Prediction of epitopes and production of monoclonal antibodies against gastric H,K-ATPase.
Monoclonal antibodies (mAbs) were produced against gastric H,K-ATPase using a theoretical and experimental strategy based on prediction of linear epitopes by molecular modelling followed by production of anti-peptide antibodies. By analysing the alpha subunit sequence, we predicted several epitopes corresponding to amino acids K519-L533, E543-Y553 and S786-L798 and produced monoclonal antibodies HK519, HK543 and HK786. All three react against gastric H,K-ATPase in RaLISA, immunohistochemistry and Western blots demonstrating that they recognize the native and the SDS-denatured ionic pump and that the epitopes are located at the surface of the native ATPase. Antibody Kd are in the range 6-10x10(-8) M. Monoclonal antibody HK519 is a competitive inhibitor of ATP, in agreement with ATP binding to K519. Neither mAb 543, nor mAb 786 inhibit the ATPase activity. Monoclonal antibody 95111, whose epitope is mapped between residues C529 and E561, competes with mAb HK543 but not with the other two. We suggest that the 95111 epitope is overlapping or very close to the HK543-553 sequence. Induction of E1 conformer by binding FITC to K519 increases the number of mAb 95111 and mAb HK543 epitopes but not that of mAb 786, supporting the fact that the fragment E543-Y553 changes accessibility, maybe during the E1-E2 transconformation.
Irnaten
M
M
INSERM U10, Hôpital C1 Bernard Bichat, Paris, France.
Gallet
X
X
Festy
F
F
Péranzi
G
G
Robert
J C
JC
Thomas-Soumarmon
A
A
Brasseur
R
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D001667:Binding, Competitive; D015153:Blotting, Western; D003429:Cross Reactions; D000939:Epitopes / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D007163:Immunosorbent Techniques; D008861:Microsomes / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D054328:Proton Pump Inhibitors; D011817:Rabbits; D013552:Swine; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010455:Peptides; D054328:Proton Pump Inhibitors; D000255:Adenosine Triphosphate; D017506:H(+)-K(+)-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate