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(The FASEB Journal. 2001;15:545-548.)
© 2001 FASEB

GPI-anchored proteins: now you see ’em, now you don’t

PETER BÜTIKOFER*, TATIANA MALHERBE*, MONIKA BOSCHUNG* and ISABEL RODITI{dagger}1

Institutes of
* Biochemistry and Molecular Biology and
{dagger} Cell Biology, University of Bern, 3012 Bern, Switzerland

1Correspondence: Institute of Cell Biology, University of Bern, Baltzerstrasse 4, 3012 Bern, Switzerland. E-mail: isabel.roditi{at}izb.unibe.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 REFERENCES
 
Many cell surface proteins are attached to membranes via covalent glycosylphosphatidylinositol (GPI) anchors that are posttranslationally linked to the carboxy-terminus of the protein. Removal of the GPI lipid moieties by enzymes such as GPI-specific phospholipases or by chemical treatments generates a soluble form of the protein that no longer associates with lipid bilayers. We have found that the removal of lipid moieties from the anchor can also have a second, unexpected effect on the antigenicity of a variety of GPI-anchored surface molecules, suggesting that they undergo major conformational changes. Several antibodies raised against GPI-anchored proteins from protozoa and mammalian cells were no longer capable of binding the corresponding antigens once the lipid moieties had been removed. Conversely, antibodies raised against soluble (delipidated) forms reacted poorly with intact GPI-anchored proteins, but showed enhanced binding after treatment with phospholipases. In the light of these findings, we have reevaluated a number of publications on GPI-anchored proteins. Many of the results are best explained by lipid-dependent changes in antigenicity, indicating this might be a widespread phenomenon. Since many pathogen surface proteins are GPI-anchored, researchers should be aware that the presence or absence of the GPI lipid moieties may have a major impact on the host immune response to infection or vaccination.—Bütikofer, P., Malherbe, T., Boschung, M., Roditi, I. GPI-anchored proteins: now you see ’em, now you don’t.


Key Words: trypanosome • Toxoplasma • CD52 • CD59 • vaccine • conformational epitope


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
REFERENCES
 
THE VARIANT SURFACE glycoprotein (VSG) is a GPI-anchored protein that covers the bloodstream form of the protozoan parasite Trypanosoma brucei. In the course of analyzing the trafficking of trypanosome surface glycoproteins, we observed that polyclonal antibodies raised against a soluble (delipidated) form of VSG showed enhanced binding after treatment of cell lysates with GPI-hydrolyzing phospholipase C (GPI-PLC; Fig. 1 ). It is well known that the removal of the lipid moiety by GPI-PLC creates a new epitope in the anchor (1) , the cross-reacting determinant (CRD). The increased reactivity was not due to the CRD, however, since it was also observed after treatment with mammalian GPI-specific phospholipase D (GPI-PLD), which does not create this epitope (Fig. 1) . These results suggested that changes in the antigenicity of the VSG might be dependent on the GPI lipid moiety.



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  		Figure 1. Delipidation increases antibody reactivity. A) Conserved core structure of the GPI anchor and the sites of enzymatic cleavage by GPI-PLC and -PLD. Some GPI anchors contain an additional fatty acid linked to inositol that renders the structure insensitive to cleavage by GPI-PLC (25) . B) SDS extracts from T. brucei bloodstream forms (4) were incubated in the absence or presence of GPI-PLC from Bacillus cereus or purified GPI-PLD from bovine serum (26) . Antigens were detected after SDS-PAGE and immunoblotting (4) using a polyclonal rabbit antiserum raised against soluble VSG MITat 1.2 (generously provided by M. L. Cardoso de Almeida, Brussels, Belgium) (top panel) or anti-CRD antibodies (Oxford GlycoSciences, Abingdon, U.K.) (bottom panel) as primary antibodies and peroxidase-conjugated second antibodies. Antibody binding was visualized using an enhanced chemiluminescence detection kit (Pierce Chemical Company, Rockford, Ill.).

