Natural Cytotoxicity Receptors NKp30, NKp44 and NKp46 Bind to

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Natural Cytotoxicity Receptors NKp30, NKp44 and NKp46 Bind to Different Heparan Sulfate/Heparin Sequences

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Marie-Lyn Hecht,†,# Benyamin Rosental,‡,# Tim Horlacher,† Oren Hershkovitz,‡ Jose L. De Paz,†,§ Christian Noti,†,| Stefan Schauer,⊥ Angel Porgador,‡ and Peter H. Seeberger*,† Laboratory for Organic Chemistry, Swiss Federal Institute of Technology (ETH) Zurich, Wolfgang-Pauli-Str. 10, 8093 Zurich, Switzerland, The Shraga Segal Department of Microbiology and Immunology and the National Institute for Biotechnology in the Negev, Ben Gurion University of the Negev, Beer Sheva 84105, Israel, Grupo de Carbohidratos, Instituto de Investigaciones Quı´micas, CSIC, Americo Vespucio, 49, 41092 Sevilla, Spain, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom, and Functional Genomics Center Zurich, UNI ETH Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland Received September 8, 2008

Natural Killer (NK) cells recognize and destroy tumors and virus-infected cells in an antibody-independent manner. The regulation of NK cells is mediated by activating and inhibiting receptors on the NK cell surface. One important family of activating receptors is the natural cytotoxicity receptors (NCRs) which include NKp30, NKp44 and NKp46. The NCRs initiate tumor targeting by recognition of heparan sulfate on cancer cells. This study aims to elucidate heparan sulfate structural motifs that are important for NCR binding. Microarray and surface plasmon resonance experiments with a small library of heparan sulfate/heparin oligosaccharides helped to clarify the binding preferences of the three NCRs. We demonstrate that the NCRs interact with highly charged HS/heparin structures, but differ in preferred modification patterns and chain lengths. The affinity of NKp30 and NKp44 for synthetic HS/heparin is approximately one order of magnitude higher than the affinity of NKp46. We further show the relevance of synthetic HS/heparin for the binding of NCRs to tumor cells and for NCR-mediated activation of natural killer cells. In conclusion, NCRs recognize different microdomains on heparan sulfate with different affinities. Keywords: natural killer cell • natural cytotoxicity receptors • heparan sulfate • heparin • carbohydrate microarrays • surface plasmon resonance

Introduction Natural killer (NK) cells are cytotoxic lymphoid cells specialized in destroying tumors and virus-infected cells.1 Unlike cytotoxic T lymphocytes, NK cells do not express antigenspecific receptors. The recognition of transformed cells occurs via the association of a multitude of cell-surface receptors with surface markers on the target cell. The NK cell surface receptors can be distinguished according to whether they activate or inhibit NK cell-mediated cytotoxicity. Numerous interactions between different receptors appear to lead to the formation of synapses between NK and target cells. The integration of activating and inhibiting signals at the synapse dictates whether or not the NK cells exert their cytolytic function on the target cell.2 Among the activating receptors, the family of Ig-like molecules is termed natural cytotoxicity receptors (NCRs). * To whom correspondence should be addressed. E-mail: seeberger@ org.chem.ethz.ch. Phone: + 41 44 633 21 03. Fax: + 41 44 633 12 35. † Swiss Federal Institute of Technology (ETH) Zurich. ‡ Ben Gurion University of the Negev. # These authors contributed equally. § Instituto de Investigaciones Quı´micas, CSIC (current address). | University of Cambridge (current address). ⊥ Functional Genomics Center Zurich.

712 Journal of Proteome Research 2009, 8, 712–720 Published on Web 02/06/2009

These natural cytotoxicity receptors include NKp30, NKp44 and NKp46 molecules. The NCRs are key triggering receptors in tumor cell recognition.3-8 All three NCRs are involved in the clearance of both tumor and virus-infected cells. The antiviral activity is initiated by the interaction of NKp44 with hemagglutinin of influenza virus or Sendai virus. NKp46 targets virusinfected cells by binding to influenza virus hemagglutinin or Sendai virus hemagglutinin-neuraminidase.9-11 In contrast, Arnon et al. showed that NK cell-mediated cytotoxicity is inhibited by binding of NKp30 to the human cytomegaloviral protein pp65.12 While several viral ligands of the NCRs are described, relatively little is known about the cellular targets of the NCRs. Protein-carbohydrate interactions are a common theme in NK cell targeting. Among the activating and inhibiting receptors are several members of the C-type lectin family. These receptors recognize both protein and sugar parts of the MHC class I molecules.13,14 In an attempt to identify cellular targets of NCRs, the three lytic receptors were probed for interaction with different carbohydrate structures. It has been shown that the NCR members interact with heparan sulfate (HS) proteoglycans.15-18 Basic surface patches on the NCRs were found. 10.1021/pr800747c CCC: $40.75

