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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 inhibitory activity of epitope tagged and untagged CD59. Stable CHO cell clones expressing similar levels of CD59  or CD59 containing an NH2-terminal peptide tag (NANPNANPNA),  were assayed for their susceptibility to complement. Expression was quantitated using anti-CD59 mAb YTH53.1 and rabbit anti-CD59 Ab.
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Figure 5: Complement inhibitory activity of epitope tagged and untagged CD59. Stable CHO cell clones expressing similar levels of CD59 or CD59 containing an NH2-terminal peptide tag (NANPNANPNA), were assayed for their susceptibility to complement. Expression was quantitated using anti-CD59 mAb YTH53.1 and rabbit anti-CD59 Ab.

Mentions: To quantitate the relative surface expression of CD59 and CD-Ly chimeric proteins, a 10–amino acid epitope tag was engineered onto the NH2 termini of the constructs. The tag did not affect the expression (Table 1) or the function (Fig. 5) of CD59. However, when the tag was placed at the NH2 terminus of Ly6E in the Ly-CD constructs, it interfered with expression and function of the chimeras. Therefore, surface expression of Ly-CD chimeras was quantitated using a rabbit Ab against a peptide corresponding to a sequence from the COOH terminus of CD59 (Table 1). This Ab reacted quantitatively with CHO cell clones stably expressing different levels of CD59 (not shown). Cell populations used in these experiments were obtained by several rounds of cell sorting, and separate CD-Ly and LyCD sets were prepared, each with a corresponding CHO cell population expressing a similar level of CD59.


Mapping the active site of CD59.

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

Complement inhibitory activity of epitope tagged and untagged CD59. Stable CHO cell clones expressing similar levels of CD59  or CD59 containing an NH2-terminal peptide tag (NANPNANPNA),  were assayed for their susceptibility to complement. Expression was quantitated using anti-CD59 mAb YTH53.1 and rabbit anti-CD59 Ab.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Complement inhibitory activity of epitope tagged and untagged CD59. Stable CHO cell clones expressing similar levels of CD59 or CD59 containing an NH2-terminal peptide tag (NANPNANPNA), were assayed for their susceptibility to complement. Expression was quantitated using anti-CD59 mAb YTH53.1 and rabbit anti-CD59 Ab.
Mentions: To quantitate the relative surface expression of CD59 and CD-Ly chimeric proteins, a 10–amino acid epitope tag was engineered onto the NH2 termini of the constructs. The tag did not affect the expression (Table 1) or the function (Fig. 5) of CD59. However, when the tag was placed at the NH2 terminus of Ly6E in the Ly-CD constructs, it interfered with expression and function of the chimeras. Therefore, surface expression of Ly-CD chimeras was quantitated using a rabbit Ab against a peptide corresponding to a sequence from the COOH terminus of CD59 (Table 1). This Ab reacted quantitatively with CHO cell clones stably expressing different levels of CD59 (not shown). Cell populations used in these experiments were obtained by several rounds of cell sorting, and separate CD-Ly and LyCD sets were prepared, each with a corresponding CHO cell population expressing a similar level of CD59.

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