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Cleavage of the Glycosylphosphatidylinositol Anchor Affects the Reactivity of Thy-1 with Antibodies1 Tova Kukulansky, Shirley Abramovitch, and Nurit Hollander2 Thy-1 protein, a member of the Ig superfamily, is bound to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. We demonstrate that following anchor cleavage by phospholipase C, the reactivity of the solubilized Thy-1 with several mAbs is lost, and its reactivity with polyclonal anti-Thy-1 Abs is markedly decreased. Hence, solubilized Thy-1 cannot be detected by a range of mAbs. In contrast, enzymatic cleavage of biotinylated Thy-1 yields an intact solubilized protein that can be detected by streptavidin. These results exclude a possible proteolytic degradation of solubilized Thy-1 and suggest that the marked decrease in Thy-1 immunoreactivity following delipidation is due to conformational changes in the Thy-1 protein. We further demonstrate that addition of phospholipase C to preformed Ab-Ag complexes causes dissociation and removal of Thy-1 from the complex, indicating that delipidation of Thy-1 induces a conformational change in Thy-1 that is sufficient to dissociate bound Ab. The possibility should therefore be considered that the GPI anchor affects the conformation of a protein to which it is linked. The Journal of Immunology, 1999, 162: 5993–5997.

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hy-1 is a surface glycoprotein of 25–29 kDa with a structure of a single variable-like Ig superfamily domain (1, 2) anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI)3 tail (3, 4). It is a major glycoprotein in rodent thymocytes and in adult neuronal cells (5–7). Although the structure and gene organization of Thy-1 are well known, its function has remained a matter of speculation. Several studies have implicated Thy-1 in cell-cell interactions. In vitro studies suggest that Thy-1 supports adhesion of thymocytes to thymic epithelium (8) and that it is involved in interactions between neural cells and astrocytes (9, 10). In vivo findings suggest that Thy-1 is involved in thymocyte differentiation (11). Thy-1 can also trigger transmembrane signaling that leads to diverse physiological outcomes in lymphocytes as well as in neurons (12–17). Our studies on the function of Thy-1 included binding assays of purified Thy-1 to different cells. We have purified Thy-1 from detergent extracts of murine thymocytes by affinity chromatography on a column of the anti-Thy-1 mAb 31-11 (18, 19). This approach requires elution of Thy-1 in detergent-containing buffers. To obtain soluble Thy-1 in detergent-free solutions, we tried to purify delipidated Thy-1. For this purpose, thymocytes were treated with phosphatidylinositol-specific phospholipase C (PIPLC) to release Thy-1 from the cell surface. Supernatants of PIPLC-treated cells were then applied to the anti-Thy-1 affinity column. Surprisingly, PI-PLC-solubilized Thy-1 did not bind to the column, suggesting that delipidation altered the immunoreactivity Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel Received for publication November 18, 1998. Accepted for publication February 18, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a grant from the Israel Academy of Sciences and Humanities. 2

Address correspondence and reprint requests to Dr. Nurit Hollander, Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel. E-mail address: [email protected] 3

Abbreviations used in this paper: GPI, glycosylphosphatidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Copyright © 1999 by The American Association of Immunologists

of Thy-1. Here we demonstrate that cleavage of the GPI anchor by PI-PLC affects the reactivity of Thy-1 with a range of mAbs.

Materials and Methods Cells Thymocytes of 4-wk-old C57BL/6 mice were used. The human B lymphoblastoid cell line JY was maintained in RPMI 1640 supplemented with 10% FCS.

Antibodies The hybridoma cell lines 31-11 (18), 42-21 (18), and G7 (20) secrete rat anti-mouse Thy-1. The hybridoma 30-H12 (21) secretes rat anti-mouse Thy-1.2. The hybridoma HO-13-4 (22) secretes mouse anti-mouse Thy1.2. Culture supernatants of these hybridoma cell lines were used as a source for anti-Thy-1 mAbs. Polyclonal rabbit anti Thy-1 Abs were a gift from the late A. F. Williams (Medical Research Council Cellular Immunology Research Unit, Oxford, England). TS2/9 is a mouse anti-human CD58 mAb (23). It was purified from ascites fluid by protein G-Sepharose (Pharmacia, Uppsala, Sweden).

Cell treatment with PI-PLC Thymocytes were incubated for 1 h at 37°C with Bacillus thuringiensis PI-PLC (kindly provided by Dr. M. G. Low, Columbia University, New York, NY) or with B. cereus PI-PLC (Boehringer Mannheim, Mannheim, Germany).

