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 immuno-gold labeled cryosections of human liver biopsies. This reactivity was not due to the presence of apo[a] in peroxisomes but to a cross-reactivity of mab 1A2. Immunoblot analysis of peroxisomal fractions and purified human catalase demonstrated that mab 1A2 reacts with catalase. Conversely, an anti-catalase antibody also recognized apo[a]. By sequence comparison we identified a 4-amino acid motif (Y-Y-P-N) that is shared between the highly repetitive kringle 4 motif of apo[a] and the carboxy-terminal third of the peroxisomal marker enzyme catalase. No other identical sequences were identified in these proteins. Results from the following experiments indicated that 1A2 recognizes this short linear epitope. i) Mab 1A2 reacted only with the 4 amino acid peptide 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. Our findings demonstrate that unknown linear epitopes in native proteins can be identified by sequence comparison between known proteins. The practical implication is that antibodies against apo[a] must be controlled for this cross-reactivity before using them for immunohistochemical studies of intracellular apo[a] in tissues or cells.
Dieplinger
H
H
Institute of Medical Biology and Human Genetics, University of Innsbruck, Austria.
Gruber
G
G
Krasznai
K
K
Reschauer
S
S
Seidel
C
C
Burns
G
G
Müller
H J
HJ
Császár
A
A
Vogel
W
W
Robenek
H
H
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001053:Apolipoproteins / Q000737:chemistry* / Q000276:immunology; D053241:Apoprotein(a); D002374:Catalase / Q000737:chemistry* / Q000276:immunology; D003429:Cross Reactions; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007150:Immunohistochemistry; D018082:Kringles / Q000276:immunology; D017270:Lipoprotein(a); D008099:Liver / Q000473:pathology / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016415:Sequence Alignment
D000911:Antibodies, Monoclonal; D001053:Apolipoproteins; D000939:Epitopes; D017270:Lipoprotein(a); D002374:Catalase; D053241:Apoprotein(a)