 2009 American Chemical Society

Natural Cytotoxicity Receptors NKp30, NKp44 and NKp46 Mutations of positively charged residues within these patches result in reduced efficiency of NK cells to recognize tumors due to failure of the killer cells to bind to the acidic HS proteoglycan. The finding that NK cells recognize HS on target cells is intriguing since aberrant glycosylation often coincides with the pathological transformation of cells. To study the role of HS/ heparin in NK cell-mediated cell lysis, details about the molecular interactions between NCRs and HS are of great interest. Structure-activity studies of HS proteoglycans have so far been hampered by the structural complexity of these macromolecules. HS and its relative heparin are characterized by repeating units of D-glucosamine (GlcN) and either L-iduronic acid (IdoA) or D-glucuronic acid (GlcA), whereby the amount of IdoA lies between 30% and 50% in HS and is greater or equal 70% in heparin. During their biosynthesis, both heparin and HS undergo multiple enzymatic modifications, including Osulfation at C2 of the uronic acids and at C3 and/or C6 of glucosamine. Additionally, glucosamine can be N-sulfated, N-acetylated or in form of a free amine. The result is a wide spectrum of heterogeneous molecules with countless different modification patterns.19 This large variety of modifications explains why heparin-type molecules are involved in the regulation of many cellular functions and interact with numerous proteins.20 The enormous structural complexity of HS/ heparin renders it difficult to establish structure-function relationships. A promising strategy to overcome these difficulties is the use of synthetic HS/heparin oligosaccharides. Structurally defined HS/heparin oligosaccharides displaying different modification patterns can provide useful structureactivity relationship information for HS. Numerous synthetic efforts yielded HS/heparin structures.21-25 Importantly, it has been shown that a small set of synthetic oligosaccharides allows for in-depth analyses of the glycosaminoglycan modification patterns at the molecular level.26,27 Here, we investigated the binding preferences of the three known NCRs, namely, NKp30, NKp44, and NKp46, for the synthetic HS/heparin oligosaccharides. The receptors were expressed as soluble NKp-Ig fusion proteins and probed for interaction with immobilized HS/heparin. Microarray and surface plasmon resonance (SPR) experiments were employed to study protein-carbohydrate interactions. The microarrays allow for multiple binding events to be determined in many replicas while using minimal amounts of material.26,28-31 SPR monitors one interaction in real-time and allows for quantification of the binding strength. We show that NKp30 and NKp46 recognize highly charged HS/heparin epitopes that are Osulfated at C2 of iduronic acid and bear one to two sulfate groups at the GlcN moiety. NKp30 preferentially binds the fully sulfated hexasaccharide, whereas NKp46 interacts stronger with the analogous tetrasaccharide. NKp44 displays a different binding pattern with 2-O-sulfation of IdoA as well as Nacetylation of GlcN contributing to the binding.

Materials and Methods General. If not otherwise specified, chemicals were purchased from Sigma-Aldrich. All aqueous solutions were made from nanopure water. Solutions used for chip hybridizations were sterile filtered through a 0.2 µm syringe filter prior to use. NHS-activated CodeLink slides were purchased from Amersham Biosciences. Microarrays were constructed using a Perkin-Elmer noncontact printer. For all microarray incubations, the protein solutions were overlaid with Hybri-slip hybridiza-

research articles tion covers for even distribution. Bound protein was detected using a goat anti-human IgG (H + L) antibody labeled with Alexa Fluor 647 (Invitrogen). Slides were scanned using a LS400 scanner (Tecan, Ma¨nnedorf, Switzerland) and the Array-Pro Analyzer software (MediaCybernetics, Bethesda, MD). Signals were quantified using Gene Spotter software (MircroDiscovery GmbH, Berlin, Germany). SPR measurements were performed on a BIAcore T100 (BIAcore, Uppsala, Sweden) operated by the BIAcore control software. HBS-N buffer, surfactant P20 and CM5 chips were purchased from BIAcore. Natural Cytotoxicity Receptors. NCRs were examined as recombinant soluble IgG Fc chimeras. NKp30 and NKp44 consisted of a single IgG-like V-type domain fused to human IgG1. NKp46D2 was an IgG1 fusion protein containing only the membrane-proximal D2 domain of NKp46. NKp30, NKp44 and NKp46D2 were expressed in CHO cells. Purification of the recombinant proteins has been described previously.15-17 Microarray Fabrication. Synthetic HS/heparin oligosaccharides 1-13 were prepared as described previously.26,32 HS/ heparin 9 was synthesized according to published procedures.33 Heparin 14 was prepared by functionalization of deaminated 5 kDa heparin with 1,11-diamino-3,6,9-trioxaundecane by reductive amination. HS/heparin oligosaccharides were dissolved in sodium phosphate buffer (50 mM, pH 8.5) and printed robotically using a piezoelectric spotting device (S11, Scienion, Berlin, Germany) onto NHS-activated CodeLink slides in 75% relative humidity at 23 °C. All samples were printed at four different concentrations (1, 0.25, 0.063, and 0.016 mM) in replicates of 10. Slides were stored in an anhydrous environment. Prior to the experiment, slides were washed three times with water to remove noncovalently attached carbohydrates from the surface. Remaining succinimidyl groups were quenched by incubating slides in 100 mM ethanolamine in sodium phosphate buffer (pH 9, 50 mM) for 1 h at 50 °C. Slides were rinsed three times with water and dried by centrifugation. Microarray Binding Assay. The arrayed slides were blocked for 1 h with 100 µL of 1% (w/v) BSA, and 0.01% (v/v) Tween20 in HBS-N (10 mM HEPES, pH 7.4, 150 mM NaCl). Slides were then washed three times with HBS-N and dried by centrifugation (5 min, 200g). Protein incubation was performed with 100 µL of protein solutions containing 20 µg of protein in HBS-N with 0.01% (v/v) Tween-20. The inhibition solution contained 5 mM deaminated heparin. Slides were incubated with the protein solutions for 1 h at room temperature under mild shaking, washed three times with HBS-N and dried by centrifugation. For detection, 100 µL of a fluorescent-labeled antibody solution was applied to the slides (2 µL of goat antihuman antibody in HBS-N containing 1% (w/v) BSA and 0.01% (v/v) Tween-20) for 1 h under the exclusion of light. Slides were washed three times with HBS-N and dried by centrifugation. Microarray Detection and Signal Processing. Slides were scanned with a LS400 scanner and analyzed by Array-Pro Analyzer. Quantification of the Alexa Fluor 647 binding signals was carried out using Gene Spotter software. For data analysis, the mean intensity of each spot was used. Spot finding was automatically performed by the software. The signal background of each slide was subtracted from the hybridization signal. For data evaluation, the average of 10 spots on the same array at 250 µM oligosaccharide concentration was selected. Errors are the standard deviations for each measurement. Each binding experiment was performed three times. SPR Immobilization Procedure. HS/heparin oligosaccharides 1, 2, and 5-7 were covalently bound to the sensor surface Journal of Proteome Research • Vol. 8, No. 2, 2009 713

