Definition of a discontinuous immunodominant epitope of intestinal alkaline phosphatase, an autoantigen in acute bacterial infections.
In patients suffering from acute bacterial infections specific autoantibodies of the immunoglobulin class G (IgG), directed against intestinal alkaline phosphatase (IAP), are transiently expressed in high titer. The epitopes on IAP which are recognized by these IAP auto-antibodies were investigated using chemical and enzymatic cleavage, followed by two-dimensional electrophoresis and immunoblotting of the fragments. Immunoreactive and nonreactive cleavage fragments were isolated and N-terminally sequenced. An epitope map was constructed by means of sequencing data, relative molecular mass of the fragments, and specific cleavage sites. To evaluate linear epitopes, the overlapping region of the immunoreactive fragments (amino acids 204-484 of the primary structure of IAP) was further analyzed by simultaneous synthesis of 95 peptides on an activated membrane. Four immunoreactive regions were revealed by immunodetection with IAP autoantibody-positive sera. Nine peptides comprising these regions were synthesized quantitatively and coupled to CH-Sepharose. However, specific IAP autoantibodies could not be isolated. This result indicated the absence of continuous epitopes at least in the analyzed region of the molecule. Binding studies with an IAP cleavage fragment suggested a discontinuous epitope involving amino acids 334-371. The results are indicative of an antigen-driven mechanism for the production of IAP autoantibodies initiated and maintained by self.
Kolbus
N
N
Neurological Department of the University, Göttingen, Germany.
Beuche
W
W
Felgenhauer
K
K
Mäder
M
M
D000208:Acute Disease; D000328:Adult; D000368:Aged; D000469:Alkaline Phosphatase / Q000276:immunology* / Q000378:metabolism; D000595:Amino Acid Sequence; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000276:immunology* / Q000378:metabolism; D001324:Autoantigens / Q000096:biosynthesis / Q000276:immunology* / Q000378:metabolism; D001424:Bacterial Infections / Q000201:enzymology / Q000276:immunology*; D001666:Binding Sites, Antibody; D003488:Cyanogen Bromide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D005260:Female; D006801:Humans; D016056:Immunodominant Epitopes / Q000032:analysis*; D007422:Intestines / Q000201:enzymology; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism
D000942:Antigens, Bacterial; D001324:Autoantigens; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D000469:Alkaline Phosphatase; D003488:Cyanogen Bromide