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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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Alignment of CD59  species homologs and HVS-15.  Amino acid sequences for the  extracellular regions of human  (22), African green monkey (11),  baboon (11), owl monkey (GenBank accession number: L22861),  marmoset (GenBank accession  number L22860), squirrel monkey  (6), and rat (23) CD59 and Herpesvirus saimiri protein HVS-15  (5) were aligned with the partial  sequences of pig (23) and sheep  (44) CD59. Residues identical in  all sequences are inverse-shaded  black and those that are conserved  in all species with one exception  are inverse-shaded gray. Putative glycosylation sites are boxed.  Lower case letters represent probable amino acid assignment, and  X represents positions with unknown sequence (23). Residues  are numbered according to the sequence of the human mature polypeptide. Every tenth residue is indicated, and both the residue number and introduced substitution are listed for each position selected for mutation. Asterisks mark mutations that disrupted the complement-inhibitory function of human CD59. The sequences were aligned using GCG software (45).
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Figure 1: Alignment of CD59 species homologs and HVS-15. Amino acid sequences for the extracellular regions of human (22), African green monkey (11), baboon (11), owl monkey (GenBank accession number: L22861), marmoset (GenBank accession number L22860), squirrel monkey (6), and rat (23) CD59 and Herpesvirus saimiri protein HVS-15 (5) were aligned with the partial sequences of pig (23) and sheep (44) CD59. Residues identical in all sequences are inverse-shaded black and those that are conserved in all species with one exception are inverse-shaded gray. Putative glycosylation sites are boxed. Lower case letters represent probable amino acid assignment, and X represents positions with unknown sequence (23). Residues are numbered according to the sequence of the human mature polypeptide. Every tenth residue is indicated, and both the residue number and introduced substitution are listed for each position selected for mutation. Asterisks mark mutations that disrupted the complement-inhibitory function of human CD59. The sequences were aligned using GCG software (45).

Mentions: The selection of particular residues for mutation was based on several criteria. First, only amino acids with side chains pointing away from the structural core of the protein in the NMR structures were chosen. Assuming that the overall conformation of CD59 in the NMR structure represents that of CD59 when bound to a putative ligand (even though the conformations of the side chains are likely to differ), only side chains of surface residues will directly participate in binding interactions. Second, given the observed lack of strict homologous restriction between certain species (9), it is likely that the active site will include some residues conserved between species so residues representing the most highly conserved of the surface amino acids were selected for mutation (Fig. 1). Third, where possible, mutations were made in regions of the protein implicated in CD59 function by others in order to test their various proposals (19, 20, 23, 31, 32).


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)

Alignment of CD59  species homologs and HVS-15.  Amino acid sequences for the  extracellular regions of human  (22), African green monkey (11),  baboon (11), owl monkey (GenBank accession number: L22861),  marmoset (GenBank accession  number L22860), squirrel monkey  (6), and rat (23) CD59 and Herpesvirus saimiri protein HVS-15  (5) were aligned with the partial  sequences of pig (23) and sheep  (44) CD59. Residues identical in  all sequences are inverse-shaded  black and those that are conserved  in all species with one exception  are inverse-shaded gray. Putative glycosylation sites are boxed.  Lower case letters represent probable amino acid assignment, and  X represents positions with unknown sequence (23). Residues  are numbered according to the sequence of the human mature polypeptide. Every tenth residue is indicated, and both the residue number and introduced substitution are listed for each position selected for mutation. Asterisks mark mutations that disrupted the complement-inhibitory function of human CD59. The sequences were aligned using GCG software (45).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Alignment of CD59 species homologs and HVS-15. Amino acid sequences for the extracellular regions of human (22), African green monkey (11), baboon (11), owl monkey (GenBank accession number: L22861), marmoset (GenBank accession number L22860), squirrel monkey (6), and rat (23) CD59 and Herpesvirus saimiri protein HVS-15 (5) were aligned with the partial sequences of pig (23) and sheep (44) CD59. Residues identical in all sequences are inverse-shaded black and those that are conserved in all species with one exception are inverse-shaded gray. Putative glycosylation sites are boxed. Lower case letters represent probable amino acid assignment, and X represents positions with unknown sequence (23). Residues are numbered according to the sequence of the human mature polypeptide. Every tenth residue is indicated, and both the residue number and introduced substitution are listed for each position selected for mutation. Asterisks mark mutations that disrupted the complement-inhibitory function of human CD59. The sequences were aligned using GCG software (45).
Mentions: The selection of particular residues for mutation was based on several criteria. First, only amino acids with side chains pointing away from the structural core of the protein in the NMR structures were chosen. Assuming that the overall conformation of CD59 in the NMR structure represents that of CD59 when bound to a putative ligand (even though the conformations of the side chains are likely to differ), only side chains of surface residues will directly participate in binding interactions. Second, given the observed lack of strict homologous restriction between certain species (9), it is likely that the active site will include some residues conserved between species so residues representing the most highly conserved of the surface amino acids were selected for mutation (Fig. 1). Third, where possible, mutations were made in regions of the protein implicated in CD59 function by others in order to test their various proposals (19, 20, 23, 31, 32).

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