KRINGLE-4 OF HUMAN APOLIPOPROTEIN[A] SHARES A LINEAR ANTIGENIC SITE WITH HUMAN CATALASE

Monoclonal antibody (mab) 1A2, directed against human apolipoprotein[a ] (apo[a]), revealed a strong reaction with peroxisomes as shown by im mune-gold labeled cryosections of human liver biopsies. This reactivit y was not due to the presence of apo[a] in peroxisomes but to a cross- reactivity of mab 1A2. Immunoblot analysis of peroxisomal fractions an d purified human catalase demonstrated that mab 1A2 reacts with catala se. Conversely, an anti-catalase antibody also recognized apo[a]. By s equence comparison we identified a 4-amino acid motif (Y-Y-P-N) that i s shared between the highly repetitive kringle 4 motif of apo[a] and t he carboxy-terminal third of the peroxisomal marker enzyme catalase. N o other identical sequences were identified in these proteins. Results from the following experiments indicated that 1A2 recognizes this sho rt linear epitope. i) Mab 1A2 reacted only with the 4 amino acid pepti de sequence in a pin-ELISA using immobilized overlapping peptides. ii) A synthetic peptide including this sequence completely inhibited the 1A2 immunoreactivity to apo[a] and catalase. iii) A recombinant fusion protein tagged with the putative epitope was recognized by mab 1A2. O ur findings demonstrate that unknown linear epitopes in native protein s can be identified by sequence comparison between known proteins. The practical implication is that antibodies against apo[a] must be contr olled for this cross-reactivity before using them for immunohistochemi cal studies of intracellular apo[a] in tissues or cells.