Identification of Heparin-binding Sites in Proteins by Selective Labeling

Identification of Heparin-binding Sites inProteins by Selective Labeling*□S

Alessandro Ori‡, Paul Free‡, Jose Courty§, Mark C. Wilkinson‡¶, and David G. Fernig‡¶�

Heparan sulfate proteoglycans are key regulators of com-plex molecular networks due to the interaction of theirsugar chains with a large number of partner proteins, whichin humans number more than 200 (Ori, A., Wilkinson, M. C.,and Fernig, D. G. (2008) The heparanome and regulation ofcell function: structures, functions and challenges. Front.Biosci. 13, 4309–4338). We developed a method to selec-tively label residues involved in heparin binding thatmatches the requirements for medium/high throughput ap-plications called the “Protect and Label” strategy. This isbased on the protection against chemical modificationgiven by heparin/heparan sulfate to the residues located inthe heparin-binding site. Thus, analysis of fibroblast growthfactor-2 bound to heparin and incubated with N-hydroxy-succinimide acetate showed that lysines involved in thesugar binding are protected against chemical modification.Moreover following release from heparin, the protected ly-sine side chains may be specifically labeled with N-hydroxy-succinimide biotin. After protein digestion, the biotinylatedpeptides were readily isolated and identified by MALDI-Q-TOF mass spectrometry. The analysis of labeled peptidesobtained from three well characterized heparin-bindingproteins with very different heparin-binding sites, fibroblastgrowth factor-2, platelet factor-4, and pleiotrophin demon-strates the success of this new approach, which thus pro-vides a rapid and reliable procedure to identify heparin-binding sites. Molecular & Cellular Proteomics 8:2256–2265, 2009.

Heparan sulfate proteoglycans are a ubiquitous componentof the extracellular space of complex organisms (1). They arecharacterized by their size and their plasticity that derivesmainly from the complex and dynamically regulated structureof the glycosaminoglycan (GAG)1 moiety.

They participate in the structural organization of the extra-cellular space (2, 3) and play an active role in molecularnetworks driving complex biological phenomena such as de-velopment (4–6), inflammation and immune response (7, 8),and disease (9). Heparan sulfate proteoglycans exert theirfunctions by interacting with a vast number of protein partnersand so regulating their activity (8, 10).

In the last decade, a number of techniques have been used toinvestigate the interaction between proteins and GAGs (for re-views, see Refs. 11–15). However, despite the identification ofmore than 200 human heparin-interacting proteins (10, 16), ourunderstanding of the structural features mediating the interac-tion remains quite poor (10, 13). Some models have been pro-posed, but they are based on the structural features of onlyrestricted groups or families of heparin-binding proteins (HBPs)(11, 17, 18). Important limitations for modeling of heparin-bind-ing sites (HBSs) are that many interactions are described solelyat a qualitative level and that a three-dimensional structure ofthe sugar-protein complex is available for less than 10% of theannotated interactions (10). The available data derive mainlyfrom x-ray crystallography (e.g. Refs. 19 and 20), NMR spec-troscopy (e.g. Ref. 21), and quantitative biophysics with site-directed mutagenesis (e.g. Ref. 22) or HBP-derived syntheticpeptides (e.g. Ref. 23). Although providing detailed structuraland kinetic information about the interaction, all these methodsare limited to the study of single protein-sugar interactions, andthey cannot be translated into a high throughput format.

In 1989 Chang (24) used a lysine-reactive chromophoricreagent to investigate the HBS of antithrombin III (ATIII). ATIIIwas modified in the presence or absence of heparin, and thecolored peptides generated by tryptic digestion were ana-lyzed by RP-HPLC to obtain important insights into the hep-arin-ATIII interaction. N-Hydroxysuccinimide (NHS) estershave a strong and selective reactivity toward primary amines,in particular �-amines on lysines, and their relative stability inneutral/weakly basic aqueous solution makes them com-pounds of choice for the investigation of HBSs, which arecharacterized by a high content of basic residues.

We developed a rapid and reliable method for the localiza-tion of HBSs that can represent an important complement toany structural investigation of heparin-protein interactions be-cause of its low requirement in terms of sample quantity andhandling time. The new method, called the “protect and label”strategy, is based on the protection against chemical modifi-cation given by heparin/HS to residues located in the HBSs.Thus, an acetyl NHS ester was used to protect residues

From the ‡School of Biological Sciences and Centre for Glycobi-ology, University of Liverpool, Liverpool L69 7ZB, United Kingdomand §Laboratoire de Recherche sur la Croissance Cellulaire, la Rep-aration et la Regeneration Tissulaires (CRRET), CNRS UMR 7149,Universite Paris XII, 94010 Creteil Cedex, France

Received, January 21, 2009, and in revised form, June 22, 2009Published, MCP Papers in Press, June 30, 2009, DOI 10.1074/

mcp.M900031-MCP2001 The abbreviations used are: GAG, glycosaminoglycan; HBS, he-

parin-binding site; HBP, heparin-binding protein; HS, heparan sulfate;NHS, N-hydroxysuccinimide; TSR, thrombospondin type-1 repeat;ATIII, antithrombin III; RP, reverse phase; FGF, fibroblast growthfactor; PTN, pleiotrophin; PF-4, platelet factor-4.

