P-glycoprotein epitope mapping. I. Identification of a linear human-specific epitope in the fourth loop of the P-glycoprotein extracellular domain by MM4.17 murine monoclonal antibody to human multi-drug-resistant cells.
A new murine monoclonal antibody (MAb), MM4.17, to human multi-drug-resistant (MDR) cells was found to be reactive in an ELISA with a synthetic 16-amino acid peptide selected from the fourth loop of the P-glycoprotein extracellular domain. Immunohistochemistry indicated that this MAb reacted in human tissues in the same pattern as that previously found with other human-specific MAbs to P-glycoprotein. For a precise definition of the MM4.17 epitope, a peptide library consisting of overlapping 4- to 10-mer residues covering the entire P-glycoprotein-fragment was synthesized on polyethylene pins and tested for MAb binding. The results of this ELISA demonstrated that the MM4.17 epitope is constituted by the continuous-linear TRIDDPET amino-acid sequence (residues 750-757 of the human MDRI-P-glycoprotein). The MAb MM4.17 recognizes only the human MDRI-P-glycoprotein isoform, and excess TRIDDPET peptide blocks the binding of the MAb to MDR variants of CEM cells. These results demonstrate that the amino-acid sequence TRIDDPET from the human MDRI gene represents the first continuous-linear epitope identified in the P-glycoprotein extracellular domain.
Cianfriglia
M
M
Laboratorio di Immunologia, Istituto Superiore di Sanità, Roma, Italy.
Willingham
M C
MC
Tombesi
M
M
Scagliotti
G V
GV
Frasca
G
G
Chersi
A
A
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology*; D002460:Cell Line; D004351:Drug Resistance / Q000235:genetics / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000032:analysis*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology*; D013329:Structure-Activity Relationship
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; D010446:Peptide Fragments