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Originally published In Press as doi:10.1074/jbc.M505738200 on July 20, 2005

J. Biol. Chem., Vol. 280, Issue 37, 32200-32208, September 16, 2005
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Chemokine-Glycosaminoglycan Binding

SPECIFICITY FOR CCR2 LIGAND BINDING TO HIGHLY SULFATED OLIGOSACCHARIDES USING FTICR MASS SPECTROMETRY*{boxs}

Yonghao Yu{ddagger}§, Matthew D. Sweeney{ddagger}, Ola M. Saad{ddagger}§, Susan E. Crown¶, Tracy M. Handel¶, and Julie A. Leary{ddagger}1

From the {ddagger}Genome Center, Departments of Chemistry and Molecular Cell Biology, University of California, Davis, California 95616 and the §Department of Chemistry, and Department of Molecular and Cell Biology, University of California, Berkeley, California, 94720

Received for publication, May 26, 2005 , and in revised form, July 15, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Glycosaminoglycans (GAGs) have recently been demonstrated to be required for the in vivo activity of several chemokines. Minimally, the interaction is thought to provide a mechanism for retention at the site of secretion and the formation of chemokine gradients that provide directional cues for receptor bearing cells, particularly in the presence of shear forces. Thus, a key issue will be to determine the sequence and structure of the GAGs that bind to specific chemokines. Herein, we describe a mass spectrometry assay that was developed to detect protein-oligosaccharide noncovalent complexes, in this case chemokine-GAG interactions, and to select for high affinity GAGs. The process is facilitated by the ability of electrospray ionization to transfer the intact noncovalent complexes from solution into the gas phase. The elemental composition as well as the binding stoichiometry can be calculated from the mass of the complex. Ligands of the chemokine receptor, CCR2 (MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, MCP-4/CCL13, and Eotaxin/CCL11), and the CCR10 ligand CTACK/CCL27 were screened against a small, highly sulfated, heparin oligosaccharide library with limited structural variation. The results revealed heparin octasaccharides with 11 and 12 sulfates as binders. Oligomerization of some chemokines was observed upon GAG binding, whereas in other instances only the monomeric noncovalent complex was identified. The results indicate that, in contrast to the apparent redundancy in the chemokine system, where several chemokines bind and activate the same receptor, these chemokines could be differentiated into two groups based on the stoichiometry of their complexes with the heparin oligosaccharides.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines are small secreted proteins that are critically involved in many biological processes, including routine immunosurveillance, inflammation, and development. Some specific chemokine receptors provide the portals by which human immunodeficiency virus gets into cells, whereas others have been implicated in inflammatory diseases associated with inappropriate cell migration (rheumatoid arthritis, multiple sclerosis, atherosclerosis, and most recently, tumor metastasis) (1, 2). Thus there is considerable interest in understanding how chemokines function, and in identifying strategies that interfere with their function.

Based on the pattern of the cysteine residues, chemokines can be divided into CXC, CC, C, and CX3C families. Once secreted, chemokines are thought to form a concentration gradient that controls the direction of the leukocyte cell migration. This process is mediated by the interactions between the chemokines and G protein-coupled seven transmembrane receptors on leukocytes; the binding event subsequently triggers downstream signaling pathways that lead to cell migration and activation (3). Approximately 45 chemokines and 19 chemokine receptors have been identified to date. Characterization of which chemokines interact with which receptors has revealed significant cross-reactivity (e.g. redundancy) in vitro; in other words multiple chemokines bind and activate the same receptor, and a given chemokine can bind multiple receptors. Nevertheless this apparent redundancy may not exist in vivo; for example, significant effects in models of inflammation have been observed using knock-out mice, or antibodies and small molecule antagonists, despite the fact that they target single receptors or chemokines (4, 5). An emerging hypothesis is that glycosaminoglycans (GAGs)2 play a role in the in vivo function of chemokines (6–9) and that specificity in these interactions could alter the apparent redundancy (10).

GAGs are linear, highly sulfated, and heterogeneous polysaccharides that are often covalently linked to core proteins residing on the membrane of cells or within the extracellular matrix. They have been classified into several major families, primarily heparin/heparan sulfate, chondroitin sulfate/dermatan sulfate, hyaluronan, and keratan sulfate (11–13). Although they are ubiquitously expressed on virtually all animal cells, the exact composition of the GAG coat of a given cell/extracellular matrix depends on the type and location of the cell and the pathophysiological state of the tissue/organism (14). GAGs are thought to influence the local cytokine network through interactions with proteins and by modulating protein-protein interactions (15), and there is accumulating evidence for specificity in these interactions. For example, the sulfation pattern on GAGs is thought to be a major determinant in their interaction with proteins, which are often highly basic (16, 17). Chemokines have been shown to bind GAGs both in vitro and in vivo, and recently it was demonstrated that this interaction is required for their function in vivo (19, 20). Immobilization of chemokines on GAGs is thought to enhance their local concentration and facilitate the formation of chemokine gradients to guide the migration of cells, especially under flow conditions (19, 20). GAG binding may also help recruit chemokines to specific populations of cells, thereby contributing to the control of cell migration in a receptor independent way.