Research

© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.2256 Molecular & Cellular Proteomics 8.10This paper is available on line at http://www.mcponline.org

exposed on the protein surface but not involved in heparinbinding, and a second reagent, NHS-biotin, was used toselectively label residues in the HBS. The identification bymass spectrometry of the biotinylated peptides, derived fromenzymatic digestion of the HBP, provides a fingerprint ofsugar binding on the protein surface.

EXPERIMENTAL PROCEDURES

Heparin-binding Proteins

Recombinant fibroblast growth factor-2 (FGF-2) (Swiss-Prot ac-cession number P09038) (residues 1–154) was expressed in C41Escherichia coli cells using a pET-14b system (Novagen, MerckChemical Ltd., Nottingham, UK) as described previously (25).Pleiotrophin (PTN), also known as heparin-binding growth-associatedmolecule or heparin affin regulatory peptide (Swiss-Prot accessionnumber P21246) (residues 33–168), was produced as described pre-viously (26). Platelet factor-4 (PF-4) (Swiss-Prot accession numberP02776) (residues 32–101) was purchased from Athens Research andTechnology Inc. (Athens, GA).

SDS-PAGE and Western Blot

Samples were separated on 15% (w/v) polyacrylamide-SDS gels.Gels were silver-stained according to Heukeshoven and Dernick (27).Samples separated by polyacrylamide-SDS gels were transferred toHybondTM nitrocellulose membrane (GE Healthcare) using a semidrysystem (Trans-Blot SD, Bio-Rad). Membranes were blocked withPBS-BSA (8.1 mM Na2HPO4, 1.2 mM KH2PO4, 150 mM NaCl, 2.7 mM

KCl, pH 7.0 (PBS) supplemented with 5 mg/ml BSA (Sigma-Aldrich))for 30 min at room temperature. The membranes were then incubatedin PBST-BSA (PBS supplemented with 0.5% (v/v) Tween 20 (PBST)and 5 mg/ml BSA) with Strep-TactinTM-horseradish peroxidase (IBAGmbH, Gottingen, Germany) (1:7500) for 30 min at room temperature.After at least five washes of 5 min with PBST, the presence ofhorseradish peroxidase was revealed with the SuperSignal West PicoChemiluminescent substrate (Pierce).

Peptide Map

Digested proteins were applied to an Agilent (Warrington, UK)Zorbax Eclipse Plus C18 RP-HPLC column (100 � 2.1 mm, 3.5 �m)equilibrated in 0.08% (v/v) TFA using a Beckman Coulter (High Wy-combe, UK) System GoldTM HPLC system. Peptides were separatedwith a 95-min gradient of 0–64% (v/v) ACN in 0.08% (v/v) TFA. Elutionwas monitored at 214 nm.

Desalting

Proteins were desalted with a 5-ml HiTrapTM desalting column (GEHealthcare) using an AKTATM HPLC system (GE Healthcare). Proteinswere isocratically eluted with the selected buffer at a flow rate of 0.5ml/min. Elution was monitored at 214 and 280 nm.

Protection and Labeling of HBS

AF-heparin beads (Tosoh Biosciences GmbH, Stuttgart, Germany;binding capacity of 4 mg of ATIII/ml of resin) were preferred to othersbased on polysaccharide matrix (e.g. Sepharose) to minimize poten-tial unspecific interactions that could increase the noise level of themethod. The loading capacity for FGF-2 was estimated at 15 mg/mlof resin. Furthermore the same resin has been successfully used in across-linking method (28).

Step 1: Binding—A 20-�l slurry of AF-heparin beads was packed ina minicolumn consisting of a P10 tip with a plastic air filter placed at

its end to retain the resin. A 5-ml sterile syringe was used to pack theminicolumn and dispense buffers. The heparin column was equili-brated with 4 � 50 �l of PB 150 buffer (17.9 mM Na2HPO4, 2.1 mM

NaH2PO4, 150 mM NaCl, pH 7.8) and loaded with either 1.2 nmol ofFGF-2, 2.6 nmol of PF-4, or 1 nmol of PTN dissolved in 200 �l of PB150 or buffer of compatible ionic strength. Loading was repeatedthree times to ensure complete binding. The minicolumn was thenwashed with 4 � 50 �l of PB 150.

