Calcium modulation of monoclonal antibody binding to phosphatidylinositol phosphate

Biochemical and Biophysical Research Communications 354 (2007) 747–751 www.elsevier.com/locate/ybbrc

Calcium modulation of monoclonal antibody binding to phosphatidylinositol phosphate Zoltan Beck a, Nicos Karasavvas b, James Tong b, Gary R. Matyas b, Mangala Rao b, Carl R. Alving b,* b

a Henry M. Jackson Foundation for Military Medical Research, USA Division of Retrovirology, Walter Reed Army Institute of Research, U.S. Military HIV Research Program, Rockville, MD 20850, USA

Received 24 December 2006 Available online 16 January 2007

Abstract The binding characteristics of two monoclonal antibodies (mAb) to phosphatidylinositol-4-phosphate (PIP) were examined: a murine IgM mAb to PIP; and a human IgG mAb (4E10) that binds both to HIV-1 envelope protein and also to neutral and anionic phospholipids, including PIP. Binding of each mAb to pure PIP was inhibited by Ca2+ as determined by ELISA. When studied by surface plasmon resonance, liposomes containing PIP could be stripped (i.e., removed) by either Ca2+ or phosphorylated haptens after binding of the liposomes to the murine anti-PIP antibody attached to a BIAcore chip. In contrast, the binding of liposomal PIP to 4E10 was irreversible and could not be stripped. We therefore conclude that Ca2+ and phosphate can modulate the initial binding of both types of antibodies to PIP. However, 4E10 binds to liposomal PIP in a two-stage process involving first Ca2+-modulated binding to the PIP polar headgroup, followed by irreversible binding to liposomal hydrophobic groups. Published by Elsevier Inc. Keywords: Phosphoinositide; Phosphatidylinositol phosphate; Liposomes; Calcium; HIV-1; 4E10; Surface plasmon resonance; Monoclonal antibodies; Antibodies to phospholipids

The specificity of an antibody for an antigen is determined by the affinity of attachment of the antigen to the antigen-binding site of the antibody, and this in turn is dependent on the contributions of all of the individual points of attachment that result in the total binding energy. Subsites in the antigen-binding site are commonly identified by cross-reactions that are observed with related haptens or antigens [1]. Certain subsites, such as subsites that bind various monosaccharides or polysaccharides, are widely distributed in numerous types of antibodies. Phosphate- or sulfate-binding subsites are also often found in monoclonal antibodies (mAbs) to carbohydrates [2] or phospholipids [3,4].

*

Corresponding author. Fax: +1 301 762 7460. E-mail address: [email protected] (C.R. Alving).

0006-291X/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.bbrc.2007.01.033

Because of the ability of anionic phospholipid phosphate to serve as a ligand for binding of Ca2+ [5,6], we examined the effects of calcium on the binding of murine and human mAbs that bind to phosphatidylinositol-4phosphate (PIP) as an antigen. Previous work has demonstrated that antibodies to PIP can be generated from mice that were immunized with liposomes containing PIP as the antigen and lipid A as an adjuvant [3]. The IgM mAbs are selected by the ability to react with liposomes containing PIP and the inability to bind to liposomes lacking PIP. Murine mAbs to PIP exhibit phosphate (or sulfate) binding subsites in that they cross-react with several different anionic phospholipids [7] and are inhibited by small soluble phosphorylated (e.g., phosphocholine, but not choline, and ATP, but not adenine), or even sulfated (sulfogalactosyl ceramide but not galactosyl ceramide) molecules [3]. In this study, we have found that Ca2+ has a strong modulatory effect on the binding of murine mAbs to PIP. In addition,