Oligomerization has also been shown to be required for some chemokines as oligomerization-deficient mutants that function like wild type (WT) in vitro, have been shown to be nonfunctional in vivo (19). Furthermore, this requirement seems to be coupled to GAG binding, because GAGs can induce some chemokines to oligomerize (19, 21). Thus, there are several sources where specificity in chemokine-GAG interactions may be derived: (i) the actual GAG-binding epitopes on the chemokines, and indeed, GAG-binding hotspots have been defined for several chemokines and show significant differences in their patterns (21), (ii) the oligomerization state that the chemokines adopt, and again differences have been reported (10, 19, 21), and (iii) the sequence of the GAGs, for which much less is known due to the difficulties in isolating and sequencing GAGs. If the argument for specificity is accurate, then the GAG interaction may significantly contribute to the localization of cells beyond that defined at the level of the chemokine-receptor interaction. The GAG interaction may, in fact, specify the use of one chemokine over another. Targeting chemokine-GAG interactions with tight binding GAG sequences or GAG mimetics that either block the interaction or compete for chemokine-receptor binding in vivo, could also form the basis of novel therapeutics. Along these lines, the objectives of this study are to develop methods for selecting and sequencing GAG ligands of a given chemokine, to determine the extent of their sequence diversity, and to establish if the "redundant" chemokines can be distinguished by their GAG-binding properties.

The characterization of chemokine-GAG interactions has been greatly facilitated by the development of reasonably efficient screening and detection techniques (22–26). However, there are still many limitations, and the resulting data have been somewhat nebulous. Affinity chromatography methods are slow and require considerable amounts of material (22), whereas oligosaccharide microarrays generally need pure and structurally defined oligosaccharides that are chemically synthesized or isolated from natural sources (25). In this report, a mass spectrometry approach was used to identify specific GAGs that bind to the chemokine ligands of CCR2. The assay is a label-free methodology that does not require chemical modification or immobilization of the protein target or the library components. The binding stoichiometry as well as the molecular formula of the oligosaccharide binders can be calculated from the masses of the protein/oligosaccharide noncovalent complex using electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry. This is possible given the fact that many structural features, including stoichiometry, ligand binding domain, and even tertiary geometry of solution noncovalent complexes can be preserved during the transition from solution to gas phase (27–31). Confirmation of the complexed oligosaccharides detected in the noncovalent complex was verified using hydrophobic trapping and elution. Identified oligosaccharides binders were spatially separated from the protein, collected, and analyzed by mass spectrometry. Specific GAG ligands were unambiguously determined by measuring the molecular mass of each ligand from a library of possible binders.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The heparin octasaccharide library was obtained from Dextra Laboratories (Reading, UK) and consists of a partial heparinase I digestion of heparin purified from porcine intestinal mucosa. The catalogue number for this library is ID1008. Sucrose octasulfate was purchased from Toronto Research Chemicals (Toronto, Canada). Thrombin and cyclodextrin sulfate were purchased from Sigma. Enterokinase was expressed and purified as described previously (32). All polymerases and restriction endonucleases were purchased from New England Biolabs (Beverly, MA).

Vector Construction, Expression, and Purification of Chemokines—Genes for the human chemokines MCP-3/CCL7, MCP-2/CCL8, Eotaxin/CCL11, and MCP-4/CCL13 were codon optimized for expression in Escherichia coli as described (33). Each gene was then cloned into modified versions of pET21, which include one or all of the following: a leader sequence for increased expression, a His tag for purification, and an enterokinase or thrombin protease site for removal of the tag (TABLE ONE).


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  		TABLE ONE
Expression conditions of the chemokines

 
MCP-1/CCL2 WT and CCL2 R18A/K19A/R24A were expressed and purified as described before (21). Both of these proteins have an additional mutation (M64I) to avoid oxidation during purification but show no effect on receptor-binding function (34). The CCL7 and CCL8 proteins were expressed as described (21).

CCL8 was lysed and purified by SP ion exchange and reverse-phase HPLC as described (21). After lyophilization, the protein was resuspended in 20 mM Tris, pH 8, 50 mM NaCl, 2 mM CaCl2 at 1 mg/ml and mixed with a 1:100 w/w ratio of enterokinase. After 48 h, the protein was acidified by addition of trifluoroacetic acid to pH 2–4 and purified by reverse-phase HPLC. Eluted CCL8 was lyophilized and stored at –20 °C.

CCL7-producing cells were lysed in 10 mM Tris, pH 8, 1 mM MgCl2 and clarified by centrifugation. The supernatant was applied to a Ni-NTA (Qiagen, Valencia, CA) column at room temperature. After washing with 20 mM imidazole, the protein was eluted with 10 mM Tris, pH 8, 300 mM NaCl, 250 mM imidazole. The eluate was then purified by reverse-phase HPLC, cleaved with a 1:250 w/w ratio of enterokinase for 48 h, and repurified as above.