Step 2: Acetylation of Exposed Lysines—The heparin column withbound protein was first equilibrated by a quick wash with 20 �l of PB150 containing 50 mM sulfo-NHS-acetate (Pierce) and then incubatedwith 20 �l of PB 150 containing 50 mM sulfo-NHS-acetate for 5 min atroom temperature to ensure complete blocking (acetylation) of ex-posed lysines. To stop the reaction, the column was extensivelywashed with 4 � 50 �l of PB 150. Acetylated protein was eluted fromheparin with 40 �l of 2 M NaCl buffered with PB buffer (44.75 mM

Na2HPO4, 5.25 mM NaH2PO4, pH 7.8).Step 3: HBS Lysine Biotinylation—Eluted protein was diluted with

200 �l of PB buffer (to give a final NaCl concentration of 0.33 M) andconcentrated with a 5-kDa-molecular mass cutoff centrifugal filter(Sartorius Ltd., Epsom, UK). After adjusting the volume to 37.2 �l withPB, biotinylation was performed by the addition of 2.8 �l of 145 mM

NHS-biotin (Pierce) in DMSO (final concentration, 10 mM) and incu-bation for 30 min at room temperature. The reaction was quenchedwith 4 �l of 1 M Tris, pH 7.5. The excess reagent was removed by fourcycles of sequential dilution with 200 �l of 10 times diluted PB (4.48mM Na2HPO4, 0.52 mM NaH2PO4, pH 7.8) and concentration with a5-kDa-molecular mass cutoff centrifugal filter. The sample was thendried by centrifugal evaporation.

Step 4: Protein Digestion—The dried sample was dissolved with 25�l of 8 M urea, 400 mM NH4HCO3, pH 7.8 and 2.5 �l of 45 mM DTT andincubated for 15 min at 56 °C. Proteins were carbamidomethylatedwith 2.5 �l of freshly made 0.1 M iodoacetamide for 15 min at roomtemperature in the dark. Samples were diluted with 70 �l of HPLCgrade water and digested overnight at 37 °C with 1.5 �g of chymo-trypsin (Sigma-Aldrich).

Step 5: Biotinylated Peptide Purification—A 40-�l slurry of Strep-Tactin Sepharose beads (IBA GmbH) were packed in a minicolumn(see above), which was equilibrated with 4 � 50 �l of 500 mM urea, 25mM NH4HCO3. Digested protein was diluted to a final volume of 400�l with HPLC grade water and applied to the column. Loading wasrepeated three times to ensure complete binding. Unbound peptideswere removed by 3 � 50-�l washes with 0.5 M urea, 25 mM NH4HCO3

followed by 2 � 50-�l washes with HPLC grade water. Biotinylatedpeptides were eluted with two lots of 20 �l of 80% (v/v) ACN, 20%(v/v) TFA, 5 mM biotin.

Steps 6 and 7: Identification of Labeled Peptides—The eluate wasconcentrated by rotary evaporation, desalted using a C18 ZipTipTM

(Millipore Ltd., Watford, UK) according to manufacturer’s instructionsand analyzed by MALDI-Q-TOF mass spectrometry.

MALDI-Q-TOF Mass Spectrometry

Samples (1 �l) were spotted 1:1 (v/v) in 5 mg/ml �-cyano-4-hy-droxycinnamic acid (Sigma-Aldrich) in 50% (v/v) ACN, 50% (v/v)HPLC grade water. Data-dependent analysis was performed on thetop 10 most intense peptides in the parent spectrum, the instrumentswitching to the next peptide for MS/MS after a minimum of 100counts had been achieved. Collision gas was argon, laser energy wasfixed at 250, the instrument was SYNAPTTM MALDI HDMS (WatersCorp., Manchester, UK) where CID can occur in either the trap or thetransfer collision cell. A ramp profile in the trap collision cell of 34–44V for m/z 700–750 up to 151–161 V for m/z 3350–3400 was used.Depending on the m/z of the peptide selected for MS/MS the corre-sponding energy ramp was applied. MS/MS spectra were processed

Selective Labeling of Heparin-binding Sites of Proteins

Molecular & Cellular Proteomics 8.10 2257

using the MassLynx v4.0 software (Waters Corp.). Peak lists weregenerated using the MaxEnt3 algorithm applying a 5% base peakintensity cutoff. Data analysis was performed using the MS-tag tool ofthe Protein Prospector package v.5.2.2 using the following parame-ters: digest, chymotrypsin; maximum missed cleavages, 5; possiblemodifications, acetyl (Lys), biotin (Lys), carbamidomethyl (Cys), andcarboxymethyl (Cys); parental ion tolerance, 100 ppm; fragment iontolerance, 300 ppm; nonspecific cleavage, at one terminus. The Uni-Prot accession number of the protein analyzed was used as a pre-search parameter. All other settings were used at default values. Inthe case of modified parental ions (e.g. sodium adduct ions), theMS-product tool of the Protein Prospector package v.5.2.2 was usedusing default settings.

