Inhibition of human SERCA3 by PL/IM430. Molecular analysis of the interaction.
The monoclonal antibody PL/IM430 has previously been reported to uncouple Ca(2+) transport from ATP hydrolysis in platelet membranes (Hack, N., Wilkinson, J. M., and Crawford, N. (1988) Biochem. J. 250, 355-361). More recently, we have demonstrated that this antibody is specific for human SERCA3 (Poch, E., Leach, S., Snape, S., Cacic, T., MacLennan, D. H., and Lytton, J. (1998) Am. J. Physiol. 275, C1449-C1458). In this paper, we have extended the analysis of the PL/IM430-SERCA3 interaction. Using HEK293 cells to express human SERCA3a, we were able to measure both ATP-mediated, oxalate-dependent (45)Ca(2+) uptake and Ca(2+)-dependent ATP hydrolysis activities due exclusively to SERCA3. Treatment with PL/IM430 inhibited both activities almost identically, with a maximal inhibition of 81 and 73% and a half-maximal concentration of 8.3 and 5.9 microg/ml, for Ca(2+) uptake and ATP hydrolysis, respectively. We conclude that PL/IM430 does inhibit SERCA3 activity but does not uncouple Ca(2+) transport from ATP hydrolysis. Using a combination of partial proteolysis, GST fusion protein expression, and mutation of residues that differ between rat and human SERCA3, we have identified human SERCA3 amino acids Pro(8) and Glu(192) as essential to forming the PL/IM430 epitope. PL/IM430 thus recognizes a linearly noncontiguous set of amino acids within the actuator domain of human SERCA3. We propose that PL/IM430 inhibits SERCA3 activity by sterically preventing movement of the actuator domain into a catalytically critical position in the E2 conformation of the enzyme.
Chandrasekera
Charukeshi P
CP
Cardiovascular Research Group, Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Lytton
Jonathan
J
D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000166:cytology; D008861:Microsomes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D014162:Transfection
D000911:Antibodies, Monoclonal; C506951:Atp2a3 protein, rat; D000939:Epitopes; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506949:ATP2A3 protein, human; D000252:Calcium-Transporting ATPases; D002118:Calcium