Detection of weak ligand interactions of leukocyte Ig-like receptor B1 by fluorescence correlation spectroscopy

Journal of Immunological Methods 320 (2007) 172 – 176 www.elsevier.com/locate/jim

Technical note

Detection of weak ligand interactions of leukocyte Ig-like receptor B1 by fluorescence correlation spectroscopy Kimiko Kuroki a , Sayoko Kobayashi b , Mitsunori Shiroishi a , Mizuho Kajikawa a , Naoaki Okamoto b , Daisuke Kohda a , Katsumi Maenaka a,⁎ a

Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, 3–1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812–8582, Japan b Life Science Group, Olympus Corp., Tokyo 192–8512, Japan Received 3 August 2006; received in revised form 27 October 2006; accepted 29 November 2006 Available online 22 December 2006

Abstract Fluorescence correlation spectroscopy (FCS) can directly and quickly detect the translational diffusion of individual fluorescence-labeled molecules in solutions. Although FCS analyses for protein–protein interactions have been performed, the very weak interactions generally observed in cell–cell recognition of the immune system have not been examined in detail. Here, we report the FCS analysis for low-affinity and fast-kinetic binding (Kd greater than μM range) of the human inhibitory immune cell surface receptor, leukocyte immunoglobulin-like receptor B1 (LILRB1), to its ligands, MHC (major histocompatibility complex) class I molecules (MHCIs) by using the single-molecule FCS detection system which requires only a small amount of sample. Since the random labeling technique for LILRB1 disturbed the MHCI binding, we performed site-specific labeling of LILRB1 by introducing a cysteine residue at the C-terminus, which could be covalently attached with the fluorescence reagent, Alexa647. This technique can be applied to other type I membrane receptors. The low-affinity binding of LILRB1-Alexa647 to MHCIs (HLACw4, and -G1) was detected by FCS, even though non-labeled MHCIs were only twice as big as the labeled LILRB1. Their dissociation constants (7.5 μM (HLA-Cw4) and 5.7 μM (HLA-G1)) could be determined and were consistent with surface plasmon resonance (SPR) data. These results indicate that the single-molecule FCS detection system is capable of analyzing the binding characteristics of immune cell surface receptors even in difficult cases such as (1) small amount of protein samples, (2) small difference in molecular weight and (3) weak affinity. Therefore, it is a powerful tool for characterization and high throughput inhibitor screening of a wide variety of cell–cell recognition receptors involved in immunologically relevant events. © 2006 Elsevier B.V. All rights reserved. Keywords: Fluorescence correlation spectroscopy; Leukocyte Ig-like receptor; MHC class I; Cell surface receptor; Weak interactions; Protein– protein interaction

Many biological events include weak protein–protein interactions, which have pivotal roles in the regulation of cellular function. For example, the cell–cell recognition

⁎ Corresponding author. Tel.: +81 92 642 6969; fax: +81 92 642 6764. E-mail address: [email protected] (K. Maenaka). 0022-1759/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2006.11.009

of immune systems is controlled by weak protein– protein interactions of cell surface receptors (e.g. Kd in the μM range). Furthermore, these cell surface receptors are key molecules as drug targets and thus biophysical techniques to analyze such interactions are important for the screening of low molecular weight inhibitor compounds.

