (LTC-ICs) mediated by MIP1 HS is a linear polysaccharide consisting of a series of hypervariable
N- and O-sulfated domains (S-domains) connected by less sulfated,
N-acetyl-rich regions. Spacing of these S-domains and their fine
structure appear to be the major determinants of the specific binding
of HS to protein ligands, including a number of
cytokines.12 The mechanism of action of HS is
controversial, but it is likely that in some cases it induces a
conformational change in the bound ligand, which enables it to be
recognized by cognate-signaling receptors. However, in the case of
MIP1 In the present study we examined whether MIP1 Materials
Radiolabeling and preparation of intact HS chains
Specific degradation of HS Nitrous acid hydrolysis was performed by the low pH method of Shively and Conrad.19 Heparinase III digestion and gel filtration column chromatography were performed as described in Stringer and Gallagher.18 Platelet heparinase digestion was performed by the addition of 4 µL platelet extract to 200 µL 3H-radiolabeled HS in 50 mM NaAc buffer (pH 5.5, 6.5, or 7.4 to create a range of oligosaccharide sizes) and 0.01% bovine serum albumin for 16 hours at 37°C. Platelet proteins were then precipitated by the addition of ice-cold trichloroacetic acid (TCA) to 10% for 15 minutes at 0°C and pelleted by microcentrifugation, 12 000 rpm, for 15 minutes. The supernatant was neutralized by the addition of 5 M NaOH. Resultant HS fragments were then size separated on a Sepharose CL-6B column,18 and the required oligosaccharide sizes were pooled and freeze dried.Preparation of MIP1  (5 µM) were preincubated for 10 minutes at room
temperature before digestion by heparinase III at 40 mIU/mL in 0.5 mM
CaAc, 50 mM NaAc, and 0.1 mg/mL bovine serum albumin, pH 7.0, in 250 µm for approximately 24 hours at room temperature. An additional 10 mIU heparinase III (in 0.5 mM CaAc, 50 mM NaAc, and 0.1 mg/mL bovine
serum albumin, pH 7.0) was added after 8 hours, and 10 mIU enzyme was
added for 2 hours at 37°C. Resultant fragments were separated on a
Biogel P10 column,18 and the void volume peak was pooled
and freeze dried. Proteins were removed by ice-cold TCA treatment as
above. The supernatant was desalted on a PD10 column eluted with
double-distilled water.
Affinity chromatography To prepare a MIP1 affinity gel column, 100 µg MIP1 was
mixed with 100 µg heparin in 500 µL coupling buffer (0.1 M HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], 80 mM
NaCl, pH 7.0) and was incubated for 20 minutes at room temperature. MIP1 was then bound to Affi-Gel 10, and the column was prepared as
previously described by Lyon et al17 alongside a control column in which the MIP1 was omitted. The final concentration of
MIP1 was deduced to be 80 µg in 0.5 mL gel, by protein assay of
the spent coupling buffer.
Affinity experiments were performed by application of radiolabeled HS
samples in 20 mM sodium phosphate buffer of physiological ionic
strength and pH level (0.15 M NaCl and pH 7.3). The sample of HS was
recirculated through the column 5 times at room temperature to maximize
its binding. The column was then washed with 2.5 mL of 0.15 M NaCl, 20 mM sodium phosphate, pH 7.3, followed by 2.5 mL each of a range of NaCl
concentrations, as described in the figure legends. Because of
interbatch variation of MIP1 Protein model building Protein models for a MIP-1 dimer were built using the program
Modeler20 interfaced to the insightII protein modeling
package (Accelrys, San Diego, CA). Two sets of protein models for
MIP-1 were created. The first set used the known protein
structure of platelet factor 4 (PF4; pdb code 1RHP chains A, B) as a
template structure, and the second set used the known structure of IL-8 (pdb code 1ICW). Typically 4 protein models were constructed for each
case. Protein models were evaluated using a scoring
function21 based on nonlocal atomic interactions.