CCL11-producing cells were lysed in 20 mM Tris, pH 8, 400 mM NaCl, 20 mM imidazole and purified on a Ni-NTA column as above. Eluate was dialyzed against 20 mM Tris, pH 8, 300 mM NaCl, 2.5 mM CaCl2. Thrombin was added at a ratio of 1:1000 w/w. After 72 h, the protein was purified by reverse-phase HPLC and lyophilized as above.

The CCL13 construct was transformed into BL21(DE3) pLysS cells that were then grown in LB at 37 °C to an A600 of 0.600. The cells were induced with 0.5 mM isopropyl 1-thio-{beta}-D-galactopyranoside and harvested after 4 h by centrifugation. The cells were lysed in 10 mM Tris, pH 8, 1 mM MgCl2, and the insoluble protein was isolated by standard methods. The final pellet was resuspended in 6 M guanidine-HCl, 20 mM Tris, pH 8, 10 mM imidazole. The extract was clarified before application to a Ni-NTA column. The protein was eluted with 6 M guanidine-HCl, 10 mM Tris, pH 8, 250 mM imidazole. Refolding was initiated by dropwise dilution into 55 mM Tris, pH 8, 264 mM NaCl, 11 mM KCl, 0.055% polyethylene glycol 3350, 1.1 mM EDTA, 0.1 mM dithiothreitol to a final protein concentration of 0.1 mg/ml. Refolded protein was dialyzed against 20 mM Tris, pH 8, 200 mM NaCl, then against 35 mM Tris, pH 8. Aminopeptidase was added at a 1:50 w/w ratio. After 120 h, cleaved protein was purified by reverse-phase HPLC and lyophilized as above.



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  		FIGURE 1.
A, ESI-FTICR mass spectrum of 5 µM MCP-1 sprayed from a solution containing 79:20:1 AcN:H2O:formic acid; B, ESI-FTICR mass spectrum of 5 µM MCP-1 sprayed from a solution of 100 mM NH4OAc. Inset, the isotopic distribution of the ions at m/z 2166. The mass to charge ratio of the dimer ion (8+ charge state) overlapped with that of the monomer ion (4+ charge state). However, the dimer ions were clearly resolved as the lower isotopic distribution in the high resolution FTICR mass spectra. No dimer was observed in the MCP-1 sprayed from the organic solvent, indicating the dimer was noncovalently associated.

 
Exact Mass Measurement of the Heparin Octasaccharide Library Components—The heparin octasaccharide library was analyzed by ESI-FTICR mass spectrometry using negative ion mode detection. Approximately 20 µM of the library was sprayed in a solvent containing 50:50 MeOH:H2O with 10 mM NH4OH. Addition of NH4OH or NH4OAc is known to prevent desulfation (35). Samples were infused into the mass spectrometer at 1 µl/min using a syringe pump (Harvard Apparatus, Holliston, MA). For exact mass determination, the octasaccharide library sample was mixed with a HPLC peptide standard mixture (Sigma), which was used as an internal calibrant. This mixture was comprised of leucine enkephalin (1–, m/z 554.2615), angiotensin II (2–, m/z 521.7594), and Val-Tyr-Val (1–, m/z 378.2029). The identified library components are shown in TABLE TWO.


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  		TABLE TWO
Exact mass measurement and identification of the octasaccharide components in the library

 
Filtration Trapping Screening Assay—For screening experiments, 40 µM of the target protein was mixed with 200 µM octasaccharide library in 100 µl of 100 mM NH4OAc (pH 6.8) at room temperature. To remove the nonspecific/low affinity binders, the protein-oligosaccharide mixture was washed three times with 1 ml of 200 mM NH4OAc (pH 6.8), and subjected each time to centrifugal ultrafiltration (5000 rpm, 40 min, 4 °C) using a molecular mass cutoff filter of 10 kDa (Millipore, Billerica, MA). After the last wash step, the solution was concentrated to ~50 µl. The retentate was diluted by addition of 1 volume of water and subjected to FTICR mass spectrometric analysis.

Hydrophobic Trapping, Elution, and Confirmation of Bound Ligands—Forty micromolar of the target protein was incubated with 200 µM octasaccharide library in 100 µl of a 100 mM NH4OAc solution (pH 6.8). The solution was then applied to an Oasis solid-phase extraction (SPE) cartridge (Waters, Milford, MA) that was previously conditioned according to the manufacturer's instructions. The nonspecific binders were removed by flushing the column three times, each with 1 ml of a 200 mM NH4OAc solution. The high affinity ligands were eluted by using 1 ml of a 760 mM NH4OAc solution. The eluate was desalted by dialyzing against water in a Bio-dialyzer unit with a molecular mass cutoff of 1 kDa (The Nest Group, Southborough, MA) and analyzed by FTICR mass spectrometry using negative ion mode detection.