RESULTS

Method Development—First the kinetics of protein modifi-cation by NHS reagents were investigated. The signal/noiseratio of the protect and label procedure was found to dependon the level of modification (acetylation) of lysines exposed onthe protein surface (Fig. 1, step 2). Despite the widespreaduse of NHS-derived reagents in chemistry and biochemistry,the kinetics of reaction with biomolecules in aqueous solutionhave been only partially elucidated (29–31). A series of exper-iments using two different model proteins were performed toestablish the optimal NHS reagent concentration and incuba-

tion time that ensured a high level of lysine modification(supplemental Figs. S1 and S2).

To demonstrate the ability of heparin to selectively protectlysines present in the HBS, a series of pilot experiments wereperformed using FGF-2 as a model protein. FGF-2 was acety-lated according to the conditions chosen in the absence or inthe presence of heparin beads (under “Experimental Proce-dures” see “Protection and Labeling of HBS” steps 1 and 2).After buffer exchange, heparin binding activity was assessedby application of the modified proteins to a heparin minicol-umn. The same amount of native FGF-2 was applied to theminicolumn as a control. A further sample, obtained by ex-posing FGF-2 acetylated in the presence of heparin beads toNHS-biotin (under “Experimental Procedures” see “Protectionand Labeling of HBS” step 3), was also applied to the heparinminicolumn. This last sample was included because it repre-sented the final product of the protect and label strategy.Lysine acetylation clearly impaired the binding of FGF-2 toheparin (Fig. 2A, sample 2). However, when the acetylationwas performed in the presence of heparin beads, the proteinretained almost completely its binding activity (Fig. 2A, sam-ple 3). This suggests a selective protection given by the sugar

1. Binding

HBP

Heparin Bead 4. Protein digestion

Biotinylated Peptide

NHS-acetate

NHS-biotin

NaClchymotrypsin

5. Purification of labeled peptides

StrepTactin™ Bead

6. Elution and MS1 analysis80 % ACN20 % TFA

5 mM biotin

TOP 10peptides

7. MS2 and assignment

2. Protection of exposed lysines

3. Dissociation and labeling of HBS lysines A

B

FIG. 1. The protect and label strategy. A, the protect and label strategy consists of three main steps. The first is the modification/blockingof exposed lysines (steps 1 and 2). HBP is bound to heparin. The protein is then modified by incubation with sulfo-NHS-acetate. This resultsin the acetylation of primary amines exposed on the protein surface, the N terminus and �-amines of lysines. The second is the labeling oflysines protected by heparin/HS (step 3). The protein is then dissociated from the sugar by increasing the ionic strength of the solution. Thefollowing incubation with NHS-biotin leads to selective labeling of lysines involved in the interaction with heparin that have not been blockedin the previous step. The third is the identification of labeled peptides (steps 4–7). The modified protein is enzymatically digested. Biotinylatedpeptides are purified by affinity chromatography using a Strep-Tactin minicolumn. Following elution captured peptides are identified byMALDI-Q-TOF mass spectrometry. B, the reactions of protection (acetylation) and labeling (biotinylation) mediated by NHS esters areschematized.


2258 Molecular & Cellular Proteomics 8.10

http://www.mcponline.org/cgi/content/full/M900031-MCP200/DC1

chain to lysines involved in heparin binding. Furthermore theretained binding activity of sample 3 (acetylated in the pres-ence of heparin beads) could be abolished by further expo-sure to NHS-biotin (Fig. 2A, sample 4).

After demonstrating that heparin can effectively prevent themodification of residues crucial for its binding, the next stepwas to show that those residues can be efficiently labeled(biotinylated) after dissociation of the protein from the sugar

(Fig. 1, step 3). FGF-2 was acetylated in the absence orpresence of heparin beads and buffer-exchanged. Both thesamples were then biotinylated as in step 3 of the protect andlabel strategy (see “Experimental Procedures”). Native andfully biotinylated FGF-2 (same conditions as above but with-out prior acetylation) were used as a negative and positivecontrol, respectively. Biotinylated proteins were identified byWestern blot. The acetylation condition chosen guaranteed a

31 33 35 37 39 41 43 45 47 49 51

Retention time (min)

0

1x104

2x104

3x104

4x104

5x104

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OD

214

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(µA

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31 33 35 37 39 41 43 45 47 49 51

Retention time (min)

0

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Strep-Tactin™ purified peptides from FGF-2:- biotinylated w/o prior acetylation (II)

- acetylated in presence of heparin and then biotinylated (IV)

