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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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Diagram of CD59Ly6E chimeric constructs. All  numbers refer to amino acid positions of CD59.
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Figure 2: Diagram of CD59Ly6E chimeric constructs. All numbers refer to amino acid positions of CD59.

Mentions: Based on the alignment of CD59 and Ly6E amino acid sequences (Fig. 1), cDNA encoding the chimeric constructs depicted in Fig. 2 were prepared. Segments of either CD59 or Ly6E cDNA were generated and joined using PCR. The general procedure used for the generation of chimeric constructs was as described (24). The 5′ and 3′ end primers, which matched an untranslated sequence of either CD59 or Ly6E, included a HindIII and ApaI site, respectively. Using either CD59 or Ly6E cloned into pCDNA3 as template, these primers were paired in a PCR with CD59-Ly6E chimeric primers that spanned the desired crossover point. The PCR products were purified from agarose gel after electrophoresis and used in a second amplification with the primers corresponding to 5′ or 3′ untranslated regions. The resulting full-length CD59-Ly6E chimeric cDNAs were cloned into the HindIII/ApaI sites of pCDNA3 for sequencing and expression. Five site-specific point mutations in CD59 (N18Q, F23D, Y36F, W40Y, and L54N) were prepared by PCR using similar techniques. In the first PCR amplification, 5′ and 3′ primers to CD59 untranslated region were paired with primers spanning the target site, and which contained a substituted codon at the target site. An epitope tag was engineered into CD59-Ly6E chimeric proteins between the first and second amino acids of mature CD59. The NH2-terminal Leu was duplicated at the COOH-terminus of the tag. A 36mer oligonucleotide encoding NANPNANPNA and with appropriate overhangs was inserted at the PstI restriction site of each point-specific mutant, and of CD59 and CD-Ly constructs (chimeras with CD59 at the NH2 terminus).


Mapping the active site of CD59.

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

Diagram of CD59Ly6E chimeric constructs. All  numbers refer to amino acid positions of CD59.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Diagram of CD59Ly6E chimeric constructs. All numbers refer to amino acid positions of CD59.
Mentions: Based on the alignment of CD59 and Ly6E amino acid sequences (Fig. 1), cDNA encoding the chimeric constructs depicted in Fig. 2 were prepared. Segments of either CD59 or Ly6E cDNA were generated and joined using PCR. The general procedure used for the generation of chimeric constructs was as described (24). The 5′ and 3′ end primers, which matched an untranslated sequence of either CD59 or Ly6E, included a HindIII and ApaI site, respectively. Using either CD59 or Ly6E cloned into pCDNA3 as template, these primers were paired in a PCR with CD59-Ly6E chimeric primers that spanned the desired crossover point. The PCR products were purified from agarose gel after electrophoresis and used in a second amplification with the primers corresponding to 5′ or 3′ untranslated regions. The resulting full-length CD59-Ly6E chimeric cDNAs were cloned into the HindIII/ApaI sites of pCDNA3 for sequencing and expression. Five site-specific point mutations in CD59 (N18Q, F23D, Y36F, W40Y, and L54N) were prepared by PCR using similar techniques. In the first PCR amplification, 5′ and 3′ primers to CD59 untranslated region were paired with primers spanning the target site, and which contained a substituted codon at the target site. An epitope tag was engineered into CD59-Ly6E chimeric proteins between the first and second amino acids of mature CD59. The NH2-terminal Leu was duplicated at the COOH-terminus of the tag. A 36mer oligonucleotide encoding NANPNANPNA and with appropriate overhangs was inserted at the PstI restriction site of each point-specific mutant, and of CD59 and CD-Ly constructs (chimeras with CD59 at the NH2 terminus).

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