FTICR Mass Spectrometry—Mass spectra were acquired on a Bruker APEX II 7-tesla FTICR mass spectrometer (Billerica, MA) equipped with an Apollo (Bruker, Billerica, MA) electrospray ionization (ESI) source. Generation of the noncovalent complex ions was performed as previously described (30). Briefly, samples were infused into the mass spectrometer at 1 µl/min using a syringe pump (Harvard Apparatus, Holliston, MA). Ions were desolvated in the nozzle-skimmer region by using a 140-V (positive mode) or 40-V (negative mode) capillary exit voltage. Following desolvation, ions were externally accumulated in a radio frequency-only hexapole for 0.5–1 s and were transferred into the ICR cell for mass analysis. Two to four ion packets were trapped using gated trapping and detected after chirp excitation. Between 8 and 100 broadband time domain transients containing 512 k or 1024 k data points were averaged before zerofill, Gaussian multiplication and fast Fourier transform. The parameters of the ESI source, ion optics, and cell were tuned for the best ion intensity. The FTICR mass spectra were internally calibrated against myoglobin or MCP-1. All the data were acquired and processed using Xmass version 6.0.0 (Bruker).

The average masses of the noncovalent complexes of MCP-1 dimer and oligosaccharide ligand were determined by using the method as previously reported by Zubarev et al. (36). Briefly, portions of the isotopic distribution above 50% relative intensity of the highest isotopic peak were used to calculate the average mass, Mave(P/L)', of the protein-ligand complex. In accordance with this methodology, a correction factor ({Delta}m50%) of 0.5 Da was then added to Mave(P/L)' to derive the true Mave(P/L). The average mass of the protein, Mave(P), was calculated from the known elemental composition. The average mass of the oligosaccharide was then determined from Mave(L) = Mave(P/L)–Mave(P).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
ESI-FTICR Analysis of WT MCP-1—The data provided below show that specific GAG-chemokine noncovalent interactions can be accurately determined from gas phase data. Various preliminary experiments were required to prove that these complexes are accurate reflections of the solution complexes. Therefore, sample preparation and parameters affecting the ionization process were critically optimized.

The ESI mass spectra of wild type (WT) MCP-1 are shown in Fig. 1. Fig. 1A represents WT MCP-1 sprayed from a solution of 79:20:1 acetonitrile:H2O:formic acid, in which the molecular mass was measured to be 8662.4615 Da. The theoretically most abundant mass of MCP-1, taking into account the fact that it and most other chemokines have two disulfide bonds, is 8662.4685 Da (1). This translates into a mass error of 0.8 ppm of the measured exact mass, clearly indicating the presence of two disulfide bonds.

When MCP-1 was sprayed from a solution of 100 mM NH4OAc (pH 6.8), a solvent environment more conducive to the folded state of the protein, a tighter charge state distribution was observed, as shown in Fig. 1B. In accordance with previous ultracentrifugation studies, MCP-1 exists in equilibrium between the monomer and dimer forms (37). The 4+, 5+, and 6+ charge states of the monomer ion and the 9+ charge state of the dimer ion were clearly observed in the mass spectra. The inset shows the overlap of the 8+ charged dimer with the 4+ charged monomer, indicating that a distinction can be made between the monomeric and dimeric states.



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  		FIGURE 2.
Mass spectrum of the heparin octasaccharide library. The octasaccharide library was sprayed from a solution containing 50:50 MeOH:H2O with 10 mM NH4OH. The mass spectrum was internally calibrated using a peptide mixture containing leucine enkephalin, angiotensin II, and Val-Tyr-Val. The 6-charge state of the octasaccharides is shown. The identified octasaccharide components are summarized in TABLE TWO. The asterisk represents the internal calibrant ion of Val-Tyr-Val. Additional smaller ions represent ammonium, water, and potassium adducts. No smaller (dp6) or larger (dp10) oligosaccharides were observed.

 
Screening of Specific MCP-1 Ligands from Heparin Octasaccharide Library—Previously, it was reported that heparin interacted with MCP-1 (6, 21). The apparent dissociation constant between unfractionated heparin and WT MCP-1 was determined to be 1.55 µM using an isothermal fluorescence titration assay. In addition, the study showed that a heparin octasaccharide mixture was able to interact with MCP-1 and to induce oligomerization (21). However, the octasaccharide library contained multiple components and the identities of the specific ligands in the library were unknown. Thus, a preliminary goal was to examine the MCP-1/GAG interaction by first identifying the specific MCP-1 oligosaccharide binding ligands in the library. Therefore, a small selection of the highly sulfated heparin structures with limited structural variation was compared in this analysis. This proof-of-principle methodology will eventually be applied to a heparan sulfate library, which is more biologically relevant.