10 kDa

20 kDa25 kDa

37 kDa50 kDa75 kDa

I II IVIII -S

biotinylation - + + +heparin - - - +acetylation - - + +

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S

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L

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FIG. 2. Heparin/HS protect residues involved in their binding against chemical modification. A, silver stain. S, standard; L, 1⁄10 of theprotein loaded onto the microcolumn; FT, 1⁄10 of the unbound fraction. 1, FGF-2; 2, acetylated FGF-2 (protein was incubated with 50 mM

sulfo-NHS-acetate for 5 min at room temperature); 3, FGF-2 acetylated in the presence of heparin beads (under “Experimental Procedures”see “Protection and Labeling of HBS” steps 1 and 2); 4, FGF-2 acetylated in the presence of heparin beads (same as 3) and then biotinylatedafter dissociation from the sugar with NHS-biotin at a final concentration of 10 mM for 30 min at room temperature. All samples werebuffer-exchanged to the same buffer (PB 150) by gel filtration chromatography before application to the heparin minicolumn. The heparinminicolumn was packed using 20 �l of AF-heparin beads and equilibrated in PB 150. B, Western blot. I, FGF-2; II, FGF-2; III, acetylated FGF-2(protein was incubated with 50 mM sulfo-NHS-acetate for 5 min at room temperature); IV, FGF-2 acetylated in the presence of heparin beads(under “Experimental Procedures” see “Protection and Labeling of HBS” steps 1 and 2). Samples were buffer-exchanged to PB 150 as in (A).Samples II–IV were then biotinylated with NHS-biotin at a final concentration of 10 mM for 30 min at room temperature. Protein concentrationswere measured after the desalting step, and the same amount of protein was subjected to biotinylation (samples II–IV) and then SDS-PAGE.C, biotinylated peptides from enzymatic digestion of samples II and IV were purified with a Strep-Tactin minicolumn using 80% (v/v) ACN, 20%(v/v) TFA as elution buffer and analyzed by RP-HPLC. Asterisks indicate peaks deriving from peptides selectively protected from acetylationby heparin and, therefore, subsequently biotinylated. HRP, horseradish peroxidase; �AU, microabsorbance units; w/o, without.



high level of protection against further biotinylation (Fig. 2B,sample III). However, the presence of heparin during theacetylation step partially prevented the modification of resi-dues that can, therefore, be biotinylated after dissociationfrom the sugar (Fig. 2B, sample IV). To establish whether thereduced protection was a generalized phenomenon involvingall lysines exposed on the protein surface, biotinylated pep-tides derived from sample II (fully biotinylated FGF-2) andsample IV (protected and labeled FGF-2) were compared byRP-HPLC (Fig. 2C). As expected, a generally reduced recov-ery of biotinylated peptides was observed in sample IV. Im-portantly the difference between the elution profiles indicatesthat the presence of heparin is selectively affecting the pro-tection of a restricted number of residues (Fig. 2C).

The last step of the implementation of the method was theoptimization of a high efficiency protocol for the purificationand identification of labeled peptides derived from the enzy-matic digestion of the product of the protect and label strat-egy (Fig. 1, steps 4–7). The high affinity of the avidin-biotininteractions allows the use of stringent conditions for thepurification of biotinylated biomolecules; however, it limits theyield of recovery that is achievable. An array of capture sup-ports and rationally designed elution buffers were comparedin a model experimental setting to maximize the efficiency ofrecovery of biotinylated peptide and ensure compatibility withthe subsequent mass spectrometric analysis (supplementalFig. S3). Strep-Tactin Sepharose was identified as the bestaffinity capture support (data not shown). Regarding the com-position of the elution buffer, the peptide recovery was en-hanced by increasing the concentration of both acid (TFA) andsolvent (ACN) up to the extreme condition where water wasexcluded from the buffer composition (80% ACN, 20% TFA)(supplemental Fig. S3B). Further improvement was achievedby the addition of a large excess of competitor (5 mM biotin) tothe elution buffer (supplemental Fig. S3B).

Identification of HBSs by the Protect and Label Strategy—The protect and label strategy was then evaluated for itsability to correctly identify HBSs in well characterized heparin-binding proteins. Labeled peptides were identified by MALDI-Q-TOF mass spectrometry. The 10 most intense ions for eachsample were automatically selected in the quadrupole, and

MS/MS data were acquired to assign chemical modificationsintroduced during the protect and label procedure (Fig. 1,steps 6 and 7, and supplemental spectra). Furthermore frag-mentation spectra allowed a further control on the affinitypurification of labeled peptides. A modification-specificmarker ion at m/z 310.16, assigned as Lys(biotin)-NH3, was infact observed in all the fragmentation spectra analyzed. Thisis consistent with the ion at m/z 126.1 previously described asa marker for lysine acetylation (biotin � acetyl � 184) (32) andwith the presence in most of the spectra of the ions m/z227.08 and 84.08 corresponding to biotin and Lys-NH3,respectively.