This scoring scheme uses a database of known crystal structures to
characterize favorable residue environments in terms of
atom-atom interaction parameters. These parameters can then be applied
to model structures to find potential misfolded regions or to
objectively select the most physically realistic model
(http://guitar.rockefeller.edu/~fmelo/anolea/anolea.html).
Heparin protein docking Docking of heparin pentasaccharide model ligands to protein model structures was performed with Autodock version 2.4.22 This program allows for flexibility in the ligand structure but uses a rigid body protein approximation to speed up the calculation. The pentasaccharides consisted of 3 GlcN2S6S residues separated by IdoA2S residues. Because the IdoA2S residue can adopt different ring conformations, 2 model ligands were used. In one, all IdoA2S residues were in the 1C4 ring form; in another, the 2S0 ring form was adopted. The model ligands had fixed glycosidic torsion angles taken from a reported heparin nuclear magnetic resonance (NMR) structure (PDB code 1HPN). Flexibility was allowed for all exocyclic torsion angles. Docking was also performed using a heparin endecasaccharide with IdoA2S residues in the 1C4 conformation, but in this case no flexibility was allowed. Partial atomic charges required for the docking calculation were obtained by ab initio quantum chemistry calculations (optimization and charge fitting using an HF/6-31G* basis set) on 1-OMe 4-OMe substituted monosaccharides. This was performed using the Jaguar program (Schrodinger, Portland, OR). Typically the 10 lowest energy (most favorable) coordinate sets were extracted for each ligand type and were used for visualization in insightII.
Interaction of aggregation variants of MIP1 , Affi-Gel 10 columns were prepared from MIP1 variants engineered to control the degree of
multimerization.15 Most bound intact 3T3 fibroblast HS
eluted from the monomeric, dimeric, and tetrameric MIP1 at
approximately 0.4 M NaCl, but there was significantly more HS binding
to the dimeric MIP1 at 0.6 M and greater than to the other
aggregation variants (Figure 1). A
control column did not exhibit any binding of HS greater than 0.15 M
NaCl (not shown). Subsequent experiments were carried out using
BB-10010 MIP1, which appears to exist predominantly in a dimeric
state in concentrations up to 1 mg/mL in phosphate-buffered saline and
across a wide pH range.14,23
Comparison of the affinity of different HS types to
MIP1 Affi-Gel 10 column were compared (Figure 2). Thirty-six
percent of the bone marrow stromal HS, 53% of the fibroblast HS, and
24% of the endothelial HS bound above physiological NaCl
concentrations. Most of the bound material had eluted by 0.8 M NaCl,
but there was significantly more HS eluting above this NaCl
concentration for both the bone marrow stromal HS (8%, A) and the
fibroblast HS (6%, C) than the endothelial cell HS (3%, B).
Therefore, fibroblast HS appeared to have a similar percentage of these
higher ionic strength binding sites for MIP1 than bone marrow stroma
HS and to have the most material binding overall, and it was used in
the following experiments to isolate and characterize the binding
site.
Effects of specific enzyme scission on MIP1 binding, HS digested
by the enzymes heparinase I or III, which cleave in regions of high and
low sulfation, respectively, was bound to MIP1 Affi-Gel 10. Heparinase I acts in the N-sulfated regions and specifically cleaves
disaccharides that contain 2-O-sulfated iduronate that is,
GlcNS(± 6S)-1,4-IdoA2S (where GlcNS indicates
-D-N-sulfated glucosamine; 6S, 6-O-sulfated; IdoA, -L-iduronic
acid; 2S, 2-O-sulfated).24 In contrast, heparinase III
cleaves GlcA-containing disaccharides,24 principally
GlcNAc/GlcNS-1,4-GlcA (where GlcA indicates  -D-glucuronic acid;
GlcNAc, -D-N-acetylglucosamine), present in regions of low
sulfation, and not contiguous N-sulfated sequences enriched in IdoA.