The heparin octasaccharide library was initially analyzed by ESI-FTICR mass spectrometry using negative ion detection. As shown in Fig. 2, at least eight unique octasaccharides were detected in the library: each compound was identified with both high precision and high mass accuracy (TABLE TWO). Five of the octasaccharides were modified only with sulfates, and varied from octa-sulfation to dodeca-sulfation. Three of the identified octasaccharides were both sulfated (octa-, nona-, or deca-) and acetylated (mono-). Exact mass measurements of the library components provided a direct indication of their degree of sulfation and acetylation, although positional isomers could not be differentiated at this stage in the analysis.

Wild type MCP-1 (40 µM) was mixed with 200 µM of the octasaccharide library (30), and the solution was loaded onto an ultrafiltration unit. After washing the retentate with 200 mM NH4OAc, the resulting solution was analyzed by ESI-FTICR mass spectrometry. As shown in Fig. 3A, two MCP-1 dimer/octasaccharide cluster ions were observed at m/z 2445 and 2455. These two ions were not present in the mass spectra of MCP-1 alone. Although MCP-1 exists in equilibrium between monomer and dimer, only the dimer was observed to bind to the octasaccharide ligands. The two WT MCP-1/octasaccharide noncovalent complexes ions were still observed after successive washings using 500 mM NH4OAc (data not shown), suggesting that these octasaccharides are specific binders (26). To evaluate whether the selection of the two octasaccharides is biased toward the ionic component of the binding forces (22), the experiment was performed using a range of salt concentrations, including 50 mM, 100 mM, 150 mM, and 200 mM NH4OAc to wash the retentate. The results of washing with 150 mM and 100 mM NH4OAc were the same compared with that of the 200 mM salt wash (data not shown). In addition to octasaccharide/11SO3 and octasaccharide/12SO3, only very minor MCP-1 dimer in complex with octasaccharide/10SO3 was observed when using 50 mM NH4OAc as the salt wash. These data clearly indicate that octasaccharide/11SO3 and octasaccharide/12SO3 preferentially bind to MCP-1. In addition, it is clear that, using 200 mM NH4OAc, a salt wash concentration that is close to the physiological ionic strength, the selection of these ligands are not biased toward the ionic component of the binding forces to any significant extent.

Screening of GAG binders was repeated using an MCP-1 triple mutant, R18A/K19A/R24A, which was shown previously to have a significantly reduced GAG binding ability (21). Incubation of the library with the mutant showed no binding with any of the heparin octasaccharides (Fig. 3B). Although this protein is considerably basic, complexation with the negatively charged GAGs was not observed, indicating that nonspecific association is not occurring using the outlined methodology.

The elemental compositions of the ligands bound to MCP-1 were determined from the masses of the noncovalent complexes. The average mass of the protein/ligand complex, Mave(P/L), can be estimated by its most abundant mass with an error of <1 Da (36). However, the determination of the most abundant mass, even in a well resolved isotopic distribution, is often complicated by the similar intensities of a set of peaks. A misidentification could result in at least 1 Da error of the molecular mass of the noncovalent complex, and, in turn, the ligand. The error in the determination of the average mass of a macromolecule can be minimized, however, by using a method described by Zubarev et al. (36) as was done herein to calculate Mave(P/L). The average mass of the noncovalent complex represented by the ion at m/z 2445 was determined to be 19,555 Da. Using 17,326 Da as the average mass of the MCP-1 dimer, the average mass of the ligand in this complex was calculated to be 2,229 Da. An octasaccharide with 11 sulfates was the only possible candidate given the identification of the library components prior to incubation with the protein. Thus, the ion at m/z 2,445 was determined to be the 8+ charge state ion of the noncovalent complex of MCP-1 dimer/octasaccharide containing 11 sulfates. Using a similar procedure, the ion at m/z 2,455 was determined to be the 8+ charge state ion of the noncovalent complex of MCP-1 dimer/octasaccharide with 12 sulfates. The theoretical isotopic distributions of these two ions were then simulated, and the mass errors were calculated to be 3.5 and 2.2 ppm, respectively (Fig. 4). The octasaccharide with 12 sulfates is the fully sulfated octasaccharide (N-, 2-O-, and 6-O-) and most likely has a structure as shown in Fig. 5A. However, the sulfate positions on the octasaccharide with 11 sulfates are unknown and may well be composed of a mixture of different positional isomers. Methods to concentrate the amount of GAG off the protein are currently being explored so that complete sequence/structural analysis can be undertaken.



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  		FIGURE 3.
ESI-FTICR mass spectra of WT MCP-1 and the RKR mutant after incubation with the heparin octasaccharide library and wash with 200 mM NH4OAc. A, WT MCP-1 was incubated with the heparin octasaccharide library and was subjected to the filtration trapping assay. Two ions corresponding to the MCP-1 dimer in complex with the octasaccharide ligands were observed. These ions were not observed when MCP-1 alone was sprayed from the same buffer. OctaX and OctaY were determined to be octasaccharide/11SO3 and octasaccharide/12SO3, respectively, as shown in Fig. 4. B, the non-GAG-binding mutant R18A/K19A/R24A was subjected to the same assay in which no complexation was observed.