research articles using primary amine coupling. HBS-N containing 0.005% (v/ v) P20 was employed as running buffer. The carboxymethylated dextran matrix (CM5 chip) was first activated at a flow rate of 10 µL/min using an 8 min injection pulse of an aqueous solution containing N-hydroxysuccinimide (NHS, 0.05 M) and N-ethyl-N′-(dimethylaminopropyl)carbodiimide (EDC, 0.2 M). Next, a solution of the oligosaccharide (50 µg/mL) containing 1 mM hexadecyltrimethylammonium chloride was flowed over the activated surface for 8 min at 5 µL/min. Remaining reactive groups on the chip surface were quenched by injection of a 1 M ethanolamine hydrochloride (pH 8.5) solution for 7.5 min at 10 µL/min. For each immobilized oligosaccharide, a parallel flow cell was coupled with HS/heparin 12 as reference cell. The following binding levels were established (in response units, RU): HS/heparin 1, 1100; HS/heparin 2, 614; HS/heparin 5, 604; HS/heparin 6, 702; HS/heparin 7, 596; HS/heparin 12, (300. SPR Binding and Inhibition Assay. HBS-N containing 0.005% (v/v) surfactant P20 was used as running buffer. Protein solutions containing 2 or 4 µM protein (200 and 400 nM for NKp44) in HBS-N buffer were injected into the analyte and the reference flow cell for 1 min at 20 µL/min. For the inhibition experiments, the protein solution contained 0.3 mM deaminated heparin (5 kDa) or 25 µM chondroitin sulfate C (60 kDa). At the end of sample injection, the running buffer was flowed over the sensor surface for 2 min to enable dissociation. The chip surface was regenerated for the next sample by injection of the following solutions for 1 min at 80 µL/min: 0.1% (w/v) SDS, 0.085% (v/v) H3PO4, 1 M NaCl and 0.1% (v/v) HCl. The response was calculated as the difference in RU between analyte and reference flow cell and monitored as a function of time (sensorgram). Kinetic Analysis by SPR. For KD determination between immobilized HS/heparins and NKp30 and NKp46, respectively, the following protein concentrations were injected at a flow rate of 35 µL/min and 25 °C: 0, 0.5, 1, 2, 5 and 10 µM. Association time was 3 min. For NKp44, the following concentrations were injected at a flow rate of 20 µL/min and 25 °C: 0, 78, 156, 312, 625 and 1250 nM. Association time was 10 min. With the above flow rates, no mass transport limitation was observed. At the end of each sample injection, running buffer was flowed over the sensor surface for 10 min to enable dissociation. After the dissociation phase, the sensor surface was regenerated for the next NCR sample using 0.1% (w/v) SDS and 0.085% (v/v) H3PO4 injected for 1 min at a flow rate of 80 µL/min of. The signal from the reference flow cell containing HS/heparin 12 was subtracted to correct for the contribution of nonspecific interactions and systematic errors. Data processing and kinetic analysis was performed using the BIAevaluation software for T100 (Version 1.1.1). Double referenced association and dissociation phase data were globally fitted to a simple 1:1 interaction model (A + B ) AB). Cells. HeLa is a human cervical adenocarcinoma cell line (ATCC no. CCL-2) expressing ligands to NCRs.15-18 The NK92 cell line, transduced by retrovirus to express high levels of wild-type NKp44 (designated as NK92-44), has been characterized in detail elsewhere.34 Flow Cytometry, Antibodies and Binding Inhibition Assays. Cells were incubated with indicated quantities (µg) of the various fusion-Igs for 2 h at 4 °C, washed, and stained with APC-conjugated-F(ab′)2 goat-anti-human-IgG-Fc (109-136-098, Jackson ImmunoResearch, West Grove, PA). Staining and washing buffer consisted of 0.5% (w/v) BSA and 0.05% sodium azide in PBS. Propidium iodide (PI) was added prior to reading 714