Binding to MIP1 of HS fragments produced by either of the enzymes
was significantly decreased in comparison with native HS (Figure
3, compare panels B and C to A). This was
most noticeable with heparinase III digestion (Figure 3C), through
which less than 10% of the HS fragments remained bound to the column
after elution with 0.15 M NaCl and bound material eluted at 0.2 M
(most) and 0.4 M NaCl compared to 56% of the intact HS binding and
elution at up to 0.7 M (Figure 3A). In contrast to heparinase III
treatment, almost 3-fold more heparinase I fragments (28%) eluted from
MIP1 between 0.2 to 0.4 M NaCl and another 6% bound greater than
0.4 M. These results indicate that both N-sulfated (S-domains) and N-acetylated regions of the HS chain are important for binding to
MIP1.
To elucidate the nature of the S-domains that constitute part of the
MIP1
Isolation of the MIP1 binding site appeared to overlap N-acetylated
and N-sulfated regions of HS, the binding site could not be isolated
from fragments produced by scission with either heparinase I or
heparinase III. Therefore, a protection assay18 was used in which MIP1 was included in a heparinase III digest to
prevent cleavage of the binding site. A prolonged digest was carried
out to ensure that only the HS fragments with strong affinity for MIP1 were left undigested. When heparinase III breakdown of HS in
the absence of MIP1 was compared by P10 gel filtration
chromatography with profiles for MIP1-protected HS (Figure 4A-B),
the most striking difference was the presence of a large void peak in
the MIP1-protected digest (Figure 4B), surmised to be the
MIP1-protected domain (MPD). There was also a marked absence of the
tetradecasaccharide and larger S-domains in the digest of the
MIP1-protected HS, presumably as these are incorporated into the
MPD. Gel-filtration chromatography of MPD on Sepharose CL6B revealed a
radioactive fraction, with a mean Kav (from several
experiments) of 0.68 (Figure 4C), equal to a mass of 8.3 kDa by
reference to the published calibration of Wasteson.25 This
is equivalent to approximately 18 disaccharides, assuming 443 Da/disaccharide (molecular weight of a nonsulfated disaccharide). By
comparison the intact fibroblast HS had a Kav of 0.38, equivalent to approximately 40 kDa (Figure 4C).
It was important to establish that MPD was indeed an MIP1 Effect of platelet heparinase on MIP1 -binding
site. Figure 5A shows the size distributions on a Sepharose CL-6B
column of oligosaccharides of mean sizes 5 kDa, 10 kDa, and 14 kDa
pooled from platelet heparinase digests carried out to completion at pH
5.2, 6.5, or 7.4. The 10-kDa (Figure 5D), 14-kDa (Figure 5C), and
20-kDa (not shown) oligosaccharides showed similar ranges of binding to
MIP1 Affi-Gel 10 as intact HS (Figure 5B), mostly eluting by 0.5 M
NaCl. In contrast, the apparent affinity of the 5-kDa oligosaccharides
for MIP1 were markedly reduced (Figure 5E), with most of the
material eluting at 0.2 M. This suggests that the size of the
MIP1-binding site is between 5 and 10 kDa, in agreement with the
8.3-kDa protected fragment (MPD) as the binding domain.
Analysis of MPD compared with native HS MPD was depolymerized by nitrous acid degradation and heparinase enzymes and then was analyzed on a P10 column to compare its structure with that of its parent fibroblast HS (Figure 6).
Treatment with low pH nitrous acid, which cleaves at GlcNS residues, yielded a distinct pattern of scission of MPD and HS, with an almost 2-fold enrichment of disaccharides in MPD and a 3-fold reduction of tetrasaccharides (Figure 6A-B). This indicates a significant increase in contiguous N-sulfated disaccharides in comparison with the original HS, consistent with MPD encompassing 2 S-domains and one GlcNAc-rich interconnecting region. The octasaccharides appeared to be the most predominant contiguous GlcNAc containing oligosaccharides (Figure 6B), with a 2-fold enrichment in MPD compared to intact HS. Heparinase III scission provides data on the size range of the S-domains in HS, elucidating in part the arrangement of the contiguous N-sulfated disaccharides identified by nitrous acid hydrolysis. There was a 5-fold enrichment of dodecasaccharide and tetradecasaccharide S-domains released from MPD by heparinase III compared to intact HS (Figure 6C-D). These oligosaccharides were confirmed to be S-domains, as opposed to incompletely digested material, by their susceptibility to low pH nitrous acid degradation (not shown). The prevalence in MPD of particular-sized fragments excised by
heparinase III and low pH nitrous acid can be rationalized in the form
of a major structural motif, with some permissive variations, which
represents this enzyme-protected binding region for MPD in HS. Such a
structure is depicted in Figure 7.