The test proteins were chosen to represent the variety ofthe heparin interactors as highlighted by the heparin interac-tome characterized so far (10). Two growth factors were se-lected, FGF-2 and PTN. The first is a prototypical member ofthe FGF family of growth factors and one of the most studiedHBPs, whereas the second is characterized by the presenceof a thrombospondin type-1 repeat (TSR) motif responsiblefor the interaction with the sugar (33). PF-4 was added as anexample of a chemokine that interacts with heparin as anoligomer and that possesses a bipartite HBS (18).

The structure of FGF-2 is described as �-trefoil, comprisinga 3-fold repeat of a four-stranded antiparallel �-sheet (34).The main HBS of FGF-2 has been extensively characterizedby different approaches including site-directed mutagenesis,synthetic peptides, x-ray crystallography, and NMR spectros-copy (19, 22, 34–37). It consists of residues Lys35, Asn36, andthe cluster of basic amino acids located in the region 128–144(22) (Table I). These residues form an extremely basic surfaceincluding strand I/strand II loop (Lys35 and Asn36), strandX/strand XI loop, strand XI, and strand XI/XII loop (Fig. 3B).The biotinylated peptides identified by the protect and labelstrategy mapped to three areas on the protein surface (TableII) (Fig. 3A). These include the previously described HBS (Ta-ble II, peptides 1, 2, 6, 7, 9, and 10), a region located in strandVI/strand VII loop (Table II, peptide 5), and the beginning ofstrand I (Table II, peptides 3, 4, and 8). The loop identified bypeptide 5 is located on the same surface of the sequence115YRSRKYSSWY124 described by Baird et al. (36) and thesequence 117SRKYTSWYVA126 described by Kinsella et al.

TABLE IHBS of protect and label strategy test proteins

The HBSs and the methods used to identify these are given. SDM, site-directed mutagenesis; x-ray, x-ray crystallography; SP, syntheticpeptide; MM, molecular modeling.

NameSwiss-Prot

accession no.Molecular

masspI HBS Method Ref.

Da

FGF-2 P09038 17,253.8 9.58 HBS-1: 35KN36, Arg90, 128KRTGQYKLGSKTGPGQK144 SDM, x-ray 19, 22HBS-2: 102FFFERLESNNYNTYRSRKYSSWYVALKR129 SP 36

PF-4 P02776 10,844.9 8.93 HBS-1: 77KNGR80, 92KKIIKK97 NMR, SDM, MM 18, 21HBS-2: 51RPRH54, Lys62, Thr56 NMR, SDM 21

PTN P21246 18,942 9.66 HBS-1: 97AECKYQFQAWGECDLNTALKTRTGSLKRALHNA129 SP 38








(23) as a possible secondary HBS. No evidence has beenpublished to date regarding the involvement of strand I inheparin binding. MS/MS spectra allowed the assignment ofmodified residues and the identification of peptides derivingfrom unspecific cleavage (Table II, peptide 4) or other chem-ical modifications (e.g. deamination (Table II, peptide 1) andsodium adduct (Table II, peptide 9)). Only in the case of the ionat m/z 1371.71 (Table II, peptide 5) was a double assignmentnecessary. Fragment ions deriving from both the isobaricpeptides 83LAMK(biotin)EDGRLL92 (MH� monoisotopicmass, 1371.7123) and 27K(biotin)DPK(biotin)RLY33 (MH� mo-noisotopic mass, 1371.6912) were in fact assigned in theMS/MS spectrum (supplemental Spectrum S5).

PF-4 belongs to the CXC family of chemokines. The mono-meric structure consists of a triple stranded antiparallel�-sheet overlaid by a C-terminal �-helix, whereas the N-terminal region is essentially disordered (18, 21). PF-4 existsas a tetramer in solution. Dimerization occurs through theantiparallel association of the first strands to form a larger�-sheet (18). The tetramer results from the further assembly of

two dimers mediated by contacts between N-terminal regions(18). The HBS has been characterized by site-directed mu-tagenesis coupled to NMR spectroscopy (21) and by molec-ular modeling (18). The HBS consists of a ring of positivelycharged residues around the surface of the tetramer formedby two pairs of lysines located within the C-terminal �-helix(92KKIIKK97), two arginines from the N terminus/strand I loop(51RPRH54), Lys77 and Arg80 from the strand II/strand III loop(77KNGR80), and the residues Lys62 and Thr56 (18, 21) (TableI) (Fig. 3D). Peptides including biotinylated Lys77 (Table II,peptides 11–13, 15–17, and 20) and Lys62 (Table II, peptide19) were identified in agreement with published data. Lys81

was found biotinylated in five cases in association with la-beled Lys77 (Table II, peptides 11–13, 15, and 16) and in onecase as a single biotinylated peptide (Table II, peptide 18).