 
Hydrophobic Trapping Mass Spectrometry—As a complementary screening approach, a hydrophobic trapping method was used to elute the specific ligands off the chemokine and verify the results from the filtration trapping screening assay. Similar to other traditional procedures (22), the proteins and protein complexes were separated from the heparin octasaccharides based on the difference between their hydrophobicity. Proteins and protein-ligand complexes are more hydrophobic and are trapped by an Oasis reverse-phase solid-phase extraction (SPE) column (38). In contrast, the heparin oligosaccharides containing multiple sulfates are hydrophilic and can be readily eluted from the column.



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  		FIGURE 4.
Elemental composition determination of the octasaccharide ligands. A, the experimental isotopic distributions of the MCP-1 dimer in complex with the octasaccharide ligands. B, the theoretical isotopic distributions of complexes, simulated using the calculated molecular formula of the octasaccharides. The mass measurement errors were calculated and shown herein.

 



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  		FIGURE 5.
Structures of polysulfated molecules. A, heparin octasaccharide with 12SO3, which was identified as a ligand of MCP-1. B, sucrose octasulfate. C, cyclodextrin sulfate.

 
A solution of 40 µM MCP-1 mixed with 200 µM octasaccharide library in 100 mM NH4OAc was applied to the hydrophobic SPE column and assayed. Nonspecific/weak binders were eluted using 200 mM NH4OAc, whereas remaining ligands that eluted using 760 mM NH4OAc were regarded as specific ligands of MCP-1. As shown in Fig. 6, the octasaccharides with 11 and 12 sulfates were detected in the 760 mM eluate and thus identified from the library as the specific ligands. These results are in agreement with those obtained from the filtration trapping assay. To ensure that the identification of the octasaccharide ligands was not the result of nonspecific binding to the hydrophobic column, control experiments were conducted in which an ammonium acetate solution containing only the heparin octasaccharide library was subjected to the same trapping assay. As shown in Fig. 6C, no octasaccharides were detected in the high salt elution fraction, which clearly indicates that the octasaccharides with 11 and 12 sulfates were bound specifically to MCP-1.



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  		FIGURE 6.
Hydrophobic trapping assay of the heparin octasaccharide library against MCP-1. The nonbinders were removed using 200 mM NH4OAc and the bound ligands were eluted using 760 mM NH4OAc. A, the octasaccharide library standard. B, the 760 mM salt elution fraction. Octasaccharide with 11 sulfates and 12 sulfates were detected, indicating that they were bound to MCP-1. C, control experiment. Library alone was subjected to the same hydrophobic trapping assay. No octasaccharides were detected in the high salt elution fraction, indicating the identification of octasaccharide with 11 and 12 sulfates as the ligands of MCP-1 was not due to the nonspecific binding between these molecules and the hydrophobic SPE column.

 
The method was also applied to detect the interactions between MCP-1 and smaller polysulfated molecules. In two parallel experiments, 40 µM MCP-1 was incubated with sucrose octasulfate and cyclodextrin sulfate, each with a concentration of 200 µM. Sucrose octasulfate was observed in the 760 mM salt elute fraction, which clearly indicated that it was a binder of MCP-1. In contrast, cyclodextrin sulfate was not detected in the high salt eluate (Supplementary Fig. S1). These data, for both of the small molecular weight compounds, were also in complete accordance with the results of the filtration trapping assay data (data not shown).

Screening of the Octasaccharide Library against Additional CCR2 Chemokines—Glycosaminoglycan binding and oligomerization characteristics were further investigated using several other chemokine ligands of CCR2, including MCP-2/CCL2, MCP-3/CCL7, MCP-4/CCL13, and Eotaxin/CCL11, as well as the CCR10 ligand CTACK/CCL27. Results of previous structural and biophysical studies have indicated that, in the absence of GAG, MCP-3 (39) and Eotaxin (40) are largely monomeric in solution, whereas MCP-2 forms a dimer (41). CTACK also forms a monomer in solution (unpublished AUC data). As observed in the data presented here for MCP-1, the MS data were completely consistent with the solution data; MCP-2 was observed to be in equilibrium between monomer and dimer, whereas MCP-3, MCP-4, Eotaxin, and CTACK existed primarily as monomers (TABLE THREE, spectra provided as supplementary material). However, the identification of the GAG ligands and the stoichiometry of the chemokine-GAG complexes were not previously known. These issues were addressed by using both the filtration trapping and hydrophobic trapping assays. Each chemokine was incubated with the heparin octasaccharide library in the NH4OAc solution. Octasaccharides with 11 and 12 sulfates were again observed to bind to all additional chemokines, however, to different extents and with different resulting oligomerization states. Minor noncovalent complexes containing the chemokines and the octasaccharide with 10 sulfates were also observed for MCP-3, MCP-4, and Eotaxin. The chemokines, however, showed significant differences in their binding stoichiometry (TABLE THREE). Although free MCP-2 exists in equilibrium between monomer and dimer in solution, only the dimer form was found to bind to octasaccharides, as observed for MCP-1. In contrast, MCP-3, MCP-4, Eotaxin, and CTACK only formed the monomer/octasaccharide complexes. Based on the intensity of the chemokine-octasaccharide complex ions, it also appeared that the octasaccharide ligands were bound to MCP-4 more tightly than MCP-3, Eotaxin, or CTACK. Although no quantitative binding constants were measured, these experiments are currently under investigation.