We have also observed GPI lipid-dependent changes in the antigenicity of procyclins, the major GPI-anchored proteins of insect forms of T. brucei. There are two types of procyclin that are classified on the basis of internal EP dipeptide repeats or GPEET pentapeptide repeats (2) . We routinely use two anti-procyclin antibodies: a mouse monoclonal antibody (mAb) that recognizes the EP repeat (3) and a rabbit polyclonal antiserum that was raised against the synthetic peptide (GPEET)3C coupled to keyhole limpet hemocyanin (2) . Both antibodies bind to the surface of living trypanosomes and recognize the corresponding procyclin on immunoblots. In contrast to the situation with VSG, however, we found that the reactivity with anti-EP (Fig. 2A ) and anti-GPEET antibodies (Fig. 2B ) was greatly reduced after treatment with GPI-PLD. Since GPEET had been labeled with 32P (4) , we could demonstrate that the amounts of protein on the blotting membrane were not affected by phospholipase treatment (Fig. 2C ). A loss of antibody reactivity was also observed with a mAb that specifically recognizes phosphothreonine epitopes on GPEET (data not shown). In addition, a panel of mAbs that bind carbohydrate epitopes on TcPRS (P. Bütikofer and I. Roditi, unpublished observations), an unrelated GPI-anchored surface molecule from Trypanosoma congolense, no longer recognized the antigen after treatment with GPI-PLD (Fig. 2D ). Incorporation of 3H-ethanolamine into the GPI anchor of TcPRS enabled the blot to be scanned after immunodetection (Fig. 2E , F ), confirming that the loss of reactivity after GPI-PLD treatment was not due to loss of the antigen from the membranes. Since amino acid, carbohydrate, and phosphothreonine epitopes were all affected, removal of the lipid(s) must have a profound effect on the overall structure that cannot be overcome by boiling in the presence of sodium dodecyl sulfate (SDS) and ß-mercaptoethanol.



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  		Figure 2. Delipidation decreases antibody reactivity. Butanol extracts from T. brucei 427 insect forms (4) expressing EP (A) and GPEET procyclins (B, C), T. congolense procyclic forms expressing TcPRS (D), human erythrocyte ghosts expressing CD59 (G), and T. brucei 427 mutant trypanosomes (4) expressing a GPEET-Fc fusion protein (H) were incubated in the absence (-) or presence (+) of purified GPI-PLD (26) . Antigens were detected as in Fig. 1 using anti-EP mAb TRBP1/247 (3) (A), anti-GPEET antiserum K1 (2) (B, H), anti-TcPRS mAb #51 (D), and anti-CD59 mAb YTH 53.1 (Biosource International, Camarillo, Calif.) (G) as primary antibodies and the corresponding peroxidase-conjugated second antibodies. Anti-EP and anti-TcPRS mAbs were gifts from T. W. Pearson (Victoria, Canada). GPEET (B, C) was labeled with 32P by an endogenous T. brucei kinase (4) . C) An autoradiograph of the blot shown in panel B demonstrating the presence of the protein in both lanes. T. congolense TcPRS (D) was labeled in the GPI anchor using [3H]ethanolamine. E, F) Radio scans of the blot shown in panel D demonstrating that equal amounts of protein were present in both lanes.

It has previously been shown that mAbs that bound to VSG on the surface of living trypanosomes frequently did not react with the same VSG on immunoblots (5) , suggesting that they bound conformational epitopes that are lost on denaturation. However, as one knows now, the membrane-bound form of VSG had an intact GPI anchor whereas the protein used for immunoblots was a delipidated soluble form generated by the action of an endogenous GPI-PLC that comes into contact with the VSG when trypanosomes are lysed (6) . Monoclonal antibodies raised against soluble VSG but that did not bind to living trypanosomes have also been described (7) . The same mAbs bound to cells that had been permeabilized by acetone fixation, however. It is possible that this procedure—which destroys biological membranes—causes the release of the parasite GPI-PLC, resulting in the (partial) delipidation of the VSG anchor. Another example comes from a publication on gp23, a GPI-anchored protein from Toxoplasma gondii. Antibodies raised against delipidated gp23 also showed much greater reactivity with the soluble form than the membrane form on immunoblots (8) . This difference was not discussed in the paper, but once again it might be explained in terms of GPI lipid-dependent changes in antibody binding.

