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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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Protection of CD59 mutant-transfected cells from lysis by  complement. (A) First-round scanning mutations; (B) second-round mutations. CHO cells transfected with mutant CD59 genes were loaded  with calcein-AM and then subjected to complement attack as described in  the Materials and Methods. Percent lysis was calculated from the fluorescence of calcein released by cell lysis as a fraction of total calcein loading  as also described in Materials and Methods. For all mutants, the total fluorescence per well was similar to the wild-type value. Mean and standard  deviations of % lysis in the presence of NHS alone (filled bars) or in the  presence of NHS + anti-CD59 blocking antibodies (open bars) were calculated from triplicate samples in a single experiment. Asterisks identify  mutants which do not protect cells from lysis. Results are from a single  experiment; independent replications gave similar results.
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Figure 2: Protection of CD59 mutant-transfected cells from lysis by complement. (A) First-round scanning mutations; (B) second-round mutations. CHO cells transfected with mutant CD59 genes were loaded with calcein-AM and then subjected to complement attack as described in the Materials and Methods. Percent lysis was calculated from the fluorescence of calcein released by cell lysis as a fraction of total calcein loading as also described in Materials and Methods. For all mutants, the total fluorescence per well was similar to the wild-type value. Mean and standard deviations of % lysis in the presence of NHS alone (filled bars) or in the presence of NHS + anti-CD59 blocking antibodies (open bars) were calculated from triplicate samples in a single experiment. Asterisks identify mutants which do not protect cells from lysis. Results are from a single experiment; independent replications gave similar results.

Mentions: The calcein-release assay was used to measure the ability of each of the scanning mutants to inhibit complementmediated lysis of transiently transfected CHO cells (Fig. 2 A). FACSĀ® analysis indicated that the mutant proteins were all expressed at the cell surface at similar levels (Table 1). When the mutant-transfected cells were tested in the calcein-release assay, most were clearly protected from lysis by the presence of the mutant gene when compared with cells transfected with the expression vector alone (Fig. 2 A, black bars). Consistent with this, co-incubation with CD59blocking antibodies produced statistically significant reversals of the protective effects of these mutants (Fig. 2 A, white bars). These results suggest that most of the scanning mutations did not disrupt the folding or the protective activity of CD59. Although mutant F47E provided statistically insignificant protection compared to cells not expressing CD59 (P = 0.059), this mutant appeared to have some activity since a significant increase in lysis occurred in the presence of blocking antibodies (P = 0.004). Only one of the scanning mutants, W40E, completely failed to provide any protection from complement-mediated lysis. Loss of the inhibitory property of this mutant is not likely to be due to misfolding of the protein since it is recognized by the conformation-sensitive monoclonal antibodies HC1 and MEM43/5 (Table 1). These results indicate that W40 forms at least part of the active site essential for inhibiting the formation of the MAC by CD59. The weak protective effect of the mutant F47E suggests that this residue may be at the periphery of the active site or that it has subtle effects on the conformation of CD59 which indirectly affect its activity.


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)

Protection of CD59 mutant-transfected cells from lysis by  complement. (A) First-round scanning mutations; (B) second-round mutations. CHO cells transfected with mutant CD59 genes were loaded  with calcein-AM and then subjected to complement attack as described in  the Materials and Methods. Percent lysis was calculated from the fluorescence of calcein released by cell lysis as a fraction of total calcein loading  as also described in Materials and Methods. For all mutants, the total fluorescence per well was similar to the wild-type value. Mean and standard  deviations of % lysis in the presence of NHS alone (filled bars) or in the  presence of NHS + anti-CD59 blocking antibodies (open bars) were calculated from triplicate samples in a single experiment. Asterisks identify  mutants which do not protect cells from lysis. Results are from a single  experiment; independent replications gave similar results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196035&req=5

Figure 2: Protection of CD59 mutant-transfected cells from lysis by complement. (A) First-round scanning mutations; (B) second-round mutations. CHO cells transfected with mutant CD59 genes were loaded with calcein-AM and then subjected to complement attack as described in the Materials and Methods. Percent lysis was calculated from the fluorescence of calcein released by cell lysis as a fraction of total calcein loading as also described in Materials and Methods. For all mutants, the total fluorescence per well was similar to the wild-type value. Mean and standard deviations of % lysis in the presence of NHS alone (filled bars) or in the presence of NHS + anti-CD59 blocking antibodies (open bars) were calculated from triplicate samples in a single experiment. Asterisks identify mutants which do not protect cells from lysis. Results are from a single experiment; independent replications gave similar results.
Mentions: The calcein-release assay was used to measure the ability of each of the scanning mutants to inhibit complementmediated lysis of transiently transfected CHO cells (Fig. 2 A). FACSĀ® analysis indicated that the mutant proteins were all expressed at the cell surface at similar levels (Table 1). When the mutant-transfected cells were tested in the calcein-release assay, most were clearly protected from lysis by the presence of the mutant gene when compared with cells transfected with the expression vector alone (Fig. 2 A, black bars). Consistent with this, co-incubation with CD59blocking antibodies produced statistically significant reversals of the protective effects of these mutants (Fig. 2 A, white bars). These results suggest that most of the scanning mutations did not disrupt the folding or the protective activity of CD59. Although mutant F47E provided statistically insignificant protection compared to cells not expressing CD59 (P = 0.059), this mutant appeared to have some activity since a significant increase in lysis occurred in the presence of blocking antibodies (P = 0.004). Only one of the scanning mutants, W40E, completely failed to provide any protection from complement-mediated lysis. Loss of the inhibitory property of this mutant is not likely to be due to misfolding of the protein since it is recognized by the conformation-sensitive monoclonal antibodies HC1 and MEM43/5 (Table 1). These results indicate that W40 forms at least part of the active site essential for inhibiting the formation of the MAC by CD59. The weak protective effect of the mutant F47E suggests that this residue may be at the periphery of the active site or that it has subtle effects on the conformation of CD59 which indirectly affect its activity.

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
Related in: MedlinePlus