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  		TABLE THREE
The heparin octasaccharide ligands and the oligomerization states of different chemokines, as determined by the filtration trapping assay

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Chemokines bind to cell-surface GAGs, the sulfation pattern of which are cell-type specific, and tightly regulated developmentally, spatially, and temporally. Our working hypothesis is that the cellular and extracellular matrix GAGs may function as modulators of chemokines in vivo by influencing their localization, presentation, and density, and they may even affect signaling properties. However, very little is known about the structures of the heparin/heparan sulfate oligosaccharides that bind to chemokines or the structures and oligomeric states of the complexes.

To detect and identify specific protein-GAG interactions, several methods have been developed, including filter trapping, affinity chromatography, and oligosaccharide microarrays. These methods possess some limitations. A direct mass spectrometry-based assay (26) for screening of protein-binding oligosaccharides has been developed by Keiser et al. (26), in which surface noncovalent affinity mass spectrometry is implemented. In this case, the target protein is chemically modified and immobilized on a hydrophobic surface. A heparin oligosaccharide library is incubated with the immobilized protein target and treated with salt washes to remove the low affinity binders. Using matrix-assisted laser desorption ionization mass spectrometry, the specific binders are detected as noncovalent complexes with a positively charged peptide, (RG)19R. Surface noncovalent affinity mass spectrometry is a screening methodology that integrates isolation, enrichment, and sequencing into one single procedure. However, this method also has limitations: the protein must be active after immobilization. In addition, an oligosaccharide ligand may elude detection, because it binds to the protein target tightly and is not abstracted by the (RG)19R peptide. Moreover, using this method, the critical information of the binding stoichiometry is lost when the carbohydrate ligands are transferred from the protein target to the (RG)19R peptide.

Label-free, mass spectrometry-based methods to screen and identify the heparin oligosaccharide ligands of chemokines, and the oligomerization states of the chemokines and chemokine-GAG complexes, are presented in this study. In the filtration trapping assay, the heparin oligosaccharide library was incubated with the target protein and, after washing, the specific binders were directly detected as noncovalent complexes with the target protein. Compared with other screening methods for detection of the protein-carbohydrate interactions, this assay has several advantages. First, it accomplishes the screening from solution, which circumvents the immobilization of either the protein target or the small molecule library. Thus, this method is not hindered by the potential complications of immobilization; e.g. protein denaturation, signal suppression, lengthy optimization of the linker length, and disruption of the ligand binding site, etc. Additionally, the chemokine/oligosaccharide binding stoichiometry can be readily extracted from the mass of the noncovalent complexes, which has proven to be critical in determining the activity of many chemokines in vivo (10). Finally, the molecular formula of the oligosaccharide ligands can be determined from the calculated average mass of the complexes. The mass spectrometry-based assays were applied to a selection of highly sulfated structures with limited structural variation; i.e. octasaccharides containing 8, 9, 10, 11, and/or 12 sites of sulfation (TABLE TWO). Based on these experiments, specific binding of octasaccharides with 10, 11, and/or 12 sulfates was observed for several different chemokines known to interact with CCR2, but not with the less sulfated oligomers present in the library.

Several steps were taken to ensure that the octasaccharides were specific binders. In one case, a centrifugal ultrafiltration unit with a molecular mass cutoff of 10 kDa was used to remove the low affinity oligosaccharides while retaining the larger protein-octasaccharide noncovalent complexes (42). It is possible that the selection of protein-binding oligosaccharides based on the concentration of the salt wash used may bias results toward the electrostatic component of the binding forces (22). To test this possibility, the screening experiment was optimized using salt washes with a series of salt concentrations, including 50, 100, 150, and 200 mM NH4OAc. The exact same ligand profile was obtained utilizing 100 or 150 mM salt wash, as compared with 200 mM NH4OAc salt wash. In addition to octasaccharide/11SO3 and octasaccharide/12SO3, only very minor octasaccharide/10SO3 was observed to be in complex with MCP-1 using 50 mM NH4OAc salt wash. These data clearly indicate that using 200 mM NH4OAc, a salt concentration that is close to the physiological ionic strength, is not biased toward the selection of these ligands. A particularly important check on specificity was a control experiment, in which the non-GAG-binding mutant R18A/K19A/R24A showed no binding, consistent with previous work (21). This was reinforced through studies with cyclodextrin sulfate using the filtration trapping assay. Although cyclodextrin sulfate is modified with 14–15 sulfates per molecule, and has the highest degree of sulfation among the molecules investigated, it showed no binding to MCP-1. This indicates that binding of the sulfated oligosaccharide ligands is not based on nonspecific charge-charge interactions. Finally, the fact that results from the hydrophobic trapping experiments were identical to the filtration trapping assay also reflects binding specificity.