Although only 17 disaccharides in length, the molecular weight of this N- and O-sulfated oligosaccharide is 8259 Da, the average size for
(TCA-treated) MPD.
Protein-docking results Two different models of the MIP-1 dimer, both based on
experimental evidence, have been proposedthe published dimeric NMR structure pdb code 1B53 and a more compact dimer with a different interface.23 We have constructed protein dimer models,
similar to those reported by Ashfield et al,23 using
monomer-monomer orientations adopted by IL-8 (pdb code1QE6) that have
a distinct quaternary structure but retain a tertiary structure similar
to that of 1B53. We have also created novel alternative dimers using PF4 (pdb code 1RHP). Both IL-8 and PF4-based dimers adopt a compact structure in contrast to the extended structure reported on the basis
of NMR data26 (pdb code 1B53). Systematic evaluation of protein structure quality according to the method of Melo and Feytmans21 indicates that the compact dimers predicted on
the basis of IL-823 or PF4 (Figure 7C) are more likely to
be physically realistic than the extended dimer proposed on the basis
of NMR data.26 The 2 compact models for the dimer are
similar in quaternary structure, with the PF4-based model having a
marginally better score than the IL-8 model. In both compact dimer
models, Lys 60 is less solvent-exposed than Lys 36 or Lys 44, whereas
in the published PDB structure 1B53, Lys 60 is the most exposed.
Ashfield et al23 have shown that of these lysine residues
Lys 60 is the most protected from methylation, indicating that Lys 60 is less exposed to solvent. In both dimer models, 1 of the 2 possible heparin-binding sites is preferred to the other by the docking calculation because amino acid side-chains are held rigid in these calculations; this suggests that the orientations of side chains are
more favorable in one of the monomer subunits.
Docking of heparin pentasaccharide ligands was performed using several protein structures. Docking was performed with the published dimeric NMR structure pdb code 1B53 (Figure 7B) and a monomer (A chain) derived from it (not shown), then it was performed with the IL-8 (not shown) and PF4-based dimers (Figure 7C). For all 3 protein models used in the docking calculations, the heparin-binding site was predicted to be in the same area of the protein surface (Figure 7C). This region includes basic residues Arg 17, Arg 45, and Arg 47 (in agreement with Koopmann and Krangel10) and occasionally Lys 44. In the published dimer 1B53 and the compact dimer model based on PF4, this area is exposed to solvent, and in both models the heparin-binding sites are at opposite ends of the dimer. No differences in predicted binding site resulted from the 2 iduronate conformations in the pentasaccharide ligands. An additional docking calculation was performed using an endecasaccharide (Figure 7D) rather than a pentasaccharide heparin model; this identified the same binding site.