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we have recently found that a broadly neutralizing human IgG mAb (4E10) that binds to an HIV-1 envelope protein and cross-reacts with neutral and anionic phospholipids [8,9], also binds to PIP [10]. Because of the hydrophobic nature of the antigen-binding site on 4E10, and the broad interactions with numerous phospholipids, it has been hypothesized that 4E10 lacks binding specificity for phospholipid headgroups, and that binding to lipid bilayers involves only nonspecific interactions with the hydrophobic fatty acyl regions of the bilayer [9]. In contrast, we have shown that binding of 4E10 to liposomal PIP involves a strong phosphate subsite in that the binding to PIP could be inhibited by phosphorylated haptens but not by nonphosphorylated haptens, thus demonstrating that the 4E10 mAb initially binds specifically to the headgroup of PIP [10]. The present work further demonstrates that the initial binding of 4E10 to PIP can be modulated by Ca2+, presumably by interfering with the binding of the phosphate subsite of 4E10 to PIP. Materials and methods Lipids. Phosphatidylinositol-4-phosphate (PIP), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), and cholesterol (CHOL) were purchased from Avanti Polar Lipids (Alabaster, AL). Liposomes. Multilamellar liposomes were prepared as previously described [11]. In brief, lipids (DMPC, CHOL, DMPG, and PIP in molar ratios of 2:1.5:0.22:0.1) dissolved in distilled chloroform were added to 50 ml pear-shaped flasks. In some preparations PIP was omitted, as indicated. Lipids were deposited as a thin film under 10 kPa vacuum at 40 °C on a rotary evaporator and dried overnight under high vacuum. Barbital buffer (0.01 M sodium barbital, 0.1 M NaCl, pH 7.4) was added to disperse the lipids to yield 50 mM phospholipid multilamellar liposome suspensions. The liposomes were freeze-thawed three times and passed twenty times through 100 nm polycarbonate filters in an Avestin Lipofast Basic extrusion apparatus to give unilamellar liposomes with mean diameters between 115 and 136 nm, as measured by a Nicomp Submicron Particle Sizer model 370. Antibodies. Murine anti-PIP IgM antibody was purified from ascites fluid as described earlier [10]. Human monoclonal IgG antibody (4E10) was purchased from Polymun Scientific (Vienna, Austria). Goat antimouse IgM antibody conjugated to HRP was purchased from Zymed Labs (San Francisco, CA), and sheep affinity-purified and HRP-linked anti-human IgG antibody was purchased from The Binding Site (San Diego, CA). ELISA. PIP was freshly dissolved in 80% ethanol and 1 nmol was added in 100 ll to wells of Immulon 2HB U bottom ELISA plates (Thermolab Systems, Franklin, MA), and allowed to dry overnight. The plates were blocked with 200 ll of 3% bovine serum albumin, 20 mM Tris, pH 7.4, 154 mM NaCl for 2 h, and appropriate antibodies (anti-PIP or 4E10) were added to the plate for 2 h. Where indicated, the mAbs were incubated with CaCl2 or 10 mM EDTA at RT for 30 min. Plates were washed 5 times with washing buffer (20 mM Tris–HCl, pH 7.4, 154 mM NaCl) using an automated plate washer (Titertek MAP-C) and the appropriate secondary antibodies were added and incubated for 1 h. Plates were washed; ABTS peroxidase substrate system (KPL, Gaithersburg, MD) was added and incubated for 1 h; and the plates were read at 405 nm with a Spectra Max 250 (Molecular Devices, Sunnyvale, CA). BIAcore studies. Surface plasmon resonance (SPR) was measured by a Biacore 2000 optical biosensor (BIAcore Inc., Uppsala, Sweden). Approximately 600–1000 response units (RU) of purified anti-PIP mAb or 4E10 were immobilized to the carboxy-methylated surface lacking a dextran matrix (BIAcore C1 chip) according to the manufacturer’s instruc-

tions using an amine coupling kit. PIP-containing liposomes were injected over the immobilized antibody surface for 150 s at a flow rate of 30 ll/min, and data were collected 30 s later. A flow cell without antibody served as a control for nonspecific binding to the C1 chip. All binding experiments were performed in barbital buffer at 25 °C at a flow rate of 30 ll/min. The anti-PIP antibody surface was regenerated by 60 ll injections of 300 mM CaCl2 in barbital buffer, and the calcium was then removed by a 50 ll pulse of 12.5 mM EDTA at a flow rate of 100 ll/min. The 4E10 antibody could not be regenerated by Ca2+, but was regenerated by 5 ll pulses of 10 mM CHAPS (Sigma–Aldrich, St. Louis, MO) at 100 ll/min. The antibody surface was stable for as long as ten days when regeneration and continuous flow of barbital buffer at 4 °C was used between experiments.