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The human inhibitory immune cell surface receptor, leukocyte Ig-like receptor B1 (LILRB1, also called as LIR1, Ig-like transcript (ILT) 2, CD85j), is expressed on the cell surface of a wide range of leukocytes including natural killer cells, T cells, B cells and myelomonocytic cells. It recognizes MHC (major histocompatibility complex) class I molecules (MHCIs) on target cells to mediate inhibitory signals and prevent the killing of normal cells expressing MHCIs. In other words, abnormal cells expressing little or no MHCIs can activate the cellular function of LILRB1-positive leukocytes due to lack of the LILRB1-mediated inhibitory signal. Our previous studies (Shiroishi et al., 2003; Kuroki et al., 2005; Shiroishi et al., 2006) using the surface plasmon resonance (SPR) technique showed that LILRB1–MHCI interactions show weak affinity with very fast dissociation rates, which are typical of cell–cell recognition receptors. Fluorescence correlation spectroscopy (FCS) (Magde et al., 1974) can determine the translational diffusion coefficient of fluorescence-labeled molecules in solutions, which depends on molecular weight, structure, and the number of the molecules. Due to recent advances in laser and microscopic technologies (Borsch et al., 1998; Eggeling et al., 1998) FCS can exhibit singlemolecule sensitivity. Thus FCS can be used for analysis of protein–nucleic acid, protein–drug and protein– protein interactions (Kinjo and Rigler, 1995; Kinjo et al., 1998; Meseth et al., 1999; Wolcke et al., 2003). However, to our knowledge, FCS analysis has not been applied to weak interactions of immune cell surface receptors, which have pivotal roles in relevant immune responses. Here we performed FCS analysis of the weak ligand binding of LILRB1, to investigate the capabilities of FCS under difficult conditions including: (1) very weak interactions, (2) when the difference in molecular weight between non-labeled ligand and labeled receptors is not very large, and (3) requirement of site-specific labeling techniques. Furthermore, we used the singlemolecule FCS detection system requiring only a small amount of sample, which has great advantages for screening high throughput inhibitors. Non-labeled recombinant ectodomains of MHCIs, HLA-Cw ⁎ 0401 (with peptide QYDDAVYKL), and HLA-G1 monomer (Cys42Ser mutant with peptide RIIPRHLQL) were produced according to our previous report (Shiroishi et al., 2003). Recombinant LILRB1 protein consisting of N-terminal D1–D2 domains in the extracellular region was expressed by E. coli as inclusion bodies, refolded and purified by gel filtration (Shiroishi et al., 2003). For FCS analysis, LILRB1 was randomly labeled by TAMRA fluorescence reagent (5-carboxyte-

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tramethyl rhodamine N-succinimidyl ester) following the standard protocol commercially provided (Molecular Probes, Eugene, OR). More than 90% of LILRB1 was chemically labeled with TAMRA. The TAMRA-labeled LILRB1 (5–10 nM) was incubated at 24 °C for 30 min with different concentrations of HLA-G1 (0–100 μM) and the single-molecule fluorescence detection FCS measurements were performed using the MF20 molecular interaction analytical system (Olympus, Tokyo, Japan) (Kobayashi et al., 2004). Only 25 μl of sample volume was required in one well of the 384-well plate for measurements. All experiments were performed with 10 s of data acquisition time per measurement, and measurements were repeated five times per sample. No interactions with HLA-G1 were detected even when the concentration was increased to 100 μM (Fig. 1A). This suggests that the random chemical modification disturbs the MHCI binding site of LILRB1. In order to label LILRB1 protein site-specifically to maintain biological function, we introduced a Cys residue with one additional Gly residue at the C-terminal of LILRB1 (hereafter, designated as LILRB1-Cys). Because the C-terminal site of membrane-bound LILRB1 is directed to the cell membrane, its modification is unlikely to interfere with the MHCI binding. Preparation of LILRB1-Cys was the same as for non-labeled LILRB1 but with 1 M L-arginine in the refolding buffer. The Cys residue can be covalently attached with the fluorescence reagent Alexa Fluor 647 C2-maleimide (Molecular Probes, Eugene, OR) near pH 7.0. The majority of the refolded LILRB1-Cys protein was purified with gel filtration as a monomer form of LILRB1-Cys (Fig. 1B). Purified LILRB1-Cys should be immediately labeled using Alexa647 C2-maleimide to avoid the formation of the LILRB1 dimers mediating the intermolecular disulfide bonds of the C-terminal Cys residues. In order to confirm whether Alexa647-labeled LILRB1 protein (hereafter, designated as Alexa647LILRB1) has functional activity, its binding activity to anti-LILRB1 mAb, HP-F1, was examined. HP-F1 mAb can directly bind to functionally active LILRB1 on the cell surface by flow cytometric analysis and can also enhance the killing activity of LILRB1-expressing T cells (Colonna et al., 1997; Saverino et al., 2000). Alexa647LILRB1 (5–10 nM) was incubated at 24 °C for 1 h with 0–330 nM of HP-F1 in 25 μl of PBS buffer (pH 7.5) containing 0.05% Tween 20. FCS measurements for Alexa647-LILRB1 were performed as for TAMRALILRB1 except that the He–Ne laser (633 nm) and a 580DF30 filter were used. HP-F1 (∼ 150 kDa) is roughly seven times bigger than Alexa647-LILRB1 (22 kDa) and shows high affinity, typical of antibody–antigen

