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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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Location and properties of the proposed active site of CD59. (A–C) Mutagenesis data. The residues which were mutated are all numbered  and those whose mutation reduced protection (* in Fig. 1) or had no effect are colored red and light blue, respectively. N18, which is N-glycosylated, is  colored green. Back (B) and side (C) views differ from the front view (A) by 180° and 90°, respectively. (D) Chemical features. Hydrophobic residues are  colored green, polar uncharged residues, light blue, positively charged residues, dark blue, and negatively charged residues, red. The view is the same as  in A. (E) Conserved residues. Non-cysteine residues that are conserved in all known CD59 sequences and HVS-15 (inverse-shaded black in Fig. 1) are  colored red and those conserved in all sequences with one exception (inverse-shaded gray in Fig. 1) are colored orange. Cysteine residues, which are also  conserved in all sequences, are colored yellow. The view is the same as in A. (F) Secondary structure. The positions of the two- and three-stranded β-sheets  (purple and red, respectively) and the α-helix (dark blue) of CD59 are shown. Loop residues are colored light blue. The view is the same as in A. All of the  experimental data are superimposed on the lowest energy NMR structure (19) drawn using Rasmol (46).
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Figure 3: Location and properties of the proposed active site of CD59. (A–C) Mutagenesis data. The residues which were mutated are all numbered and those whose mutation reduced protection (* in Fig. 1) or had no effect are colored red and light blue, respectively. N18, which is N-glycosylated, is colored green. Back (B) and side (C) views differ from the front view (A) by 180° and 90°, respectively. (D) Chemical features. Hydrophobic residues are colored green, polar uncharged residues, light blue, positively charged residues, dark blue, and negatively charged residues, red. The view is the same as in A. (E) Conserved residues. Non-cysteine residues that are conserved in all known CD59 sequences and HVS-15 (inverse-shaded black in Fig. 1) are colored red and those conserved in all sequences with one exception (inverse-shaded gray in Fig. 1) are colored orange. Cysteine residues, which are also conserved in all sequences, are colored yellow. The view is the same as in A. (F) Secondary structure. The positions of the two- and three-stranded β-sheets (purple and red, respectively) and the α-helix (dark blue) of CD59 are shown. Loop residues are colored light blue. The view is the same as in A. All of the experimental data are superimposed on the lowest energy NMR structure (19) drawn using Rasmol (46).

Mentions: The sidechain of W40 partially fills a cleft formed by the packing of the α-helix against the three-stranded β-sheet on the glycosylated face of CD59 (19, 20). In a second round of mutagenesis, residues in and around this cleft, specifically K38, K41, R53, L54, and E56, were mutated. Residue D24, which is adjacent to W40 but projects in the opposite direction, was also mutated because it forms part of a conserved loop consisting of residues 20-24 (20, 23). Of these mutants, D24R, R53E, and E56R failed to provide any protection from complement that was reversible by co-incubation with CD59-blocking antibodies (Fig. 2 B). All of the mutations that were tested are represented on the NMR structure (19) in Fig. 3, A–C.


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)

Location and properties of the proposed active site of CD59. (A–C) Mutagenesis data. The residues which were mutated are all numbered  and those whose mutation reduced protection (* in Fig. 1) or had no effect are colored red and light blue, respectively. N18, which is N-glycosylated, is  colored green. Back (B) and side (C) views differ from the front view (A) by 180° and 90°, respectively. (D) Chemical features. Hydrophobic residues are  colored green, polar uncharged residues, light blue, positively charged residues, dark blue, and negatively charged residues, red. The view is the same as  in A. (E) Conserved residues. Non-cysteine residues that are conserved in all known CD59 sequences and HVS-15 (inverse-shaded black in Fig. 1) are  colored red and those conserved in all sequences with one exception (inverse-shaded gray in Fig. 1) are colored orange. Cysteine residues, which are also  conserved in all sequences, are colored yellow. The view is the same as in A. (F) Secondary structure. The positions of the two- and three-stranded β-sheets  (purple and red, respectively) and the α-helix (dark blue) of CD59 are shown. Loop residues are colored light blue. The view is the same as in A. All of the  experimental data are superimposed on the lowest energy NMR structure (19) drawn using Rasmol (46).
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Figure 3: Location and properties of the proposed active site of CD59. (A–C) Mutagenesis data. The residues which were mutated are all numbered and those whose mutation reduced protection (* in Fig. 1) or had no effect are colored red and light blue, respectively. N18, which is N-glycosylated, is colored green. Back (B) and side (C) views differ from the front view (A) by 180° and 90°, respectively. (D) Chemical features. Hydrophobic residues are colored green, polar uncharged residues, light blue, positively charged residues, dark blue, and negatively charged residues, red. The view is the same as in A. (E) Conserved residues. Non-cysteine residues that are conserved in all known CD59 sequences and HVS-15 (inverse-shaded black in Fig. 1) are colored red and those conserved in all sequences with one exception (inverse-shaded gray in Fig. 1) are colored orange. Cysteine residues, which are also conserved in all sequences, are colored yellow. The view is the same as in A. (F) Secondary structure. The positions of the two- and three-stranded β-sheets (purple and red, respectively) and the α-helix (dark blue) of CD59 are shown. Loop residues are colored light blue. The view is the same as in A. All of the experimental data are superimposed on the lowest energy NMR structure (19) drawn using Rasmol (46).
Mentions: The sidechain of W40 partially fills a cleft formed by the packing of the α-helix against the three-stranded β-sheet on the glycosylated face of CD59 (19, 20). In a second round of mutagenesis, residues in and around this cleft, specifically K38, K41, R53, L54, and E56, were mutated. Residue D24, which is adjacent to W40 but projects in the opposite direction, was also mutated because it forms part of a conserved loop consisting of residues 20-24 (20, 23). Of these mutants, D24R, R53E, and E56R failed to provide any protection from complement that was reversible by co-incubation with CD59-blocking antibodies (Fig. 2 B). All of the mutations that were tested are represented on the NMR structure (19) in Fig. 3, A–C.

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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