Currently, we do not know whether the heparin octasaccharides with 10 and 11 sulfates show any positional preferences among the different chemokines; future sequencing experiments will focus on this question. So although significant selectivity of GAGs based on composition alone is apparent from this current study, exact specificity as it relates to sequence and isomeric preference has yet to be determined. Heparin is also not the most relevant GAG with respect to cell surface localization, and applying this methodology to heparan sulfate GAGs may well be very informative. Heparin GAGs may be more involved in protection of chemokines from proteolysis, storage, or inhibition of chemokines from premature receptor binding. They are also not as heterogeneous as heparan sulfate and thus may be less likely to show major differences in specificity. However, use of the heparin GAGs was chosen initially for methods development and proof of principle, because it is commercially available in size-defined forms and is inexpensive.

Nevertheless, even with heparin, differences in the chemokine octasaccharide interactions were observed in their oligomerization properties. The first group includes MCP-1 and MCP-2, which bind as dimers to heparin octasaccharides with 11 and 12 sulfates. The second group of chemokines, consisting of MCP-3, MCP-4, Eotaxin, and CTACK, bind to the same highly sulfated octasaccharides, however, they remain monomeric. Minor binding to heparin octasaccharide with 10 sulfates was also observed for MCP-3, MCP-4, and Eotaxin. These results indicate that despite the receptor-binding redundancy of these chemokines, they can be distinguished with respect to GAG binding based on their oligomerization states. This is quite important, and it is the first reported observation of such specificity in vitro. Future studies with heparan sulfate may reveal further differences, in both oligomerization and sequence preferences.

The results also add to the accumulating evidence that glycosaminoglycan binding and oligomerization are coupled for some chemokines. The first evidence for GAG-induced oligomerization involved a solid phase assay where it was observed that addition of cold chemokine, to a mixture of radiolabeled chemokine incubated in the presence of heparin-Sepharose beads, recruited additional radiolabel (7). Later work showed that both GAG binding-deficient mutants and oligomerizationdeficient mutants of MCP-1/CCL2, RANTES/CCL5, and MIP-1{beta}/CCL4, were all nonfunctional in an in vivo intraperitoneal cell recruitment assay, even though they could stimulate cell migration in vitro (19). In additional studies that indicated a mechanistic connection between GAG binding and oligomerization, heparin octasaccharides were shown to induce tetramer formation of MCP-1 by AUC (21).

However, it appears that not all chemokines need to oligomerize to function. The naturally monomeric chemokines MCP-3, Eotaxin, and IP-10 are all active in vivo. Although one could argue that they could oligomerize when bound to GAGs, the data presented here suggest that at least for MCP-3 and Eotaxin, this is not the case, and AUC data support the MCP-3 results.3 Thus, the MS assay provides a powerful high throughput screen for determining oligomerization states of chemokines in solution by themselves and in the presence of GAGs.

Based on these results, we have been able to distinguish between two classes of CCR2 chemokines. Importantly, the MS recapitulated solution based results for the oligomerization states of MCP-1, -2, -3, Eotaxin, CTACK, and the non-GAG-binding mutant of MCP-1 in the absence of GAGs, and it provided new information on the GAG-bound states, including complexes that could not be studied by AUC due to precipitation (e.g. Eotaxin plus octasaccharide).

In future studies we will expand these filtration trapping assays to determine the sequence of the tight binding GAGs. This will pave the way for chemical synthesis of homogeneous GAGs that can be used for structural and other biophysical studies of chemokine-GAG complexes. In addition, the importance of the chemokine-GAG interaction suggests a new strategy for blocking chemokine function in the treatment of disease. Indeed, soluble heparin has been shown to inhibit chemokine activity in vivo (8). However, low molecular weight, homogeneous, tight binding GAGs, selected for particular chemokines, could provide much more potent and specific alternatives.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants GM63581 (to J. A. L.) and AI37113-09 (to T. M. H.), and by U.C. Discovery (Grant Bio03–10367, to T. M. H.) and U.C. AIDs (Grant 1D03-B-005, to T. M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S6. Back

1 To whom correspondence should be addressed. Tel.: 530-754-4987; Fax: 530-754-9658; E-mail: jaleary{at}ucdavis.edu.

2 The abbreviations used are: GAG, glycosaminoglycan; AUC, analytical ultracentrifugation; HPLC, high performance liquid chromatography; FTICR, Fourier transform ion cyclotron resonance; ESI, electrospray ionization; SPE, solid-phase extraction; CCR, CC chemokine receptor; WT, wild type; Ni-NTA, nickel-nitrilotriacetic acid; MCP-1, monocyte chemoattractant protein-1; MS, mass spectrometry. Back

3 Y. Yu, M. D. Sweeney, O. M. Saad, S. E. Crown, T. M. Handel, and J. A. Leary, unpublished data. Back



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