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1.
Biochemistry ; 35(10): 3263-9, 1996 Mar 12.
Article in English | MEDLINE | ID: mdl-8605162

ABSTRACT

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C9 component of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The complement-inhibitory activity of CD59 is species-selective, and is most effective toward C9 derived from human or other primate plasma. The species-selective activity of CD59 was recently used to map the segment of human C9 that is recognized by this MAC inhibitor, using recombinant rabbit/human C9 chimeras that retain lytic function within the MAC [Husler, T., Lockert, D. H., Kaufman, K. M., Sodetz, J. M., & Sims, P. J. (1995) J. Biol. Chem. 270,3483-3486]. These experiments suggested that the CD59 recognition domain was contained between residues 334 and 415 in human C9. By analyzing the species-selective lytic activity of recombinant C9 with chimeric substitutions internal to this segment, we now demonstrate that the site in human C9 uniquely recognized by CD59 is centered on those residues contained between C9 Cys359/Cys384, with an additional contribution by residues C-terminal to this segment. Consistent with its role as a CD59 recognition domain, CD59 specifically bound a human C9-derived peptide corresponding to residues 359-384, and antibody (Fab) raised against this C9-derived peptide inhibited the lytic activity of human MAC. Mutant human C9 in which Ala was substituted for Cys359/384 was found to express normal lytic activity and to be fully inhibited by CD59. This suggests that the intrachain Cys359/Cys384 disulfide bond within C9 is not required to maintain the conformation of this segment of C9 for interaction with CD59.


Subject(s)
CD59 Antigens/metabolism , Complement C9/metabolism , Complement Inactivator Proteins/metabolism , Amino Acid Sequence , Animals , CD59 Antigens/pharmacology , Complement C9/antagonists & inhibitors , Complement C9/genetics , Complement Inactivator Proteins/pharmacology , Complement Membrane Attack Complex , Disulfides , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Fragments/metabolism , Protein Binding , Protein Conformation , Recombinant Fusion Proteins/metabolism , Species Specificity , Structure-Activity Relationship , Transfection
2.
J Biol Chem ; 270(34): 19723-8, 1995 Aug 25.
Article in English | MEDLINE | ID: mdl-7544344

ABSTRACT

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The inhibitory function of CD59 derives from its capacity to interact with both the C8 and C9 components of MAC, preventing assembly of membrane-inserted C9 polymer. MAC-inhibitory activity of CD59 is species-selective and is most effective when both C8 and C9 derive from human or other primate plasma. Rabbit C8 and C9, which can substitute for human C8 and C9 in MAC, mediate virtually unrestricted lysis of human cells expressing CD59. In order to identify the segment of human C8 that is recognized by CD59, recombinant peptides containing human or rabbit C8 sequence were expressed in Escherichia coli and purified. CD59 was found to specifically bind to a peptide corresponding to residues 334-385 of the human C8 alpha-subunit, and to require a disulfide bond between Cys345 and Cys369. No specific binding was observed to the corresponding sequence from rabbit C8 alpha (residues 334-386). To obtain functional evidence that this segment of human C8 alpha is selectively recognized by CD59, recombinant C8 proteins were prepared by co-transfecting COS-7 cells with human/rabbit chimeras of the C8 alpha cDNA, and cDNAs encoding the C8 beta and C8 gamma chains. Hemolytic activity of MAC formed with chimeric C8 was analyzed using target cells reconstituted with CD59. These experiments confirmed that CD59 recognizes a conformationally sensitive epitope that is within a segment of human C8 alpha internal to residues 320-415. Our data also suggest that optimal interaction of CD59 with this segment of human C8 alpha is influenced by N-terminal flanking sequence in C8 alpha and by human C8 beta, but is unaffected by C8 gamma.


Subject(s)
Antigens, CD/metabolism , Complement C8/metabolism , Membrane Glycoproteins/metabolism , Amino Acid Sequence , Animals , Binding Sites , CD59 Antigens , Cell Line , Complement C8/chemistry , Complement C8/genetics , Complement Inactivator Proteins/metabolism , Epitopes , Humans , In Vitro Techniques , Molecular Sequence Data , Protein Conformation , Rabbits , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Species Specificity
3.
J Biol Chem ; 270(8): 3483-6, 1995 Feb 24.
Article in English | MEDLINE | ID: mdl-7533152

ABSTRACT

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C9 component of the C5b-9 membrane attack complex, thereby protecting human cells from lysis by human complement. The complement-inhibitory activity of CD59 is species-selective and is most effective toward C9 derived from human or other primate plasma. By contrast, rabbit C9, which can substitute for human C9 in the membrane attack complex, mediates unrestricted lysis of human cells. To identify the peptide segment of human C9 that is recognized by CD59, rabbit C9 cDNA clones were isolated, characterized, and used to construct hybrid cDNAs for expression of full-length human/rabbit C9 chimeras in COS-7 cells. All resulting chimeras were hemolytically active, when tested against chicken erythrocytes bearing C5b-8 complexes. Assays performed in the presence or absence of CD59 revealed that this inhibitor reduced the hemolytic activity of those chimeras containing human C9 sequence between residues 334-415, irrespective of whether the remainder of the protein contained human or rabbit sequence. By contrast, when this segment of C9 contained rabbit sequence, lytic activity was unaffected by CD59. These data establish that human C9 residues 334-415 contain the site recognized by CD59, and they suggest that sequence variability within this segment of C9 is responsible for the observed species-selective inhibitory activity of CD59.


Subject(s)
Antigens, CD/metabolism , Complement C9/metabolism , Membrane Glycoproteins/metabolism , Amino Acid Sequence , Animals , Binding Sites , CD59 Antigens , Cell Line , Chickens , Complement C9/genetics , Complement Inactivator Proteins/metabolism , DNA, Complementary , Humans , Molecular Sequence Data , Protein Binding , Rabbits , Recombinant Fusion Proteins
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