Flow cytometry Cells were incubated sequentially at 4°C for 30 min with primary antiThy-1 Abs and with secondary FITC-conjugated mouse anti-rat IgG (Jackson ImmunoResearch, West Grove, PA) or goat anti-mouse IgM (Zymed Laboratories, South San Francisco, CA), with three washes after each step. The cells were then fixed with 1% paraformaldehyde and analyzed on a FACSort flow cytometer (Becton Dickinson, Mountain View, CA).

Cell labeling Cells were surface labeled with biotin as described (24). The cells were incubated for 30 min on ice in PBS (pH 8.0) containing 0.1 mg/ml sulfosuccinimidobiotin (Pierce, Rockford, IL), and then washed three times.

Immunoprecipitation, gel electrophoresis, and Western blot analysis Thy-1 was immunoprecipitated from supernatants of PI-PLC-treated thymocytes or from cell lysates prepared with 1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). Cell supernatants were centrifuged at 100,000 3 g for 1 h. Thy-1 was immunoprecipitated by 0022-1767/99/$02.00

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31-11 or by 30-H12 mAb prebound to goat anti-rat IgG-coupled Sepharose, by HO-13-4 mAb prebound to goat anti-mouse IgM-coupled Sepharose, by 42-21 mAb prebound to goat anti-rat IgM-coupled Sepharose, and by G7 mAb or by rabbit anti-Thy-1 prebound to protein A-Sepharose. CD58 was immunoprecipitated by TS2/9-Sepharose from supernatants of PI-PLCtreated JY cells or from JY cell lysates prepared with 1% Triton X-100. Sepharose beads were washed, and immunoprecipitates were eluted from beads by boiling in sample buffer. The proteins were subjected to SDSPAGE under nonreducing conditions and were then transferred to nitrocellulose membranes. For detection of unlabeled Thy-1, membranes were probed sequentially either with H0-13-4 mAb and with donkey anti-mouse IgM conjugated to HRP (Jackson ImmunoResearch), or with G7 mAb and with protein A conjugated to HRP (Sigma, St. Louis, MO), or with rabbit anti Thy-1 and with goat anti-rabbit IgG conjugated to HRP (Sigma). For detection of unlabeled CD58, membranes were probed with TS2/9 mAb and with rabbit anti-mouse IgG conjugated to HRP (Zymed Laboratories). Biotinylated proteins were probed with streptavidin conjugated to HRP (Jackson ImmunoResearch). The probed proteins were visualized by enhanced chemiluminescence.

Results Our initial observation was that Thy-1 released from PI-PLCtreated thymocytes could not be detected by immunoprecipitation and Western blot analysis, a method which was routinely used by us for detection of cell-bound Thy-1. As shown in Fig. 1A, immunoprecipitation with mAb 31-11, 30-H12, and 42-21, followed by Western blot analysis with the mAb HO-13-4, was inefficient in the detection of solubilized Thy-1. The three mAbs used for immunoprecipitation are directed against distinct epitopes of Thy-1 (19, 25). Similar results were obtained when Thy-1 was immunoprecipitated with the mAbs G7 and HO-13-4 (data not shown). Reactivity of solubilized Thy-1 with polyclonal Abs was affected as well. Although not entirely lost, it was markedly reduced (Fig. 1B). It should be noted that Thy-1 could not be detected even when 1% CHAPS was added to PI-PLC-treated cell supernatants, indicating that the observed differences in Ab binding to intact and to delipidated Thy-1 did not result from the presence or absence of detergent in cell lysates and cell supernatants (data not shown). Cleavage of the GPI anchor by two different PI-PLC preparations (derived from B. thuringiensis and B. cereus) yielded Thy-1 molecules that could not be detected by immunoprecipitation and Western blot analysis (Fig. 1C). In contrast, the GPI-anchored isoform of CD58 (26 –29) was readily detected in supernatants of JY cells, which were treated with the two phospholipases (Fig. 1D). To ensure immunoprecipitation of soluble rather than membranebound proteins, cell supernatants were centrifuged at 100,000 3 g for 1 h before immunoprecipitation. Since the two types of PI-PLC yielded similar results, further studies were performed with PIPLC of B. thuringiensis. A possible explanation for our inability to detect Thy-1 in PIPLC-treated cell supernatants is that thymocyte Thy-1 was resistant to PI-PLC. To test this possibility, Thy-1 expression before and after treatment with PI-PLC was followed by flow cytometry. As shown in Fig. 2, PI-PLC treatment of thymocytes resulted in marked reduction of Thy-1 expression, indicating that the GPI anchor of thymocyte Thy-1 was susceptible to PI-PLC cleavage. It was further determined whether anti Thy-1 Abs could not bind and immunoprecipitate native delipidated Thy-1, or whether they did not recognize the delipidated molecule after denaturation in SDS-PAGE. Thymocytes were incubated in the absence or presence of PI-PLC. Nonimmunoprecipitated samples of PI-PLCtreated or -untreated cell supernatants and of untreated cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with HO-13-4 or with G7 anti Thy-1 mAb. Fig. 3 shows that the Abs did not bind to delipidated Thy-1 in Western blots. The amount of either lysates or supernatants loaded in each lane of the gel represented an equal number of cells. Moreover, no Thy-1

