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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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Western-blot analysis of mutant N18Q. CHO cells transfected with the expression vector alone or with the vector expressing wildtype or mutant N18Q CD59 were solubilized and subjected to SDS-PAGE  along with purified erythrocyte CD59 and then Western-blotted with antiCD59 antibody MEM43 as described in the Materials and Methods.  Lanes 1, 2, and 3 contained 500, 100, and 50 ng of erythrocyte CD59, respectively. Lanes 4, 5, and 6 contained aliquots of the cell lysate corresponding to ∼5 × 105 CHO cells transfected with the expression vector  alone or with the vector expressing mutant N18Q or wild-type CD59,  respectively.
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Figure 5: Western-blot analysis of mutant N18Q. CHO cells transfected with the expression vector alone or with the vector expressing wildtype or mutant N18Q CD59 were solubilized and subjected to SDS-PAGE along with purified erythrocyte CD59 and then Western-blotted with antiCD59 antibody MEM43 as described in the Materials and Methods. Lanes 1, 2, and 3 contained 500, 100, and 50 ng of erythrocyte CD59, respectively. Lanes 4, 5, and 6 contained aliquots of the cell lysate corresponding to ∼5 × 105 CHO cells transfected with the expression vector alone or with the vector expressing mutant N18Q or wild-type CD59, respectively.

Mentions: To test the role of the glycans at the highly conserved N-glycosylation site in the complement-inhibitory function of CD59, N18 was mutated to Q. The N18Q mutant form of CD59 runs as a single sharp band of ∼14 kD on SDS-PAGE, at least 5 kD smaller than wild-type CD59 expressed in CHO cells, which runs as a ladder of bands in the molecular mass range 20–25 kD, and erythrocyte CD59, which runs as a broad smear in the molecular mass range 19–25 kD (Fig. 5). The mobility and banding patterns of all of the other mutants were indistinguishable from those of wild-type CD59 (data not shown). The mobility of N18Q CD59 is similar to that of deglycosylated CD59 (28), indicating that the mutant is not glycosylated. The expression of mutant N18Q at the cell surface, where it binds all of the polyclonal and monoclonal antibodies (Table 1), demonstrates that N-glycosylation is not required for native-like folding and expression of human CD59. Mutant N18Q retains wild-type levels of complement-inhibitory activity (Fig. 2 B) and binds all of the blocking antibodies at levels similar to that of wild-type CD59 (Table 1), suggesting that the N-glycan does not contribute to the active site. This is consistent with the ability of YTH53.1 and BRIC 229 to immunoblot CD59 from endoglycosidase F-treated erythrocytes (37). These data suggest that N-glycosylation is not required for the maintenance of the structure or complement-inhibitory function of human CD59.


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)

Western-blot analysis of mutant N18Q. CHO cells transfected with the expression vector alone or with the vector expressing wildtype or mutant N18Q CD59 were solubilized and subjected to SDS-PAGE  along with purified erythrocyte CD59 and then Western-blotted with antiCD59 antibody MEM43 as described in the Materials and Methods.  Lanes 1, 2, and 3 contained 500, 100, and 50 ng of erythrocyte CD59, respectively. Lanes 4, 5, and 6 contained aliquots of the cell lysate corresponding to ∼5 × 105 CHO cells transfected with the expression vector  alone or with the vector expressing mutant N18Q or wild-type CD59,  respectively.
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Related In: Results  -  Collection

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Figure 5: Western-blot analysis of mutant N18Q. CHO cells transfected with the expression vector alone or with the vector expressing wildtype or mutant N18Q CD59 were solubilized and subjected to SDS-PAGE along with purified erythrocyte CD59 and then Western-blotted with antiCD59 antibody MEM43 as described in the Materials and Methods. Lanes 1, 2, and 3 contained 500, 100, and 50 ng of erythrocyte CD59, respectively. Lanes 4, 5, and 6 contained aliquots of the cell lysate corresponding to ∼5 × 105 CHO cells transfected with the expression vector alone or with the vector expressing mutant N18Q or wild-type CD59, respectively.
Mentions: To test the role of the glycans at the highly conserved N-glycosylation site in the complement-inhibitory function of CD59, N18 was mutated to Q. The N18Q mutant form of CD59 runs as a single sharp band of ∼14 kD on SDS-PAGE, at least 5 kD smaller than wild-type CD59 expressed in CHO cells, which runs as a ladder of bands in the molecular mass range 20–25 kD, and erythrocyte CD59, which runs as a broad smear in the molecular mass range 19–25 kD (Fig. 5). The mobility and banding patterns of all of the other mutants were indistinguishable from those of wild-type CD59 (data not shown). The mobility of N18Q CD59 is similar to that of deglycosylated CD59 (28), indicating that the mutant is not glycosylated. The expression of mutant N18Q at the cell surface, where it binds all of the polyclonal and monoclonal antibodies (Table 1), demonstrates that N-glycosylation is not required for native-like folding and expression of human CD59. Mutant N18Q retains wild-type levels of complement-inhibitory activity (Fig. 2 B) and binds all of the blocking antibodies at levels similar to that of wild-type CD59 (Table 1), suggesting that the N-glycan does not contribute to the active site. This is consistent with the ability of YTH53.1 and BRIC 229 to immunoblot CD59 from endoglycosidase F-treated erythrocytes (37). These data suggest that N-glycosylation is not required for the maintenance of the structure or complement-inhibitory function of human CD59.

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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