Changes in antibody reactivity on GPI lipid removal are not peculiar to parasite proteins. We have also observed a reduction in antibody binding after GPI-PLD treatment of CD59, a GPI-anchored protein that protects mammalian cells from complement-mediated lysis (Fig. 2G ). Although a systematic survey of the literature is difficult—since similar observations are often mentioned only in passing—several publications on mammalian cells (9 10 11) and one on Paramecium (12) document that antibody binding is strongly influenced by the hydrophobic portion of the GPI anchor. The most thoroughly investigated molecule is the lymphocyte surface molecule Thy-1 (10) . Removal of the GPI lipid moiety by either GPI-PLC or GPI-PLD caused a marked increase in the dissociation constants of a range of antibodies, including some recognizing amino acid epitopes, which was manifested as a loss of reactivity. It was suggested by Barboni et al. (10) that the conformational changes that accompanied delipidation and could be detected by circular dichroism were responsible for the changes in antigenicity (10) . A loss of reactivity has also been observed with CD52, an unusually small GPI-anchored lymphocyte surface antigen of 12 amino acids (also known as Campath-1 antigen). Once again, removal of the GPI lipid moiety abolished binding of Campath-1 antibodies to the polypeptide (11) .

One possible explanation is that lipids in the GPI anchor might affect the conformation of a protein by acting as a constraint at its carboxyl terminus. When we replaced the GPI anchor of GPEET procyclin by the Fc portion of immunoglobulin G, the hybrid protein reacted with the anti-GPEET antibody (4) and was not affected by GPI-PLD (Fig. 2H ). In the case of CD52, a synthetic peptide with a two amino acid extension could restore reactivity of the Campath-1 epitope (13) . However, the antibodies did not bind the peptide unless it was covalently linked via the carboxyl terminus to derivatized cellulose membranes or to bovine serum albumin, both of which might impose the necessary constraints on the polypeptide.

Numerous studies demonstrate that removal of the GPI lipid moiety in vitro can cause significant alterations in enzymatic activities (14 15 16 17) or ligand binding properties (18 19 20) , which again might be explained in terms of conformational changes. The delipidation of proteins by GPI-PLC or -PLD in vivo, be it through direct cleavage from the cell surface or removal of the GPI anchor from shed proteins, might be an effective means of defusing activities or interactions that might otherwise occur at the wrong time or out of context.

The effect of delipidation on antigenicity might also be one reason why GPI-anchored proteins are the most abundant surface proteins of many parasites. The release and delipidation of these proteins, either by endogenous parasite phospholipases (6 , 21) or by host enzymes, might serve as an antigenic decoy if antibodies against the soluble form do not react with the membrane-bound protein on the living organism. Although it is not always the case that antibodies against the GPI-anchored form of a protein do not bind the soluble form or vice versa (6 , 22 23 24) , the antigen used and the method of selection of the antibody might be critical determinants in the success and/or interpretation of many experiments. By the same token, potential vaccine candidates that are expressed as recombinant proteins in bacteria, and thus are not GPI-anchored, might be inappropriate antigens for a protective immune response, precisely because of their altered conformation.


   ACKNOWLEDGMENTS
 
We thank Dirk Dobbelaere and Paul Englund for constructive comments on the manuscript and Anant Menon for scanning the blots and for helpful discussions. This work was supported by grants from the Swiss National Science Foundation (3100–050587.97 and 3100–050932.97).

Received for publication July 7, 2000. Revision received August 7, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 REFERENCES
 

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