FIGURE 1. Immunoreactivity of Thy-1 is modified following cleavage of the GPI anchor. A, Thymocytes were incubated for 1 h at 37°C in PBS containing PI-PLC of B. thuringiensis or in PBS. Thy-1 was immunoprecipitated from supernatants of PI-PLC-treated (lane 1) or -untreated (lane 2) thymocytes and from lysates of untreated cells (lane 3). Immunoprecipitation was performed with the anti-Thy-1 mAbs 31-11, 30-H12, or 42-21. Immunoprecipitates were subjected to nonreducing SDS-PAGE and Western blot analysis with the anti-Thy-1 mAb HO-13-4. B, As in A, but immunoprecipitation and Western blot analysis were performed with rabbit anti-Thy-1 polyclonal Abs. C, Thy-1 was immunoprecipitated by the mAb 31-11 from supernatants of thymocytes incubated for 1 h at 37°C with PI-PLC of B. cereus in triethanolamine buffer (lane 1) or with triethanolamine buffer (lane 2), and with PI-PLC of B. thuringiensis in PBS (lane 3) or with PBS (lane 4). Thy-1 was similarly precipitated from lysates of untreated cells (lane 5). Immunoprecipitates were subjected to nonreducing SDS-PAGE and Western blot analysis with the anti-Thy-1 mAb HO-13-4. D, CD58 was immunoprecipitated by the mAb TS2/9 from supernatants of JY cells incubated with triethanolamine buffer (lane 1) or PBS (lane 2), and with PI-PLC of B. cereus in triethanolamine buffer (lane 3) or PI-PLC of B. thuringiensis in PBS (lane 4). Immunoprecipitates were subjected to nonreducing SDS-PAGE and Western blot analysis with the mAb TS2/9. The bar on the right marks the boundaries of the broad CD58 band (29).

was detected in supernatants that were derived from cell numbers that were ten times higher than the cell numbers used for preparation of lysates (data not shown). To determine whether Abs react with delipidated Thy-1 in its native form, Thy-1 was immunoprecipitated from cell lysates or from supernatants of biotinylated cells. Immunoprecipitation was

FIGURE 2. Expression of Thy-1 by PI-PLC-treated thymocytes. Thymocytes were incubated for 1 h in the presence (solid histograms) or in the absence (open histograms) of PI-PLC. Cells were then stained with the mAb 31-11 (A), 30-H12 (B), or HO-13-4 (C), and analyzed by flow cytometry.

The Journal of Immunology

FIGURE 3. Solubilized Thy-1 is not recognized by mAbs in Western blots. Thymocytes were incubated for 1 h at 37°C in PBS or in PBS containing PI-PLC. Supernatants of thymocytes incubated in the absence (lane 1) or in the presence (lanes 2 and 4) of PI-PLC, and lysates of untreated cells (lanes 3 and 5) were subjected to nonreducing SDS-PAGE and transfer to nitrocellulose. Thy-1 was detected in Western blots by the anti-Thy-1 mAbs G7 (lanes 1-3) or HO-13-4 (lanes 4 and 5).