Results Binding of mAbs to PIP and liposomes containing or lacking PIP As shown by SPR in Fig. 1A, anti-PIP mAb bound PIPcontaining liposomes in a concentration-dependent manner, but binding was not observed with liposomes lacking PIP. After binding of the liposomal PIP to the mAb, the liposomes could be stripped from the antibody by soluble glucose-1-phosphate or adenosine monophosphate, but not by adenosine (Fig. 1B). Thus, despite the binding site specificity of the mAb for the PIP headgroup, the mAb contains a strong subsite specificity for soluble phosphate. It can be assumed that the binding site of the antibody must also have accommodated some or all of the larger inositol-4-phosphate headgroup of PIP as part of the binding specificity. The human 4E10 mAb exhibited binding to pure PIP as determined by ELISA (Fig. 2A). However, on examination by SPR, the 4E10 mAb recognized liposomes containing PIP to the same extent that it recognized anionic liposomes lacking PIP (Fig. 2B). This was in contrast to the murine anti-PIP mAb which only bound to anionic liposomes containing PIP (Fig. 1A). The data with 4E10 are in accordance with previous observations that 4E10 recognizes a variety of neutral and anionic lipids and liposomes [8,9]. Effects of calcium on binding of murine and human mAbs to PIP or liposomes containing PIP Purified murine mAb to PIP was pre-incubated with 10 mM EDTA to remove any protein-bound Ca2+, and the EDTA was removed by dialysis. The antibody was then tested for binding to PIP by ELISA in the presence of different concentrations of Ca2+. As shown in Fig. 3A, Ca2+ concentrations ranging from 10 6 to 100 mM had little effect on the binding of the antibody to PIP. However, in the presence of 101 mM Ca2+, a profound inhibitory effect on the binding to PIP was observed. The inhibitory effect of 30 mM Ca2+ was completely reversed by EDTA, and EDTA actually increased the binding more than four-fold above the initial baseline in the absence of added Ca2+ (Fig. 3B). The increased binding above the baseline presumably occurred because the ELISA blocking reagent

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Fig. 1. Binding of liposomes containing PIP [L(PIP)] or lacking PIP [(L)] to murine anti-PIP mAb and displacement of the binding by soluble haptens, as measured by SPR. (A) Binding of liposomes to antibody. Liposomes were diluted two-fold from 3.9 mM phospholipid. (B) Displacement of liposomes by phosphorylated haptens. PIP-containing liposomes (0.244 mM liposomal phospholipid) were injected over the antibody surface for 150 s. The antibodyliposome complex was subsequently treated with 60 ll of adenosine, AMP, or glucose-1-phosphate.

Fig. 2. Binding of human 4E10 mAb to PIP and to liposomes containing PIP as determined by ELISA (A) or SPR (B). See text for details.

Fig. 3. Effects of Ca2+ on binding of mAbs to PIP. (A) Inhibitory effects of Ca2+. (B) Reversal of inhibitory effect of Ca2+ on binding of murine anti-PIP mAb to PIP by treating the ELISA plate and the antibody with 12.5 mM EDTA. The error bars represent the SD values of three experiments with triplicate measurements of each point.