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Hecht et al. for exclusion of dead cells. Flow cytometry was performed using a FACSCanto flow cytometer (Becton Dickinson, Mountain View, CA) and fluorescence data were acquired using logarithmic amplification. Results are shown either as overlay staining histograms (X-axis represents fluorescence intensity and Y-axis represents cell counts) or as the normalized geometric mean fluorescence intensity (MFI) of the stained populations. For binding inhibition experiments, indicated quantities (µg) of NCR-Ig fusion proteins were premixed with different synthetic oligosaccharides and added to cells for staining as above. Activation of NK92-44 by Anti-NKp44 and Synthetic Oligosaccharides. Plates (96U) were pre-coated with 2 µg/mL anti-NKp44 mAb (R&D&D SYSTEMS, MAB22491) for 3 h at 37 °C (diluted with PBS, final volume 100 µL). After 3 h at 37 °C, wells were washed 3 times with 200 µL of PBS before NK9244 cells were added. A total of 3 × 104 NK92-44 cells/well mixed with heparan sulfate or with different synthetic oligosaccharides were incubated for 24 h at 37 °C. Heparan sulfate15 was kindly provided by John T. Gallagher (University of Manchester, Manchester, U.K.). IFNγ concentrations in the supernatants were then assayed by standard ELISA according to the manufacturer’s instructions (BioLegend, San Diego, CA).

Results HS/Heparin Microarray. To determine the binding preferences of the NCRs, the three receptors were probed for interaction with the oligosaccharides of a synthetic HS/heparin library.26 The library contained 13 different HS/heparin-type sugars of varying lengths and modification patterns (Chart 1). The synthetic HS/heparin fragments of the library were all equipped with an amine linker. Functionalized deaminated heparin from natural sources with an average molecular weight of 5 kDa was also included in the library. Since all of the synthetic oligosaccharides, especially the hexasaccharides, are not easily accessible, this HS/heparin library provides a unique tool for HS/heparin structure-function relationship analyses. The oligosaccharides were covalently immobilized on Nhydroxysuccinimide (NHS) activated glass slides by an arraying robot using 1 nL of carbohydrate solution for one spot. Each compound was spotted in 10 replicas of four different concentrations. The average spot size was 200 µm in diameter. Immobilization of the structures on CodeLink activated ester slides yielded a signal/noise ratio of g10. In summary, we followed the strategy of Noti et al.26 to prepare miniaturized synthetic HS/heparin arrays that can be used for binding experiments with the NCRs. Specific Interaction of NCRs with Synthetic HS/Heparin Oligosaccharides. To investigate the binding specificity of the NCRs, we expressed the extracellular domains of NKp30, NKp44 and NKp46 as recombinant receptor-immunoglobulin Fc chimeras. In the case of NKp46, only the membrane proximal D2 domain was analyzed since this immunoglobulin fold of NKp46 was sufficient for tumor cell binding35 and heparin binding.17,18 For microarray binding experiments, 20 µg of protein was used for each slide. The slides were incubated with the protein solutions, washed and finally incubated with fluorescently labeled anti-human IgG antibody. The fluorescence signal was obtained by reading the slides by an array scanner. The microarray data revealed that all the NCRs bound specifically to some of the synthetic structures with different binding preferences (Figure 1). Binding was strongly inhibited by addition of 5 mM deaminated heparin. Together with the decreasing signal intensity along the HS/heparin concentration

Natural Cytotoxicity Receptors NKp30, NKp44 and NKp46 Chart 1.

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HS/Heparin Library Containing 13 Synthetic Oligosaccharides and Heparin from Natural Sources

gradient on the array, we ruled out nonspecific association between NCRs and HS/heparin. Importantly, the slides also confirmed binding of all three NCRs to natural heparin 14. This result is in accordance with the finding that all the NCRs, notably NKp30, interact with the carbohydrate moiety of

Figure 1. Fluorescence images of HS/heparin microarrays after incubation with NKp30, NKp44 and NKp46D2 obtained from the array scanner. Each sugar was printed on the slide at four different concentrations ranging from 1000, 250, 63, and 16 µM in 10 replicas. Bottom images show inhibition of the binding by 5 mM heparin from natural sources.

heparan sulfate proteoglycans. There has been conflicting data on the NKp30-heparin interaction because NKp30 molecules with excessive N-glycosylation do not bind HS/heparin.15,36 Our experiment performed with nonexcessively glycosylated NKp30 clearly supported a binding interaction between NKp30 and heparin from natural sources. Binding Patterns of NKp30, NKp44 and NKp46D2. We quantified the signal intensities of the microarray experiment in order to compare the individual binding patterns of the NCRs (Figure 2). NKp30 and NKp46D2 displayed similar binding patterns as both proteins bound to the highly sulfated oligosaccharides 1, 2, 5, 7 and 10. These structures bear two to three sulfate groups per disaccharide unit and represent heparin-type molecules. Heparin displays on average 2.7 sulfate modifications per disaccharide, whereas heparan sulfate is structurally more diverse and carries fewer sulfate groups. HS/ heparin 2 displays a modification pattern that cannot be found in nature. Glucuronosyl C5 epimerases do not act on glucuronic acids adjacent to N-acetylated glucosamine moieties.37 The binding pattern of NKp30 and NKp46D2 overlapped, but the relative intensities between the individual interactions were different. NKp30 interacted strongest with the fully sulfated hexasaccharide 1, whereas NKp46D2 bound most tightly to the analogous tetrasaccharide 7. The NKp44 binding pattern differed from those of NKp30 and NKp46D2: (1) NKp44 did not only bind to oligosaccharides 1, 2, 5, 7 and 10, but also 3 and 6, and (2) the interaction between NKp44 and HS/heparin 2 generated the most intense signal. All three NCRs bound to iduronic acid carrying O-sulfate groups at the C2 and C4 position (HS/heparin 11). Notably, NCRs did not bind to any other monosaccharides in the library Journal of Proteome Research • Vol. 8, No. 2, 2009 715