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Fig. 1. Site-specific labeling of recombinant LILRB1 protein and its binding ability to anti-LILRB1 mAb (HP-F1). (A, C) Titration experiments to determine the binding affinity of anti-LILRB1 mAb, HP-F1 to randomly (A) and site-specifically (C) labeled LILRB1. (B) Gel filtration of LILRB1Cys with Superdex 75 26/60 (Amersham Biosciences). (D) Binding data of (C) fitted according to the simple Langmuir 1:1 binding model using the Origin 7.0 software package.

interactions, and is therefore suitable for FCS analysis. Fig. 1C shows the increase in diffusion time in a dosedependent manner, indicating the formation of the functional LILRB1–HP-F1 complex. The counts per particle (CPP) of the LILRB1–HP-F1 complex is about 60 kHz, much larger than that of the background (0.8 kHz). The dissociation constants for this binding are determined by fitting with the standard Langmuir 1:1 binding model (Fig. 1D): Dinc ¼

C  Dmax C þ Kd

where Dinc is the increment of the measured diffusion constant from the unbound diffusion constant; Dmax is the bound diffusion constant; C is the concentration of the non-labeled sample; and Kd is the dissociation constant. The dissociation constant for this binding is estimated to be 1.6 nM, which is consistent with that previously

reported by SPR (Kd ∼ 2.5 nM) (Kuroki et al., 2005). Furthermore, two component fitting was also performed, providing similar binding parameters (data not shown). Therefore, Alexa647-LILRB1 has the same functional activity as the non-labeled protein. The site-specific fluorescence labeling of LILRB1D1D2 is required for FCS analysis. Previous studies clearly showed that LILRB1–MHCI binding exhibits very low affinity and fast kinetics (Shiroishi et al., 2003, 2006; Kuroki et al., 2005). Moreover, MHCIs (HLA-Cw4 and -G1) are twice as large (43 kDa) as labeled LILRB1 (22 kDa). In order to examine whether FCS is applicable for such binding events, the binding of Alexa647-LILRB1 with classical MHCI (HLA-Cw4) and non-classical MHCI (HLA-G1) was examined. Alexa647-LILRB1 (5 nM) was incubated at 24 °C for 1 h with 0–85 μM of HLA-Cw4 or 0–123 μM of HLA-G1 in 25 μl of PBS buffer (pH 7.5) containing

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0.05% Tween 20 and FCS analysis was performed. Fig. 2 shows the dose-dependent increase in the diffusion time for LILRB1 binding to HLA-Cw4 and HLA-G1 with only about 3% difference in CPP upon complex formation. The Kd values for LILRB1–HLA-Cw4 and -HLA-G1 interactions, estimated by fitting with the standard Langmuir 1:1 binding model, are 7.5 μM and 5.7 μM, respectively. These values are essentially the same as those determined by SPR (Kd ∼ 6.5 μM and 2 μM for HLA-Cw4 and HLAG1, respectively). These results indicate that FCS can detect weak protein–protein interactions typical of cell surface receptors using small non-labeled proteins. In comparison with the other popular biophysical technique, surface plasmon resonance (SPR) analysis, FCS has some advantages: (1) the measurement time is fast (∼1 min per sample), (2) a 384-well plate suitable for high throughput screening can be used, (3) samples can be easily set up and recovered in batch wells, (4) crude samples can be used

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for the detection, and (5) the binding stoichiometry can be determined. In conclusion, we have established a site-specific labeling system for LILRB1, which can be applied to other proteins, especially type I membrane receptors. The FCS analysis can provide binding constants for lowaffinity MHCI binding of LILRB1. These results suggest that FCS can be used for a wide range of protein–protein interactions, because its limitation for binding affinity and molecular weight differences between labeled and non-labeled samples can be extended. FCS requires only a small amount of labeled and non-labeled proteins even in low-affinity binding systems. Therefore, the automated single-molecule FCS measurement system has a great potential for high throughput screening of inhibitors for low-affinity immune cell surface receptor recognition, which is one of the main targets for drug discovery in the post-genome era.