PTN is a heparin-dependent growth factor characterized bythe presence of two TSR-I motifs. It consists of two disor-dered basic clusters at its N and C termini and two �-sheetscontaining the TSR-I motifs (N-TSR, residues 45–71; C-TSR,residues 97–129) (33, 38). The two TSR motifs have been

FIG. 3. Labeled peptides and heparin-binding site: test proteins. A, FGF-2 (residues 25–153) is shown using overlaid schematic andsurface representations (Protein Data Bank code 1FQ9). Heparin-derived octasaccharide is shown as sticks. B, FGF-2 (residues 25–153) isshown using schematic representation only (Protein Data Bank code 1FQ9). Heparin-derived octasaccharide is shown as sticks. C, primarystructure of PTN (residues 1–167). D, tetrameric PF-4 (residues 38–101) is shown using overlaid schematic and surface representations (ProteinData Bank code 1F9Q). Previously described HBS lysines are shown in red (see Table I). Lysines labeled by the protect and label strategy areshown in green (see Table II). Yellow indicates an overlap between labeled amino acids and previously described HBS residues. Other residuesare shown in gray. E, tetrameric PF-4 is shown as in C but in addition the HBS sequence 51RPRH54 of each monomer is highlighted in orange.Structures are represented using PyMol.




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proposed as possible HBSs (33); however, Hamma-Kourbaliet al. (38) recently demonstrated the involvement of only theC-TSR in heparin binding (Table I). Labeled peptides mappedlysines located at the beginning (Table II, peptides 24–28) andin the center (Table II, peptide 30) of the C-TSR (Fig. 3C).Biotinylated lysines deriving from the region between the twoTSR domains (Table II, peptides 21–23 and 28) (Fig. 3C) werealso identified.

DISCUSSION

One of the main limitations on the study of heparin/HS-protein interactions is the lack of rapid and reliable methodsfor the identification of HBSs. A new strategy for the identifi-cation of HBSs was developed. Reaction conditions able toensure a high level of protein modification by NHS reagentswere established. A high efficiency protocol for the recoveryof labeled (biotinylated) peptides was optimized. Automatedtandem mass spectrometry and partially manually curateddata interpretation allowed the assignment of labeled resi-dues in 28 of the 30 peptides selected for the three samplesanalyzed. Data obtained were used to evaluate the ability ofthe new method to correctly predict HBSs in well character-ized heparin-binding proteins.

The results show that (i) heparin can effectively prevent themodification of residues in a protein that are crucial for itsbinding to the polysaccharide (Fig. 2A, sample 3), (ii) furtherexposure to NHS-biotin after dissociation of the protein fromthe sugar can abolish the retained heparin binding activity(Fig. 2A, sample 4), (iii) the residues that remain unmodifiedduring acetylation in the presence of heparin can be effec-tively labeled (biotinylated) (Fig. 2B, sample IV), and (iv) thelabeling is not a generalized phenomenon but is restricted toa specific set of lysines (Fig. 2C). Thus, the application of theprotect and label strategy (steps 1–3) allows the specificlabeling of lysines crucial for heparin binding that, by defini-tion, will represent the HBS.

The protect and label strategy allowed the correct localiza-tion of the main HBS of all the test proteins, providing a strongrationale for the validity of the method. Some important pointshighlight the advantages and limitations of the new method. Inthe case of FGF-2, the canonical HBS was successfullymapped by the biotinylated peptides, including the loop be-tween strand I and strand II (34CKNGGFF40) even though thecontribution of the lysine to the binding is relatively minor (22).The second region identified, strand VI/strand VII loop(83LAMKEDGRLL92), is adjacent to a proposed secondaryheparin-binding site (23, 36, 39) of approximately millimolaraffinity. The region including residues Lys119, Tyr120, andTrp123 has been proposed also as a secondary receptor-binding site (40). Thus the FGF-2 mutants K119E, Y120A, andW123A displayed decreased mitogenic activity on Swiss 3T3fibroblasts when compared with wild type FGF-2 (40). Theseresidues were shown to be responsible for a secondary bind-ing event between FGF-2 and its receptor that is dependent

on the presence of heparin (40). Residue Lys86 and residuesArg118 and Lys119 are located in two adjacent loops (distanceof Lys86 C� to Lys119 C�, 9.3 Å) (Fig. 3, A and B), forming abasic patch on the protein surface that could better describethe secondary binding site proposed with the use of syntheticpeptides. More difficult is the interpretation of the signal de-riving from the N terminus of the protein. The N terminus ofFGF-2 is a highly disordered and mobile region (34, 41). Itsinvolvement in heparin binding has been excluded by deletionmutagenesis (39) because mutants lacking 23 (N14 FGF-2)and 49 (N41 FGF-2) N-terminal amino acids displayed unal-tered affinity for heparin compared with a 146-amino acidform (FGF-2-(11–157)) as judged by heparin affinity chroma-tography (39). However, this analysis is qualitative, and boththe mutants were less efficient in stimulating DNA synthesis inBALB/c3T3 cells when compared with FGF-2-(11–157) (39),supporting a functional role for these residues. Alternatively,labeling of the N terminus may arise due to protection derivingfrom a conformational change induced upon binding toheparin.