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Hecht et al. tures as binding partners. Still, the interactions between the NCRs and HS/heparin were not purely electrostatic in nature. Different overall binding patterns with significant variations in the relative affinities were identified. Binding Specificities of NKp30, NKp44 and NKp46D2. We sought to understand the different NCR binding patterns at a molecular level. For that purpose, we first conducted qualitative surface plasmon resonance (SPR) experiments. SPR experiments were useful to validate the microarray data. Furthermore, SPR measurements allowed for real-time analysis of proteinligand interactions. In the SPR technique, visible light is cast on a metal surface and the change in refractive index on the chip surface is monitored. For the NCR binding analyses, we immobilized HS/heparin oligosaccharides 1, 2, 5, 6 and 7 on carboxymethylated dextran chips. Parallel to each analyte flow cell, monosaccharide 12 was immobilized in the reference flow cell. HS/heparin 12 was chosen as control because it did not interact with any of the NCRs in the microarray or SPR experiments (SPR: data not shown). Moreover, HS/heparin 12 displayed a typical heparin sulfation pattern and was comparatively readily accessible by chemical synthesis. Therefore, we considered HS/heparin 12 a good compound to quantify the nonspecific contribution of the NCR-HS/heparin interaction.

Figure 2. Quantification of the fluorescence signal for each sugar of the HS/heparin microarray probed with NKp30, NKp44 and NKp46D2. Data shown are the average of 10 spots on the same array at 250 µM sugar concentration; errors are standard deviations for each measurement.

(HS/heparins 12 and 13). Monosaccharide 11 is an artificial HS/heparin mimetic because of the 4-O-sulfation. The finding that all NCRs bound to this highly charged molecule emphasizes the importance of ionic interactions between HS/heparin and the cytotoxicity receptors. Noticeable differences in the absolute fluorescence were measured between the strongest signals of NKp30, NKp44 and NKp46D2. This result suggests that the NCRs have different kinetic properties. However, the difference in total fluorescence does not reflect different binding affinities. Binding affinities can only be reliably compared by determination of the equilibrium dissociation constants (see below). In summary, we found that the NCRs prefer highly sulfated HS/heparin struc716

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First, we investigated the interaction of NKp30 with synthetic HS/heparin structures. The microarray experiments predicted a high affinity for the highly sulfated structures. SPR experiments were performed to elucidate molecular details of the interactions using oligosaccharides 1, 2, 5 and 6. Each binding experiment comprised two protein injections of different concentrations (2 and 4 µM) as well as an injection of 4 µM protein containing 0.3 mM natural 5 kDa heparin. The sensorgrams revealed binding interactions between NKp30 and HS/heparins 1, 2 and 5 but not 6 (Figure 3A). These results indicated that O-sulfation at C2 IdoA was necessary but not sufficient for binding. Additionally, GlcN requires one negative charge, either N-sulfation or O-sulfation at C6. Comparison of the sensorgrams indicated slightly tighter binding to hexasaccharide 5 than hexasaccharide 2 as the dissociation rate of the protein from this chip was slower. Thus, N-sulfation of GlcN appears to be more important than O-sulfation at C6. Comparisons of sensorgrams are only appropriate when comparable amounts of synthetic structures are immobilized on the chip, as was the case for HS/heparins 2 and 5. To exclude nonspecific association of the fusion-Ig to the surface, binding of human killer cell immunoglobulin receptor KIR2DS4-Ig to the chips was determined. There was no association between KIR2DS4Ig and the HS/heparin oligosaccharides observed (data not shown). Furthermore, heparin strongly inhibited binding of NKp30 to the HS/heparin-modified chips, which indicates that the interactions between NKp30 and HS/heparins 1, 2 and 5 are specific. Next, we analyzed the interaction between NKp44 and HS/ heparins 2 and 6 employing SPR. The microarray data revealed a strong interaction between NKp44 and hexasaccharide 2 and weak affinity of NKp44 for hexasaccharide 6. Both oligosaccharides carry a sulfate group at C2 of IdoA and are acetylated at the GlcN. Thus, we hypothesized whether a motif of alternating sulfate and acetyl groups contributed to binding. The sensorgram confirmed a strong affinity of NKp44 for hexasaccharide 2 (Figure 3B). The injection of 400 nM protein resulted in a strong binding signal. However, we were not able to demonstrate binding of NKp44 to HS/heparin 6 at these low protein concentrations.