(bovine serum albumin) contributed Ca2+ that was eliminated by EDTA. The binding of mAb 4E10 to pure PIP was inhibited by similar levels of Ca2+ that inhibited the murine anti-PIP mAb as shown by ELISA. When examined by SPR, the liposomes containing PIP were displaced from the murine anti-PIP mAb by increasing concentrations of Ca2+, and displacement occurred in a biphasic manner (Fig. 4A). In contrast to the murine anti-PIP mAb, the PIP-containing liposomes could not be stripped off the 4E10 mAb either by the addition of Ca2+ or by addition of a soluble phosphorylated hapten (Fig. 4B). Thus, it appears that 4E10 resembles the murine anti-PIP mAb in that the initial binding of 4E10 to pure PIP is inhibited by Ca2+, but it differs from the anti-PIP mAb in that the binding of liposomal PIP cannot be reversed by Ca2+.

Discussion It is well known that Ca2+ binds to inositol phosphatides. Hauser and Dawson demonstrated electrostatic binding of 45Ca2+ to a monolayer of phosphatidylinositol in a Langmuir trough, and binding was maximal when the separation of the phospholipid headgroups approximated the diameter of a hydrated Ca2+ ion [5]. The calcium could have been complexed either to the phosphate or to the adjacent cyclitols, or both. The Ca2+ interaction with phosphate could be relevant in that Ca2+ might have been binding mainly to the 4-phosphate of PIP in the present study, and binding of the anti-PIP antibody is known to involve at least one antigen-binding subsite that recognizes phosphate [3,4]. It is therefore likely that at least part of the observed inhibitory effects of Ca2+ in this study were due

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Fig. 4. Displacement by Ca2+ of liposomal PIP bound to murine anti-PIP as measured by SPR, and lack of displacement of liposomal PIP from human 4E10 mAb by Ca2+ or phosphorylated hapten. (A) PIP-liposomes (0.244 mM liposomal phospholipid) were bound to the anti-PIP mAb. The antibody-liposome complex was subsequently treated with 60 ll of 10 3–103 mM of CaCl2, as indicated and data points were collected 30 s thereafter. (B) PIP-liposomes (5 mM liposomal phospholipid) were bound to the 4E10 mAb. The antibody-liposome complex was subsequently treated with 60 ll of 10 3–103 mM of CaCl2 or AMP, as indicated and data points were collected 30 s later.

to electrostatic interactions of the divalent cation with the anionic phospholipid, with resultant inhibition of binding of the antibody to the phospholipid headgroup of PIP. A previous study also demonstrated that Ca2+ had complex effects on the binding of syphilitic anti-cardiolipin antibodies to cardiolipin [12]. However, the suggestion by the latter authors that the extent of antibody binding was influenced by changes in phospholipid phase structures initiated by Ca2+ seems to us to be less likely than simple displacement of the antibody due to competitive electrostatic interactions with the phosphate headgroups. Recently, a novel subsite, consisting of a Ca2+ coordination site, was mapped to the antigen-binding site in a monoclonal antibody to CD4, a protein present on T-helper lymphocytes [13]. This report was the first study to demonstrate the presence of a Ca2+-binding subsite in the antigen-binding site that contributed to the specificity of an antibody for a larger antigen. The possibility exists that a Ca2+-binding subsite might also be present in our mAbs, or indeed in many antibodies to phospholipids or in other types of antibodies that have phosphate- or sulfate-binding subsites. This might not necessarily be surprising in that phospholipids used for immunization would be likely to have Ca2+ bound to their headgroups, and the Ca2+ might contribute to specific recognition of the phospholipid antigen. Regardless of whether inhibitory effects of Ca2+ in this study were due to electrostatic binding of Ca2+ to the phospholipid or to a calcium coordination site in the antibody, or both, it is apparent that the specificities and affinities of the antibodies to the PIP antigen were strongly influenced by the presence of Ca2+. It is also apparent that Ca2+ could possibly have dramatic effects on the binding of other types of antibodies that have the ability to bind to phosphorylated or sulfated antigens or haptens. The human IgG mAb 4E10 is a broadly neutralizing HIV-1-binding antibody that is known to bind to the HIV-1 gp41 envelope protein at a site on the protein that has considerable hydrophobicity [9,14]. The 4E10 mAb has also been reported to exhibit binding properties to a variety of pure phospholipids, including cardiolipin, phosphatidylserine, phosphatidylcholine, phosphatidylethanol-

