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Mutational analysis of the active site and antibody epitopes of the complement-inhibitory glycoprotein, CD59.

Bodian DL, Davis SJ, Morgan BP, Rushmere NK - J. Exp. Med. (1997)

Bottom Line: The putative active site includes residues conserved across species, consistent with the lack of strict homologous restriction previously observed in studies of CD59 function.Competition and mutational analyses of the epitopes of eight CD59-blocking and non-blocking monoclonal antibodies confirmed the location of the active site.Additional experiments showed that the expression and function of CD59 are both glycosylation independent.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biophysics, Oxford, United Kingdom.

ABSTRACT
The Ly-6 superfamily of cell surface molecules includes CD59, a potent regulator of the complement system that protects host cells from the cytolytic action of the membrane attack complex (MAC). Although its mechanism of action is not well understood, CD59 is thought to prevent assembly of the MAC by binding to the C8 and/or C9 proteins of the nascent complex. Here a systematic, structure-based mutational approach has been used to determine the region(s) of CD59 required for its protective activity. Analysis of 16 CD59 mutants with single, highly nonconservative substitutions suggests that CD59 has a single active site that includes Trp-40, Arg-53, and Glu-56 of the glycosylated, membrane-distal face of the disk-like extra-cellular domain and, possibly, Asp-24 positioned at the edge of the domain. The putative active site includes residues conserved across species, consistent with the lack of strict homologous restriction previously observed in studies of CD59 function. Competition and mutational analyses of the epitopes of eight CD59-blocking and non-blocking monoclonal antibodies confirmed the location of the active site. Additional experiments showed that the expression and function of CD59 are both glycosylation independent.

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The epitopes of anti-CD59 function-blocking mAb cluster  in the region of the proposed active site of CD59. Residues whose mutation disrupted the binding of each antibody (inverse shaded black in Table 1)  are shaded black and those whose mutation led to reduced levels of binding (shaded gray in Table 1) are shaded gray. Only antibodies HC1 and  MEM43/5 have no CD59-blocking ability. In the last panel, all of the  residues that were mutated are shaded gray except for the visible residues  whose mutation disrupted the function of CD59 which are shaded black  and labeled. In each panel the protein face shown is the one containing  the proposed active site shown in the same view as in Fig. 3 A. The experimental data are superimposed on the lowest energy NMR structure  (19) drawn using Rasmol (46).
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Figure 4: The epitopes of anti-CD59 function-blocking mAb cluster in the region of the proposed active site of CD59. Residues whose mutation disrupted the binding of each antibody (inverse shaded black in Table 1) are shaded black and those whose mutation led to reduced levels of binding (shaded gray in Table 1) are shaded gray. Only antibodies HC1 and MEM43/5 have no CD59-blocking ability. In the last panel, all of the residues that were mutated are shaded gray except for the visible residues whose mutation disrupted the function of CD59 which are shaded black and labeled. In each panel the protein face shown is the one containing the proposed active site shown in the same view as in Fig. 3 A. The experimental data are superimposed on the lowest energy NMR structure (19) drawn using Rasmol (46).

Mentions: The antibody epitopes were mapped by measuring the ability of each antibody to bind to CHO cells transfected with each of the mutants. Although this approach cannot delineate their borders precisely, the data show that the epitopes of the blocking antibodies are overlapping but distinct (Table 1 and Fig. 4). All include W40, plus differing portions of continuous surface defined by R53 and other residues adjacent to or in close proximity to W40, suggesting that this region overlaps with the active site of CD59. This is consistent with the results of the calcein-release assay, in which mutants W40E and R53E were unable to protect cells from complement-mediated lysis. It may be significant, however, that mutation of D24 to R did not prevent the binding of any of the blocking antibodies, although this change disrupts CD59 activity. The binding data also indicate that the epitopes of the non-blocking antibodies both lie in a distinct region of the protein which includes L33. However, mutation of E56 to R, which disrupts the function of CD59, also prevents binding of the non-blocking antibody HC1. This implies that the epitope of HC1 and the active site of CD59 partially overlap but in such a way that antibody binding does not completely block CD59 activity. Although the epitope for HC1 appears discontinuous in the figure, this cannot be concluded since the role of intervening residues in antibody binding was not tested.