All the lysines that are affected by major chemical shiftchanges upon binding of heparin to a tetrameric form of PF-4(21) were successfully identified by the protect and labelstrategy with the exception of the two pairs of lysine locatedin the C-terminal helix. The sequence 92KKIIKK97 was firstproposed as the center of the HBS of PF-4 and also used forthe development of a heparin-binding consensus sequenceby Cardin and Weintraub (17). However, investigation by NMRspectroscopy and site-directed mutagenesis revealed a minorcontribution of this region to sugar binding (21). Side chainresonances of residues Arg53, His54, Thr56, Lys62, and Arg80

were more perturbed upon heparin binding than those ofC-terminal lysines (21). Furthermore substitution of all fourlysines to alanines reduced the affinity of the mutant PF-4 forheparin only by a factor 4 as assessed by a fluorescencebinding assay (21). In agreement with these data, the protectand label strategy supports the AD interface of the PF-4tetramer (Fig. 3E) as the core of the heparin-binding regionwith C-terminal lysines involved in secondary and more tran-sitory contacts, which are unable to ensure protection ofthese residues against chemical modification.

Finally the selective labeling confirmed recent data (38)indicating the C-TSR domain as the main HBS of PTN (Fig.3C). However, the biotinylation of other residues includingLys81, Lys86, Lys92, and Lys93, located at the boundariesbetween N-TSR and C-TSR, suggest a possible involvementof this area, which has not been investigated previously, inheparin binding.

In summary, the examples cited above indicate that theprotect and label strategy can represent a useful tool for thelocalization of unknown HBSs in heparin-binding proteins.The method displayed a great sensitivity for the detection ofheparin-binding surfaces together with a good level of spec-ificity despite the high lysine content of the protein tested (14



for FGF-2, eight for PF-4, and 28 for PTN) with the possibleexception of the highly flexible N-terminal region of FGF-2.The main limitation of the technique remains the identificationof regions involved in the sugar binding that do not containlysines (e.g. the 51RPRH54 region of PF-4; Fig. 3E). The pro-tect and label strategy can be considered as an in vitrodocking that, because of its low requirements in terms ofmaterial (nanomolar range) and sample handling time (2bench days), can find applications as a hypothesis generatorfor further mutagenesis studies or as a complementary tech-nique for structural studies, e.g. in silico docking.

In 2004 Vives et al. (28) reported a new method for defin-ing amino acid residues involved in heparin binding basedon cross-linking between HBP and heparin beads and N-terminal sequencing of the captured peptides. This method,recently updated to an “in-solution” version (42), sharesadvantages with the protect and label strategy, such as thelow requirement of material and its rapidity, as well asdrawbacks, such as being based on NHS chemistry. How-ever, the cross-linking reaction occurs upon binding of theprotein to a preactivated and hence modified sugar,whereas the protect and label strategy introduces chemicalmodification on protein-heparin complexes assembled inpseudophysiological conditions. Furthermore the combina-tion of the selective labeling of peptides with MS/MS se-quencing techniques allows the translation of the protectand label strategy into a medium/high throughput formatwhere a semipurified mixture of HBPs are analyzed at thesame time, and their HBSs are mapped due to the uniquemass signatures deriving from the labeled peptides. Theapplication of the method could possibly be expanded toother electrostatic interactions (protein-GAG, protein-pro-tein, and protein-nucleic acid) provided there is the involve-ment of lysines in at least one of the binding partners. Finallythe development of new protein modification reagents ableto selectively target different amino acids, e.g. guanido-specific compounds (43), could further expand the applica-bility of protect and label strategies.

Acknowledgments—We thank Dr. Liam Brady (Waters Corp.), Dr.D. A. Ward and M. C. Prescott (Functional Proteomics facility, Uni-versity of Liverpool) for MS data acquisition and Professor J. E.Turnbull for reviewing the manuscript.

* This work was supported by the European Union Marie CurieEarly Stage Training Programme (MolFun) (to A. O.), the Cancer andPolio Research Fund and the North West Cancer Research Fund (toA. O., M. C. W., and D. G. F.), and “Agence National de la Recher-che” Grant P002754 and “Institut National du Cancer” Grant PL06-093 (to J. C.).

□S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.

¶ Both authors contributed equally to this work.� To whom correspondence should be addressed. Tel.: 44-151-

795-4471; Fax: 44-151-795-4406; E-mail: [email protected].

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Identification of Heparin-binding Sites in Proteins by Selective Labeling