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Natural Cytotoxicity Receptors NKp30, NKp44 and NKp46

Table 1. Equilibrium Dissociation Constants for NCR-HS/ Heparin Interactions

Figure 3. SPR sensorgrams for NCR-HS/heparin interactions. (A) Interaction of NKp30 with HS/heparin 1, 2, 5 and 6. The protein samples measured contained 4 µM NKp30, 2 µM NKp30 as well as 4 µM NKp30 and 0.3 mM heparin from natural sources. (B) Interaction of NKp44 with HS/heparin 2 and 6. The following samples were analyzed: 0.4 µM NKp44 and 0.2 µM NKp44 as well as 0.4 µM NKp44 and 0.3 mM heparin from natural sources. (C) Interaction of NKp46D2 with HS/heparin 1 and 7. The protein samples measured were 4 µM NKp46D2 and 2 µM NKp46D2 as well as 4 µM NKp46D2 and 0.3 mM heparin from natural sources.

We probed NKp46D2 for interaction with HS/heparins 1 and 7 by SPR in order to validate and refine the microarray data. Interestingly, NKp46D2 had significantly higher affinity for the fully sulfated tetrasaccharide 7 than the fully sulfated hexasaccharide 1 on the microarray. Identical results were obtained with SPR experiments (Figure 3C). NKp46D2 bound tetrasaccharide 7 tighter than hexasaccharide 1. These results demonstrate that in the case of NKp46D2 the chain length is critical for the interaction. A tetrasaccharide is significantly tighter bound than an analogous hexasaccharide. To demonstrate further the specificity of the interaction between the NCRs and the HS/heparin oligosaccharides, inhibition experiments with chondroitin sulfate were performed. Chondroitin sulfate belongs to the family of the glycosaminoglycans; thus, it is structurally related to heparan sulfate/heparin. However, chondroitin sulfate differs from HS/ heparin regarding monosaccharide composition and sulfation pattern. By SPR measurements, the interaction between the NCR proteins and immobilized HS/heparin structures was monitored in the presence of chondroitin sulfate or heparin from natural sources. To compare the inhibition efficiency of chondroitin sulfate and heparin, equimolar concentrations of sugar residues of the two glycosaminoglycans were used. Addition of 25 µM chondroitin sulfate to the reaction mixture had only little effect on binding, whereas equivalent amounts

NCR

oligosaccharide

KD

NKp30 NKp44 NKp46D2

HS/heparin 1 HS/heparin 2 HS/heparin 7

7 × 10-7 M 3 × 10-7 M 2 × 10-6 M

of heparin led to a complete inhibition of the binding interaction (Supporting Information Figure 1). The minimal inhibition observed with chondroitin sulfate is explained by unspecific ionic interactions. These data reinforce specific binding of NKp30, NKp44 and NKp46D2 to HS/heparin. Binding Affinities of NKp30, NKp44 and NKp46D2. To quantify the NCR binding affinities for the synthetic HS/heparin structures, we conducted kinetic analyses by surface plasmon resonance (SPR). The strongest interacting synthetic HS/ heparin for each NCR based on microarray experiments was selected and the equilibrium dissociation constant determined (Table 1). The KD values of NKp30 (to HS/heparin 1), NKp44 (to HS/heparin 2) and NKp46D2 (to HS/heparin 7) were 0.7, 0.3, and 2 µM, respectively. The dissociation constants for the interactions of NKp30 and NKp44 with HS/heparin were in the high nanomolar range, whereas the dissociation constant of NKp46D2 with HS/heparin was approximately one order of magnitude higher. These values correlate nicely with the dissociation constants for the interactions of NKp30, NKp44 and NKp46D2 with heparin from natural sources, that are 50, 40, and 400 nM.15,16 In both cases, NKp30 and NKp44 exhibit comparable affinities to HS/heparin that are significantly higher than those of NKp46D2. The fact that the values of the interactions with the synthetic structures are 10-fold lower than with natural heparin indicates that the selected HS/heparins are not identical to the actual interacting heparan sulfate epitopes. Still, the affinities are comparatively high for lectin-carbohydrate interactions and compare well to the affinities for natural heparin. These data suggest that the HS/ heparin oligosaccharides represent fragments of the NCR epitopes. Inhibition of NCR Binding to Tumor Cells by the Synthetic Oligosaccharides. We further investigated whether the chipconjugated synthetic oligosaccharides that strongly bound to NCRs can inhibit NCRs’ binding to their natural ligands expressed on tumor cells. We compared the fully sulfated tetrasaccharide 7 and the fully sulfated hexasaccharide 1. Hexasaccharide 4, which did not bind to any NCR in the microarray experiments, was employed as a negative control. Natural heparan sulfate (HS) was employed as a positive control for inhibition capacity. Heparin/HS 1 was the most potent inhibitor of the binding of all three NCRs to HeLa tumor cells (Figures 4 and 5A). In contrast, HS/heparin 7 inhibited the binding of NKp46 and NKp30 but not the binding of NKp44 to HeLa tumor cells. HS/heparin 4, the negative control, showed none to dull inhibition of NCRs binding to tumor cells. HS, the positive control, was by far the better inhibitor in all experiments (Figures 4 and 5A). HS/heparin 2 inhibited the binding of NKp44 to tumor cells but to a lesser degree than the inhibition manifested by HS/heparin 1 (Figure 5A). To test whether the synthetic oligosaccharides could influence NK cell activation through NKp44, we employed natural killer cells that express high levels of transfected wild-type NKp44 (designated as NK92-44).34 NK92-44 cells were plated in wells that were precoated with an anti-NKp44 mAb. For Journal of Proteome Research • Vol. 8, No. 2, 2009 717