performed with four anti Thy-1 mAbs. Proteins of immune complexes were resolved by SDS-PAGE and probed in Western blots with streptavidin. This approach circumvents the inability of Abs to recognize delipidated Thy-1 in Western blots. As shown in Fig. 4, although the four Abs immunoprecipitated Thy-1 from detergent lysates of biotin-labeled cells, they did not bind Thy-1 that was cleaved from the cell membrane by PI-PLC. These results indicate that the reactivity of Thy-1 with mAb is altered as a result of anchor cleavage, and that the loss of reactivity is expressed both in immunoprecipitation and in Western blot analysis. To ascertain that sensitivity of Thy-1 to PI-PLC was not lost due to its biotinylation, biotin-labeled thymocytes were treated with PI-PLC. Detergent extracts of cells before and after PI-PLC treatment, as well as supernatants of PI-PLC-treated and -untreated cells, were subjected to SDS-PAGE and Western blot analysis with streptavidin. A protein with an apparent molecular mass of Thy-1 was present in lysates but not in supernatants of untreated cells. Following PI-PLC treatment, this protein was present in supernatants but not in lysates of treated cells, indicating its release from cell membranes (Fig. 5, lanes 1-4). Since polyclonal anti-Thy-1 Abs, in contrast to our mAbs, retained some reactivity with delipidated Thy-1 (Fig. 1), the released biotinylated protein was identified as Thy-1 by its immunoprecipitation with rabbit anti-Thy-1 before SDS-PAGE and Western blot analysis with streptavidin (Fig. 5, lanes 5– 6). Similar to the SDS-PAGE in Fig. 3, the SDSPAGE in Fig. 5 was also performed in such a way that each lane represented an equal number of cells. It was further tested whether anchor cleavage affects preformed associations of Thy-1 with Abs. Thy-1 was immunoprecipitated with 31-11-bound Sepharose from lysates of biotin-labeled cells. The Sepharose beads were incubated for 1 h at 37°C with or without PI-PLC before protein elution with sample buffer, SDS-PAGE, and Western blot analysis with streptavidin. Supernatants of incubated beads were similarly resolved by SDS-PAGE and analyzed by Western blotting. As expected, when beads were incubated in

FIGURE 4. Solubilized Thy-1 is not recognized by mAbs in immunoprecipitation assays. Biotin-labeled thymocytes were incubated for 1 h at 37°C in PBS containing PI-PLC or in PBS. Thy-1 was immunoprecipitated from supernatants of treated (PI-PLC) or untreated (PBS) cells as well as from lysates of untreated cells. Immunoprecipitation was performed with the anti-Thy-1 mAb 31-11 (lane 1), 30-H12 (lane 2), G7 (lane 3), or HO13-4 (lane 4). Immunoprecipitated proteins were subjected to nonreducing SDS-PAGE and Western blot analysis with streptavidin.

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FIGURE 5. Biotinylated Thy-1 is released from the cell surface by PIPLC. Biotin-labeled thymocytes were incubated in PBS containing PI-PLC (treated) or in PBS (untreated). Lysates of untreated cells (lane 1) or PIPLC-treated cells (lane 2) and supernatants of untreated cells (lane 3) or PI-PLC-treated cells (lane 4) were subjected to nonreducing SDS-PAGE. Thy-1, immunoprecipitated with rabbit Abs from supernatants of untreated cells (lane 5) or PI-PLC-treated cells (lane 6), was also subjected to SDSPAGE. Following transfer to nitrocellulose, proteins were detected by probing with streptavidin.

PBS following immunoprecipitation, Thy-1 was recovered from beads, but not from bead supernatants. However, when beads were incubated with PI-PLC before protein elution, Thy-1 could not be recovered neither from beads nor from their supernatants (Fig. 6, lanes 1– 4). This was surprising because it was not expected that anchor cleavage would alter reactivity of biotin-labeled Thy-1 with streptavidin. Since the beads were washed three times with buffer containing 1% detergent in between PI-PLC treatment and protein elution, it was hypothesized that anchor cleavage increased the dissociation rate of the preformed Ab-Ag complex in a way that caused removal of the dissociated Thy-1 by washing the beads. This was verified by saving the wash buffer, concentrating it by Centricon-10 concentrator (Amicon, Beverly, MA) and testing it for the presence of Thy-1. As demonstrated (Fig. 6, lanes 5– 6), Thy-1 could be recovered from the wash buffer, indicating that the preformed Thy-1/Ab complex was dissociated following its treatment with PI-PLC.

Discussion The present study demonstrates that delipidation leads to a marked alteration in Thy-1 immunoreactivity. Proteolytic destruction of Thy-1 could explain the effect of PI-PLC treatment on Ab binding. However, this explanation is unlikely due to the following: 1) Delipidation by two different preparations of PI-PLC, a commercial enzyme derived from B. cereus and a noncommercial enzyme derived from B. thuringiensis, resulted in a marked change in immunoreactivity of Thy-1. Although it has been previously demonstrated that CD58 is sensitive to proteolytic cleavage (24), the two preparations of PI-PLC had no proteolytic effect on CD58 (Fig. 1).