amine, and even liposomes, as detected by ELISA (also confirmed by us in Fig. 2), while another mAb, 2F5, that similarly binds both gp41 and phospholipid, recognizes only cardiolipin [8,9]. Based on this, it has been proposed that 4E10 and 2F5 utilize different mechanisms in binding to the HIV-1 envelope in that 2F5 specifically recognizes the phospholipid headgroup while 4E10 binds nonspecifically by hydrophobic interactions with the phospholipid fatty acids in the liposomal bilayer. According to this latter view, 4E10 recognizes a specific HIV-1 gp41 amino acid sequence and simultaneously has nonspecific hydrophobic interactions with membrane phospholipids [9]. From studies on the hydrophobic profiles of the antibody, it was suggested that the complementarity determining region of 4E10 actually becomes embedded and partially sequestered in the hydrophobic region of the phospholipid bilayer. The effect of Ca2+ in this study is consistent with our previous demonstration that 4E10 contains a phosphate-binding subsite [10]. We therefore conclude that the interaction of 4E10 with the phospholipid bilayer is a two-stage process, in which binding first occurs to a phospholipid headgroup (such as PIP), including interactions of a 4E10 antigenbinding subsite with the phospholipid phosphate, and this is then followed by strong hydrophobic interactions with the phospholipid fatty acids that cannot be reversed by blocking the phospholipid headgroups with Ca2+. As noted above, the 4E10 antibody binds to a variety of purified phospholipids, but it is not known which of the many HIV-1 membrane phospholipids are most important for the initial interactions of 4E10 with the HIV-1 envelope lipid bilayer. Nor is it known which of the membrane phospholipids might be preferentially recognized by 4E10 during the HIV-1 neutralization process. Although a variety of lipid candidates might be considered [14], the lipid binding characteristics of the 4E10 mAb resemble those of murine IgM anti-PIP mAbs. The present study therefore suggests the theoretical possibility that PIP, or a similar inositol phosphatide, such as phosphatidylinositol or phosphatidylinositol-bis-phosphate (PIP2), could represent a possible initial binding site for 4E10. Although PIP2 is normally found on the inner lamella of plasma membranes, the

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host cells of HIV-1 become apoptotic during the maturation of the virus, and apoptotic cells exhibit phospholipid scramblase activity that eliminates phospholipid bilayer asymmetry by inducing bidirectional movements of phospholipids across the membrane, resulting in randomized distribution of all major classes of phospholipid across both inner and outer leaflets of the bilayer [15,16]. It would therefore seem possible that inner leaflet phospholipids, including PIP and PIP2, could become exposed on the outer leaflet both in the infected apoptotic cells and in the budded virus. Regardless of the exact HIV-1 gp41-associated initial phospholipid target of 4E10, or the targets of other antibodies that bind to phospholipids or sulfolipids, the present study demonstrates that Ca2+ can be used as a probe to identify the two-phase interaction of the 4E10 antibody first with head groups of phospholipids, and followed by hydrophobic interactions with the phospholipids. Ca2+ therefore might represent an important modulating factor in the initial binding of the 4E10 antibody in vivo. Acknowledgments This work was supported through Cooperative Agreement No. DAMD17-93-V-3004 between the Henry M. Jackson Foundation for the Advancement of Military Medicine and the U.S. Army Medical Research and Material Command, working together with the Division of AIDS, National Institute for Allergy and Infectious Diseases, NIH, Bethesda, MD. The views and opinions expressed herein are the private opinions of the authors and do not necessarily reflect the views of the U.S. Army or the US Department of Defense. References [1] E.A. Kabat, The antibody combining site, Prog. Immunol. 5 (1983) 67–85. [2] E.A. Kabat, J. Kiao, W.H. Sherman, E.F. Osserman, Immunochemical characterization of the specificities of two human monoclonal IgM’s reacting with chondroitin sulfates, Carbohydr. Res. 130 (1984) 289–297.

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