Mutational analysis of the active site and antibody epitopes of the complement-inhibitory glycoprotein, CD59.

Bodian DL, Davis SJ, Morgan BP, Rushmere NK - J. Exp. Med. (1997)

The epitopes of anti-CD59 function-blocking mAb cluster  in the region of the proposed active site of CD59. Residues whose mutation disrupted the binding of each antibody (inverse shaded black in Table 1)  are shaded black and those whose mutation led to reduced levels of binding (shaded gray in Table 1) are shaded gray. Only antibodies HC1 and  MEM43/5 have no CD59-blocking ability. In the last panel, all of the  residues that were mutated are shaded gray except for the visible residues  whose mutation disrupted the function of CD59 which are shaded black  and labeled. In each panel the protein face shown is the one containing  the proposed active site shown in the same view as in Fig. 3 A. The experimental data are superimposed on the lowest energy NMR structure  (19) drawn using Rasmol (46).
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Related In: Results  -  Collection

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Figure 4: The epitopes of anti-CD59 function-blocking mAb cluster in the region of the proposed active site of CD59. Residues whose mutation disrupted the binding of each antibody (inverse shaded black in Table 1) are shaded black and those whose mutation led to reduced levels of binding (shaded gray in Table 1) are shaded gray. Only antibodies HC1 and MEM43/5 have no CD59-blocking ability. In the last panel, all of the residues that were mutated are shaded gray except for the visible residues whose mutation disrupted the function of CD59 which are shaded black and labeled. In each panel the protein face shown is the one containing the proposed active site shown in the same view as in Fig. 3 A. The experimental data are superimposed on the lowest energy NMR structure (19) drawn using Rasmol (46).
Mentions: The antibody epitopes were mapped by measuring the ability of each antibody to bind to CHO cells transfected with each of the mutants. Although this approach cannot delineate their borders precisely, the data show that the epitopes of the blocking antibodies are overlapping but distinct (Table 1 and Fig. 4). All include W40, plus differing portions of continuous surface defined by R53 and other residues adjacent to or in close proximity to W40, suggesting that this region overlaps with the active site of CD59. This is consistent with the results of the calcein-release assay, in which mutants W40E and R53E were unable to protect cells from complement-mediated lysis. It may be significant, however, that mutation of D24 to R did not prevent the binding of any of the blocking antibodies, although this change disrupts CD59 activity. The binding data also indicate that the epitopes of the non-blocking antibodies both lie in a distinct region of the protein which includes L33. However, mutation of E56 to R, which disrupts the function of CD59, also prevents binding of the non-blocking antibody HC1. This implies that the epitope of HC1 and the active site of CD59 partially overlap but in such a way that antibody binding does not completely block CD59 activity. Although the epitope for HC1 appears discontinuous in the figure, this cannot be concluded since the role of intervening residues in antibody binding was not tested.

Bottom Line: The putative active site includes residues conserved across species, consistent with the lack of strict homologous restriction previously observed in studies of CD59 function.Competition and mutational analyses of the epitopes of eight CD59-blocking and non-blocking monoclonal antibodies confirmed the location of the active site.Additional experiments showed that the expression and function of CD59 are both glycosylation independent.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biophysics, Oxford, United Kingdom.

ABSTRACT
The Ly-6 superfamily of cell surface molecules includes CD59, a potent regulator of the complement system that protects host cells from the cytolytic action of the membrane attack complex (MAC). Although its mechanism of action is not well understood, CD59 is thought to prevent assembly of the MAC by binding to the C8 and/or C9 proteins of the nascent complex. Here a systematic, structure-based mutational approach has been used to determine the region(s) of CD59 required for its protective activity. Analysis of 16 CD59 mutants with single, highly nonconservative substitutions suggests that CD59 has a single active site that includes Trp-40, Arg-53, and Glu-56 of the glycosylated, membrane-distal face of the disk-like extra-cellular domain and, possibly, Asp-24 positioned at the edge of the domain. The putative active site includes residues conserved across species, consistent with the lack of strict homologous restriction previously observed in studies of CD59 function. Competition and mutational analyses of the epitopes of eight CD59-blocking and non-blocking monoclonal antibodies confirmed the location of the active site. Additional experiments showed that the expression and function of CD59 are both glycosylation independent.

Show MeSH