Structure-function relationships of the complement regulatory protein, CD59.
CD59 (membrane inhibitor of reactive lysis, protectin) is a membrane protein whose functions include the inhibition of the insertion of the ninth component of complement into the target membrane. It belongs to a superfamily of proteins including Ly-6, elapid snake venom toxins, and urokinase receptor (UPAR); the members of the superfamily have a similar structure that includes four (in mammals five) disulfide bridges that maintain a three-dimensional conformation consisting of a central core, three finger-like "loops" extending from it and a small loop near the coboxyl end. We have used site directed mutagenesis to explore three aspects of the structure of CD59: 1) the role of the disulfide bridges in expression and function of the molecule; 2) the location of epitopes reacting with monoclonal antibodies to the molecule; and 3) the parts of the molecule that are critical to its function in inhibiting complement lysis. Mutant molecules in which the disulfides maintaining the finger-like loops (Cys3-Cys26, Cys19-Cys39, and Cys45-Cys63) were removed were not expressed on the cell surface. The mutation of the disulfide (Cys6-Cys13) resulted in no change in expression or function. The mutation of Cys64-Cys69 maintaining the small loop resulted in an expressed molecule with increased functional activity. The major epitope for 6 of 7 monoclonal antibodies was centered on Arg53 as the mutation 53Arg-->Ser resulted in a loss of interaction with these antibodies, as did the deletion of four nearby residues (Leu54-Asn57). The alteration 55Arg-->Ser resulted in loss of reactivity for some but not other antibodies. The reactivity with one monoclonal antibody, H19, was abrogated by the mutations 61Tyr-->Gly and 61Tyr-->Ala. Functional activity of the molecule was not adversely altered by mutations in the first and second loops; however, the 61Tyr-->Gly mutation was non-functional. The mutation of 61Tyr-->His diminished function but changes 61Tyr-->Ala and 61Tyr-->Phe had no effect on function. We conclude that the functional site of CD59 is located in this region of the molecule.
Petranka
J
J
Department of Medicine, Duke University Medical Center, Durham, NC 2710, USA.
Zhao
J
J
Norris
J
J
Tweedy
N B
NB
Ware
R E
RE
Sims
P J
PJ
Rosse
W F
WF
D000818:Animals; D018957:CD59 Antigens / Q000737:chemistry*; D016466:CHO Cells; D015938:Complement Membrane Attack Complex / Q000037:antagonists & inhibitors; D006224:Cricetinae; D018604:Epitope Mapping; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D018957:CD59 Antigens; D015938:Complement Membrane Attack Complex