Mapping of an epitope of human leukocyte alpha interferon A which is recognized by the murine monoclonal antibody NK2.
An epitope of human leukocyte alpha interferon A (IFN-A), which is recognized by the murine monoclonal antibody NK2, has been mapped by using four successive approaches. Limited proteolysis of the IFN-A chain, followed by electrophoresis, Western blotting, and probing of the proteolytic fragments with NK2 showed that an epitope was located within the sequence residues 110-140. A panel of human IFN subtypes bearing substitutions within the sequence 110-140 was tested for reactivity with NK2 in enzyme-linked immunosorbent assays. The results from these assays suggested that the epitope is within the sequence 112-121. Analysis of a hybrid protein IFN-A(1-92)/F(93-166) revealed that the N-terminal region of IFN-A played no significant role in NK2 binding. Three residues of IFN-F (Asn113, Val114, and Lys121) were substituted for the corresponding residues from IFN-A (Lys113, Glu114, and Arg121) by site-directed mutagenesis of the gene encoding IFN-F. NK2 was able to bind the mutated protein, IFN-F(A 113, 114, 121), as well as unmodified IFN-A. The data show that the epitope recognized by NK2 is located within the C-terminal region of IFN-A (residues 112-121). This epitope consists of the essential residues 114 and 116, and residues 112, 113, 115, 117, and 121 presumably contribute the configuration of the epitope.
Alexenko
A P
AP
All-Union Research Institute of Genetics and Selection of Industrial Microorganisms, Ministry of Medical Industry of the USSR, Moscow.
Izotova
L S
LS
Kostrov
S V
SV
Strongin AYa
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D010450:Endopeptidases; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D006868:Hydrolysis; D016898:Interferon-alpha / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010449:Peptide Mapping; D011817:Rabbits
D000911:Antibodies, Monoclonal; D000939:Epitopes; D016898:Interferon-alpha; D010446:Peptide Fragments; D010450:Endopeptidases