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Figure 4. Effect of synthetic HS/heparin oligosaccharides on binding of recombinant NKp46 and NKp30 to HeLa tumor cells. NCRs were premixed for 0.5 h with different carbohydrates and exposed to 5 × 104 cells for 2 h at 4 °C. After incubation, cells were washed and incubated with FITC-anti-Fc secondary antibody. PI was added to exclude dead cells when staining results were analyzed with FACSCanto II. (A) Effect of titrated concentrations (1-32 µg/mL) of carbohydrates on NKp46 (12 µg/mL) binding to HeLa cells. (B) FACS histograms overlay comparing the inhibition of NKp46 staining of HeLa by HS and oligosaccharides 1, 4 and 7. 2Ab is staining of HeLa with only the secondary antibody. (C) Effect of titrated concentrations (1-32 µg/mL) of carbohydrates on NKp30 (16 µg/mL) binding to HeLa cells. Staining results (geometric mean fluorescence intensity) for (A) and (C) were normalized according to the staining of HeLa by NCR without the presence of carbohydrates.

some of the samples, the experimental culture medium was additionally supplemented with heparan sulfate or synthetic oligosaccharides. After 24 h, IFNγ concentration in the supernatants was assayed by standard ELISA (Figure 5B). Incubation of NK92-44 cells with anti-NKp44 mAb resulted in IFNγ secretion, while resting cells did not secret detectable amounts of IFNγ. Significant enhancement of IFNγ secretion was observed for mAb-activated NK92-44 cells cocultured with heparan sulfate or with hexasaccharide 2, but not with the 718

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Figure 5. Effect of synthetic HS/heparin oligosaccharides on binding of recombinant NKp44 to tumor cells and on NKp44mediated activation of NK cells. (A) Effect of titrated concentrations (1-32 µg/mL) of carbohydrates on NKp44 (2 µg/mL) binding to HeLa cells. Cells were stained and analyzed as described in the legend to Figure 4. Staining results (geometric mean fluorescence intensity) were normalized according to the staining of HeLa by NCR without the presence of carbohydrates. (B) Effect of hexasaccharide 2 on the secretion of IFNγ by NK92-44 cells activated with antiNKp44 mAb. A total of 3 × 104 NK92-44 cells were incubated for 18 h in wells precoated with PBS (-) or precoated with anti-NKp44 (+). Culture medium contained 16 µg/ mL of HS, hexasaccharide 2, hexasaccharide 4, or did not contain any carbohydrate. IFNγ concentrations in the supernatants were then assayed by standard ELISA. Results are the summary of two experiments normalized according to IFNγ secretion from anti-NKp44 activated cells without any carbohydrates. Asterisks (*, **), P < 1 × 10-5 as analyzed by the ANOVA single factor. Bars ( SD.

negative control hexasaccharide 4 (Figure 5B). However, antiNKp44 activation was imperative, since coculturing with either heparan sulfate or hexasaccharide 2 and without anti-NKp44 did not induce IFNγ secretion by NK92-44 cells.

Discussion Here, we demonstrate that NKp30, NKp44 and NKp46 recognize highly sulfated HS/heparin-type structures with differing individual specificities. The general preference for molecules containing two to three sulfate groups per disaccharide unit suggests that the NCRs interact with microdomains on heparan sulfate that are sulfated above average. Comparison of the NKp30, NKp44 and NKp46 binding patterns obtained from microarray measurements reveals significant differences in binding specificities when variables such as the positions of the sulfate groups and the chain length are altered. This finding is illustrated by the fact that the three proteins do not share any sequence homology and NKp46 is located on a different chromosome than NKp30 and NKp44.