Fig. 2. Characterization of MHCI–LILRB1 interactions by FCS. (A, B) Left: protein titration experiments to determine the binding affinity of LILRB1 (5 nM Alexa647-LILRB1) for HLA-Cw4 (A) and HLA-G1 (B). Right: fitting analysis according to the simple Langmuir 1:1 binding model using the Origin 7.0 software package.

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Acknowledgments MS was supported by a JSPS postdoctoral fellowship for young researchers. KM and DK were supported in part by the Ministry of Education, Science, Sports, Culture and Technology of Japan, and the Protein 3000 project. KK, MK and KM were supported by the Japan Bio-oriented Technology Research Advancement Institute (BRAIN). References Borsch, M., Turina, P., Eggeling, C., Fries, J.R., Seidel, C.A., Labahn, A., Graber, P., 1998. Conformational changes of the H+-ATPase from Escherichia coli upon nucleotide binding detected by single molecule fluorescence. FEBS Lett. 437, 251. Colonna, M., Navarro, F., Bellon, T., Llano, M., Garcia, P., Samaridis, J., Angman, L., Cella, M., Lopez-Botet, M., 1997. A common inhibitory receptor for major histocompatibility complex class I molecules on human lymphoid and myelomonocytic cells. J. Exp. Med. 186, 1809. Eggeling, C., Fries, J.R., Brand, L., Gunther, R., Seidel, C.A., 1998. Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy. Proc. Natl. Acad. Sci. U. S. A. 95, 1556. Kinjo, M., Rigler, R., 1995. Ultrasensitive hybridization analysis using fluorescence correlation spectroscopy. Nucleic Acids Res. 23, 1795. Kinjo, M., Nishimura, G., Koyama, T., Mets, R., Rigler, 1998. Singlemolecule analysis of restriction DNA fragments using fluorescence correlation spectroscopy. Anal. Biochem. 260, 166. Kobayashi, T., Okamoto, N., Sawasaki, T., Endo, Y., 2004. Detection of protein–DNA interactions in crude cellular extracts by fluorescence correlation spectroscopy. Anal. Biochem. 332, 58.

Kuroki, K., Tsuchiya, N., Shiroishi, M., Rasubala, L., Yamashita, Y., Matsuta, K., Fukazawa, T., Kusaoi, M., Murakami, Y., Takiguchi, M., Juji, T., Hashimoto, H., Kohda, D., Maenaka, K., Tokunaga, K., 2005. Extensive polymorphisms of LILRB1 (ILT2, LIR1) and their association with HLA-DRB1 shared epitope negative rheumatoid arthritis. Hum. Mol. Genet. 14, 2469. Magde, D., Elson, E.L., Webb, W.W., 1974. Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers 13, 29. Meseth, U., Wohland, T., Rigler, R., Vogel, H., 1999. Resolution of fluorescence correlation measurements. Biophys. J. 76, 1619. Saverino, D., Fabbi, M., Ghiotto, F., Merlo, A., Bruno, S., Zarcone, D., Tenca, C., Tiso, M., Santoro, G., Anastasi, G., Cosman, D., Grossi, C.E., Ciccone, E., 2000. The CD85/LIR-1/ILT2 inhibitory receptor is expressed by all human T lymphocytes and down-regulates their functions. J. Immunol. 165, 3742. Shiroishi, M., Tsumoto, K., Amano, K., Shirakihara, Y., Colonna, M., Braud, V.M., Allan, D.S., Makadzange, A., Rowland-Jones, S., Willcox, B., Jones, E.Y., van der Merwe, P.A., Kumagai, I., Maenaka, K., 2003. Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G. Proc. Natl. Acad. Sci. U. S. A. 100, 8856. Shiroishi, M., Kuroki, K., Tsumoto, K., Yokota, A., Sasaki, T., Amano, K., Shimojima, T., Shirakihara, Y., Rasubala, L., van der Merwe, P.A., Kumagai, I., Kohda, D., Maenaka, K., 2006. Entropically driven MHC class I recognition by human inhibitory receptor leukocyte Ig-like receptor B1 (LILRB1/ILT2/CD85j). J. Mol. Biol. 355, 237. Wolcke, J., Reimann, M., Klumpp, M., Gohler, T., Kim, E., Deppert, W., 2003. Analysis of p53 “latency” and “activation” by fluorescence correlation spectroscopy. Evidence for different modes of high affinity DNA binding. J. Biol. Chem. 278, 32587.