Mapping of multiple binding domains of the superantigen staphylococcal enterotoxin A for HLA.
Multiple binding sites on the staphylococcal enterotoxin A (SEA) molecule which interact with class II MHC Ag have been suggested by previous studies comparing SEA binding with that of another superantigen, toxic shock syndrome toxin-1. Using the synthetic peptide approach we have identified multiple regions of the SEA molecule which are responsible for binding to HLA Ag on Raji cells. Overlapping peptides were synthesized corresponding to the complete amino acid sequence of SEA: SEA(1-45), SEA(39-66), SEA(62-86), SEA(83-104), SEA(102-124), SEA(121-149), SEA(146-173), SEA(166-193), SEA(187-217), and SEA(211-233). Like the native SEA molecule, all of the peptides exhibited relatively high beta-sheet and low alpha-helical structure as determined by circular dichroism spectroscopy. A direct competition assay was employed with peptide blockage of 125I-SEA binding to MHC Ag. SEA(1-45), SEA(39-66), SEA(62-86), and SEA(121-149) but none of the other peptides blocked binding to Raji cells. The relative potency of the peptides in blocking SEA binding was determined with SEA(39-66) much greater than SEA(1-45) = SEA(62-86) = SEA(121-149). Peptide competition was seen at concentrations as low as 55 microM. Further, antibodies were produced to all of the peptides and tested for their ability to bind to SEA and inhibit SEA binding to HLA. Consistent with the direct inhibition of binding, antisera to SEA(1-45), SEA(39-66), and SEA(62-86) reduced the ability of SEA to bind Raji cells, whereas, antisera to the remaining peptides failed to block binding. The data suggest that the binding of the superantigen SEA to MHC molecules involves several N-terminal regions on SEA as well as an additional internal domain. This allows for the presence of multiple binding sites in an extended N-terminal region of the SEA molecule or a discontinuous binding epitope.
Griggs
N D
ND
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611.
Pontzer
C H
CH
Jarpe
M A
MA
Johnson
H M
HM
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins; D001665:Binding Sites; D001667:Binding, Competitive; D002942:Circular Dichroism; D004768:Enterotoxins / Q000378:metabolism*; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D007106:Immune Sera / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011817:Rabbits; D018089:Superantigens
D001427:Bacterial Toxins; D004768:Enterotoxins; D000949:Histocompatibility Antigens Class II; D007106:Immune Sera; D010446:Peptide Fragments; D018089:Superantigens; C030107:enterotoxin F, Staphylococcal; C026066:enterotoxin A, Staphylococcal