Natural Cytotoxicity Receptors NKp30, NKp44 and NKp46 Binding experiments performed with heparin from natural sources suggest that the epitopes recognized by NKp30 and NKp46 are cross-reactive, yet different from the NKp44 epitope.15 Consistent with these data, NKp30 and NKp46 bind the same synthetic HS/heparin structures. However, the epitopes are certainly not identical. NKp30 preferentially binds to hexasaccharide 1, whereas NKp46 binds more tightly to the analogous tetrasaccharide 7. Keeping in mind that dense immobilization on chips allows for multivalent interactions, these data must not be misinterpreted as reflecting the actual length of the epitope. For instance, one NKp46 protein might interact with multiple oligosaccharides 7 on the chip. The NKp46 heparan sulfate epitope contains one or multiple tetrasaccharide fragments containing a 2-O-sulfated iduronic acid moiety and a glucosamine that is sulfated at the 6-O- and N-positions. Most likely, the same repeating motif can be found in the epitope of NKp30. The NKp44 binding pattern obtained by microarray emphasizes the importance of 2-O-sulfation at the iduronic acid moiety and N-acetylation at the glucosamine moiety for the interaction. Opposing data concerning the importance of N-acetylation were obtained by SPR, where oligosaccharide 6 does not bind to NKp44. One possible explanation is that the different degree of immobilization density on the microarray versus SPR chip has an influence on weak interactions such as NKp44-HS/heparin 6. Microarrays contain more oligosaccharides per area unit, and thus, the probability for multivalent interactions is higher. Multivalent interactions might strengthen the association between NKp44 and HS/heparin 6 on the microarray. Although microarray and SPR are inherently different testing systems, the combination of the two techniques allows in most cases for cross-validation of the data. We consider the microarray technique particularly useful for the fast analysis of multiple protein-carbohydrate binding events. In contrast, SPR is a powerful method for the study of realtime protein-carbohydrate interactions and the determination of binding strengths. The KD values for the interactions of the NCRs with the synthetic HS/heparin structures fit nicely to the values obtained with natural heparin and confirm a stronger affinity of NKp30 and NKp44 for synthetic HS/heparin compared to NKp46. The oligosaccharides in our library represent only fragments of the epitopes, which explains the overall 10-fold diminished affinity of the NCR-HS/heparin interactions compared to natural heparan sulfate. The library contains a variety of oligosaccharides with different typical modification patterns and chain lengths. However, the molecules are of restricted length, lack the infrequent 3-O-sulfation at the glucosamine portion, and HS/heparin 9 is the only structure that contains a glucuronic acid moiety. Furthermore, hexasaccharides 2 and 6 are not found in nature in exactly this composition since glucuronic acid adjacent to acetylated glucosamine is not epimerized to iduronic acid.37 The glucosamine needs to be modified by a GlcNAc N-deacetylase/GlcN N-sulfotransferase38,39 to allow for epimerization of the neighboring GlcA. Despite some limitations regarding length and sequence variation, our synthetic HS/heparin library presents a powerful tool to elucidate the structure-function relationship of heparan sulfate glycosaminoglycans. The analysis of structurally defined oligosaccharides allows for comparison of different modification patterns and for determination of absolute affinities between the binding proteins and defined oligosaccharide structures. In contrast, studies with natural heparin can only

research articles estimate affinities, and structure-activity experiments are complicated by the heterogeneity and the high charge density of these molecules. Using the library, we found that the NCRs interact specifically with heparin and some of the synthetic HS/ heparin oligosaccharides. The observation that ionic interactions play an important role indicates that the NCRs bind to highly charged stretches on heparan sulfate. Nevertheless, we were able to discern differences in the binding preferences of NKp30, NKp44 and NKp46. The identified binding patterns of the individual NCRs provide an indication on the composition of the natural heparan sulfate epitope. Testing the synthetic HS/heparin for inhibition of NCR binding to natural ligands expressed on the membrane of tumor cells revealed both similarities and dissimilarities to the pattern observed for NCR binding to the chip-conjugated synthetic HS/heparin. On the chip, NKp30 preferentially binds to hexasaccharide 1, whereas NKp46 binds more tightly to the analogous tetrasaccharide 7. Yet, for NKp46, hexasaccharide 1 suppresses binding to tumor-expressed natural ligands better than tetrasaccharide 7. Tetrasaccharide 7 does not block NKp44 binding to tumor cells while suppressing binding of NKp46 and NKp30 to the same cells. The difference might be due to the length of the natural HS epitopes recognized by the NCRs. For example, an octameric epitope has been predicted for NKp46 based on the structure of its HS-binding site.17 That the currently tested synthetic HS/heparin oligosaccharides do not fully represent the exact HS natural ligands is clear from the significantly higher inhibition potency of natural HS compared to the synthetic HS/heparin tested. Hexasaccharide 2 was further tested for its function in NKp44-mediated activation of NK cells. We published that soluble HS augment NK function induced by mAb specific for NKp44.16 Soluble hexasaccharide 2 is able to significantly induce NKp44-mediated NK activation, yet to a lesser extent than HS. This is in accordance with the reduced potency of hexasaccharide 2 as compared to HS, when its inhibition of NKp44 binding to HeLa tumor cells was studied. Heparan sulfate is among the most informationally rich biopolymers in nature. Heparan sulfate glycosaminoglycans play an essential role in key biological processes and are of particular importance to the survival and progress of various forms of cancer.40 Changed levels of heparan sulfate in tumor cells could result in altered recognition, eventually in combination with other ligand molecules. Structural alteration of heparan sulfate due to biosynthetic reasons or tumor-induced expression of modifying enzymes could also lead directly to altered recognition by NCRs or to altered binding properties for protein ligands, which in combination could give new epitopes for NCRs. In this study, we investigated the NCR-HS/ heparin interactions using HS/heparin microarrays and SPR. For the first time, detailed molecular information about the NCR-HS/heparin structure-activity relationship was obtained that provides now a basis for investigations of the poorly understood process of NK cell target recognition. Carbohydraterecognizing receptors constitute a significant part of the innate immune system and its typical pattern recognition features.41 NK cells recognize patterns apparent on transformed cells; the observed differences between the HS epitopes recognized by the three different NCRs further emphasize the complexity of the transformed cell pattern recognized by NK cells.

Acknowledgment. This work was supported by ETH Zurich and by the Cooperation Program in Cancer Research Journal of Proteome Research • Vol. 8, No. 2, 2009 719

research articles of the Deutsches Krebsforschungszentrum (DKFZ) and Israel’s Ministry of Science (MOS). We thank Dr. J. Sobek for assistance in the construction of the microarrays and the Functional Genomics Centre Zurich for providing technical support.

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