FIGURE 6. Complexes of Thy-1 with mAb are dissociated following cleavage of the GPI anchor. Thy-1 was immunoprecipitated with 31-11bound Sepharose from lysates of biotin-labeled thymocytes. The Sepharose beads were then incubated for 1 h in the presence or absence of PI-PLC, and were then washed with lysis buffer containing 1% CHAPS. Proteins eluted from PI-PLC-treated beads (lane 1) or from untreated beads (lane 2), supernatants harvested following incubation of beads with (lane 3) or without (lane 4) PI-PLC, and the buffer that was saved following washes of untreated beads (lane 5) or PI-PLC-treated beads (lane 6) were subjected to nonreducing SDS-PAGE and Western blot analysis with streptavidin. Samples of washing buffer (lanes 5 and 6) were concentrated to the volume of samples 1– 4 (100 ml) before SDS-PAGE analysis.

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2) As participants of the Fourth International Workshop on Human Leukocyte Differentiation Ags, we have screened a “blind” panel of 141 mAbs for binding to untreated and PI-PLC-treated B cells (30). The PI-PLC in that screening was of the same noncommercial preparation used in the present study. Only four mAbs revealed reduced binding activity to cells treated with the enzyme. These were later defined as Abs to the GPI-anchored molecules CD73 and CD48 (30). The fact that the same PI-PLC did not affect Ab binding to a large panel of transmembrane proteins supports the notion that the PI-PLC preparation was devoid of a proteolytic contaminant. In addition, following treatment of thymocytes with the same enzyme preparation, we presently analyzed by flow cytometry the expression of various surface proteins. No effect on expression of transmembrane surface molecules could be demonstrated (data not shown). 3) Following PI-PLC treatment of Sepharose beads carrying preformed complexes of biotinylated Thy-1 and Abs, the dissociated Thy-1 was detected by streptavidin (Fig. 6). Thus, GPI cleavage was not accompanied by proteolysis. 4) Thy-1 is relatively insensitive to proteolysis (3). The finding that delipidated Thy-1 lost reactivity with several Abs implies that delipidation induces a marked conformational change in the Thy-1 protein. The mAbs 30-H12 and HO-13-4 recognize the Thy-1.2 allelic determinant specified by a Gln residue at position 89 (31). The mAbs 31-11, 42-21, and G7 recognize nonpolymorphic determinants of Thy-1 (19, 20). It has been demonstrated that 31-11 and 42-21 recognize distinct antigenic determinants (25). Thus, since the anti-Thy-1 mAbs recognize at least three distinct epitopes, their loss of reactivity indicates that delipidation triggers a major change in Thy-1 that alters the regions of the protein specifying the epitopes for at least three Abs. The demonstration that PI-PLC caused dissociation of preformed Ag-Ab complexes implies that delipidation induces a conformation change in Thy-1 that is sufficient to dissociate bound Abs. However, some epitopes are probably preserved since certain antiThy-1 mAbs, such as H155-124, were shown to bind PI-PLC solubilized Thy-1 (8), and since we demonstrated partial activity of polyclonal Abs with solubilized Thy-1 (Fig. 1). These Abs may recognize linear rather than conformational determinants. A mechanism by which the GPI anchor, when attached to cell membranes or to detergent micelles, imposes a specific conformation on Thy-1, may operate as well for other GPI-linked proteins. For instance, Durbin et al. (32) described a mAb that binds carcinoembryonic Ag when it is anchored to the cell membrane, but not when it is released by PI-PLC. We and others reported that when the GPI-anchored form of CD58 is immunoprecipitated by the mAb TS2/9, a greater recovery is obtained after cleavage of the GPI anchor by PI-PLC (28, 29). This may be accounted for by more efficient binding of the Ab to delipidated CD58 due to altered conformation. In summary, the possibility should be considered that the GPI anchor affects the conformation of the protein to which it is linked. This may have functional implications for GPI-anchored proteins. It has been proposed that GPI anchors are not the sole membrane anchor of proteins to which they are attached, and that GPI-containing proteins have other hydrophobic domains that may interact with cell membranes (33). These proteins would not be released from cells upon enzymatic cleavage of the anchor. In addition, acylation of the inositol in GPI anchors confers resistance to release following cleavage by PI-PLC (34). Such multipoint attachment to membranes has been also proposed for acylated proteins as rhodopsin and the b2-adrenergic receptor (35, 36). Detachment of these two molecules from the acyl groups leads to conformational and functional changes in the protein without release from the membrane. It has been proposed that such a mechanism may be

useful in maintaining an active or inactive conformation (37). According to this model, phospholipase action may mediate an on/off switch. On the other hand, cleavage and release of GPI-anchored molecules, such as Thy-1, may control cell surface expression and production of their soluble forms. Altered conformation of the soluble forms may be responsible for modulation of their activity.

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