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Mapping the active site of CD59.

Yu J, Abagyan R, Dong S, Gilbert A, Nussenzweig V, Tomlinson S - J. Exp. Med. (1997)

Bottom Line: CD59 is a widely distributed membrane-bound inhibitor of the cytolytic membrane attack complex (MAC) of complement.We present data obtained from a combination of molecular modeling and mutagenesis techniques, which together indicate that the active site of CD59 is located in the vicinity of a hydrophobic groove on the face of the molecule opposite to a "hydrophobic strip" suggested earlier.In addition, removal of the single N-linked glycosylation site at Asn18 of CD59 resulted in an enhancement of complement inhibitory activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, New York University Medical Center, New York 10016, USA.

ABSTRACT
CD59 is a widely distributed membrane-bound inhibitor of the cytolytic membrane attack complex (MAC) of complement. This small (77 amino acid) glycoprotein is a member of the Ly6 superfamily of proteins and is important in protecting host cells from the lytic and proinflammatory activity of the MAC. CD59 functions by binding to C8 and/or C9 in the nascent MAC and interfering with C9 membrane insertion and polymerization. We present data obtained from a combination of molecular modeling and mutagenesis techniques, which together indicate that the active site of CD59 is located in the vicinity of a hydrophobic groove on the face of the molecule opposite to a "hydrophobic strip" suggested earlier. In addition, removal of the single N-linked glycosylation site at Asn18 of CD59 resulted in an enhancement of complement inhibitory activity.

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Complement resistance of CHO cells expressing  CD59 and site-specific CD59  mutants. Complement resistance  of CHO cell populations expressing similar levels of either  CD59 or CD59 containing the  indicated amino acid substitutions was determined. Cells were  exposed to 10% NHS.
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Figure 8: Complement resistance of CHO cells expressing CD59 and site-specific CD59 mutants. Complement resistance of CHO cell populations expressing similar levels of either CD59 or CD59 containing the indicated amino acid substitutions was determined. Cells were exposed to 10% NHS.

Mentions: The three conserved residues (shown in green, Fig. 7 b) and the conserved hydrophobic residue at position F23 within the putatively identified binding groove of CD59 were individually targeted for site-specific mutagenesis. The four mutant CD59s prepared were F23D (Phe at position 23 changed to Asp), Y36F, W40Y and L54N. The amino acids used for the substitutions were chosen to have similar shape but different physico-chemical properties. The W40Y mutant was not stably expressed on the surface of transfected CHO cells. Of the three expressed mutant proteins, L54N had almost no function, F23D retained some but significantly reduced function, and Y36F had normal function (Fig. 8). For unknown reasons, only relatively moderate levels of the L54N mutant protein could be expressed on CHO cells, but activity was compared with CHO cells expressing similar levels of wild-type CD59 (see Table 1). In an attempt to assess whether the mutant proteins were correctly folded, we studied their reactivity with three different mAbs that each bind to nonoverlapping conformational epitopes on CD59. All mutant proteins were recognized by these antibodies (Table 1).


Mapping the active site of CD59.

Yu J, Abagyan R, Dong S, Gilbert A, Nussenzweig V, Tomlinson S - J. Exp. Med. (1997)

Complement resistance of CHO cells expressing  CD59 and site-specific CD59  mutants. Complement resistance  of CHO cell populations expressing similar levels of either  CD59 or CD59 containing the  indicated amino acid substitutions was determined. Cells were  exposed to 10% NHS.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196154&req=5

Figure 8: Complement resistance of CHO cells expressing CD59 and site-specific CD59 mutants. Complement resistance of CHO cell populations expressing similar levels of either CD59 or CD59 containing the indicated amino acid substitutions was determined. Cells were exposed to 10% NHS.
Mentions: The three conserved residues (shown in green, Fig. 7 b) and the conserved hydrophobic residue at position F23 within the putatively identified binding groove of CD59 were individually targeted for site-specific mutagenesis. The four mutant CD59s prepared were F23D (Phe at position 23 changed to Asp), Y36F, W40Y and L54N. The amino acids used for the substitutions were chosen to have similar shape but different physico-chemical properties. The W40Y mutant was not stably expressed on the surface of transfected CHO cells. Of the three expressed mutant proteins, L54N had almost no function, F23D retained some but significantly reduced function, and Y36F had normal function (Fig. 8). For unknown reasons, only relatively moderate levels of the L54N mutant protein could be expressed on CHO cells, but activity was compared with CHO cells expressing similar levels of wild-type CD59 (see Table 1). In an attempt to assess whether the mutant proteins were correctly folded, we studied their reactivity with three different mAbs that each bind to nonoverlapping conformational epitopes on CD59. All mutant proteins were recognized by these antibodies (Table 1).

Bottom Line: CD59 is a widely distributed membrane-bound inhibitor of the cytolytic membrane attack complex (MAC) of complement.We present data obtained from a combination of molecular modeling and mutagenesis techniques, which together indicate that the active site of CD59 is located in the vicinity of a hydrophobic groove on the face of the molecule opposite to a "hydrophobic strip" suggested earlier.In addition, removal of the single N-linked glycosylation site at Asn18 of CD59 resulted in an enhancement of complement inhibitory activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, New York University Medical Center, New York 10016, USA.

ABSTRACT
CD59 is a widely distributed membrane-bound inhibitor of the cytolytic membrane attack complex (MAC) of complement. This small (77 amino acid) glycoprotein is a member of the Ly6 superfamily of proteins and is important in protecting host cells from the lytic and proinflammatory activity of the MAC. CD59 functions by binding to C8 and/or C9 in the nascent MAC and interfering with C9 membrane insertion and polymerization. We present data obtained from a combination of molecular modeling and mutagenesis techniques, which together indicate that the active site of CD59 is located in the vicinity of a hydrophobic groove on the face of the molecule opposite to a "hydrophobic strip" suggested earlier. In addition, removal of the single N-linked glycosylation site at Asn18 of CD59 resulted in an enhancement of complement inhibitory activity.

Show MeSH
Related in: MedlinePlus