Studies by Hoogewerf et al13 imply that
glycosaminoglycans such as heparin mediate the oligomerization of
chemokines, including MIP1 MIP1 As might be expected the S-domains of HS, which are enriched in
O-sulfation, appeared to be important for MIP1 Another striking difference with a number of previously characterized
binding sites on HS is the large molecular mass (8.3 kDa, equivalent to
approximately 17 disaccharides) of the MIP1 The depolymerization profiles of MPD, after specific enzyme and chemical scission, were used to determine the key structural features shown in the diagram in Figure 7. The heparinase III depolymerization profile (Figure 6C) indicates that murine 3T3 fibroblast HS, in common with human fibroblast HS,34 largely consists of blocks of 3 to 7 N-sulfated disaccharides (S-domains) spaced apart by extended N-acetylated sequences. The 34-saccharide MPD has a 5-fold enrichment of the rare dodecasaccharide and tetradecasaccharide S-domains (Figure 6D) compared to the parent HS. MPD has approximately twice the level of contiguous N-sulfated disaccharides (compare Figure 6B with 6A), which affirms the presence of 2 S-domains and is consistent with the ratio of approximately 2 to 1 N-sulfated to N-acetylated disaccharides in the proposed average structure (Figure 7A). Internal IdoA residues flanked by GlcNS in fibroblast heparinase III-resistant S-domains are consistently modified by 2-O-sulfation of C2,32 and 1 or 2 6-O-sulfate groups are seen more frequently toward the center of longer fibroblast S-domains, such as those in MPD. All heparinase III-derived S-domains that have been sequenced end in GlcNAc.32 The most prevalent contiguous N-acetylated region in MPD appears to be an octasaccharide (Figure 6B), which separates the 2 S-domains. The molecular weight of the predicted structure (Figure 7A) containing these features is almost exactly 8.3 kDa, the average size of MPD. The motif of 2 S-domains separated by an N-acetylated region is
emerging as a common structure for multimeric cytokines binding to HS
and is thought to enable simultaneous interaction with 2 or more
binding sites on the proteins. The PF418
IFN- An MPD model was constructed using the coordinates of the solution
structure of heparin (1 HPN) to form the S-domains (Figure 7A) because
it has been shown that variations in sulfate substitution have
relatively minor effects on the overall conformation of the polysaccharide chain.37 The unsulfated middle region of
alternating GlcA and GlcNAc residues was modeled as an extended chain
in which glycosidic linkages adopt sterically allowed conformations.
The iduronate-containing S-domains such as heparin can be expected to
adopt a relatively well-defined conformation, but there is no reason to
suppose that the unsulfated GlcA-containing regions are similarly
constrained.38 The GlcA-GlcNAc (cellobioselike) and
GlcNAc-GlcA (maltoselike) linkages may adopt more than one low-energy
conformation,39,40 leading to flexibility in this region.
Binding sites face outward in opposite directions, so some flexibility
of the HS fragment is necessary if each terminal S-domain is to bind to
one of the 2 sites. The unsulfated central region of the MPD fragment
measures approximately 40 Å in length when fully extended, similar to
the end-to-end distance between the 2 heparin-binding sites of the
dimer of 30 to 40 Å (for both the NMR structure 1B53 and the compact
dimer modeled here). The overall length of the extended MPD fragment is
more than 140 Å, which would be ample to wrap around 3 faces of the
MIP1 Heparin probes were used to model where the S-domains of MPD were most
likely to interact with the MIP1 Mutation of the key basic heparin-binding residues in MIP1
We thank Erika de Wynter (Leeds University, United Kingdom) and members of the Haematology Group (Paterson) for their continued support with this project.
Submitted November 16, 2001; accepted April 17, 2002.
Supported by Cancer Research UK.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Sally E. Stringer, Drug Development Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Rd, Withington, Manchester, M20 4BX, United Kingdom; e-mail: sallyelizabethstringer{at}yahoo.co.uk.
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 K. Kwong, R. A. Vaishnav, Y. Liu, N. Subhedar, A. J. Stromberg, M. L. Getchell, and T. V. Getchell Target ablation-induced regulation of macrophage recruitment into the olfactory epithelium of Mip-1{alpha}-/- mice and restoration of function by exogenous MIP-1{alpha} Physiol Genomics, December 15, 2004; 20(1): 73 - 86. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
), dimeric (
),
or tetrameric (
) MIP1
) in 0.15 M
NaCl. The same types of HS were also applied to a control column (
).
Bound material was eluted with a stepwise NaCl gradient
(. . . . . . .). Graphs are representative of duplicate
experiments.
, dodecasaccharides; and 
, tetradecasaccharides. Each graph is
representative of 2 or more experiments.
), 10 kDa (
(IFN-