Analysis of amino acid sequence variations and immunoglobulin E-binding epitopes of German cockroach tropomyosin.
The allergenicities of tropomyosins from different organisms have been reported to vary. The cDNA encoding German cockroach tropomyosin (Bla g 7) was isolated, expressed, and characterized previously. In the present study, the amino acid sequence variations in German cockroach tropomyosin were analyzed in order to investigate its influence on allergenicity. We also undertook the identification of immunodominant peptides containing immunoglobulin E (IgE) epitopes which may facilitate the development of diagnostic and immunotherapeutic strategies based on the recombinant proteins. Two-dimensional gel electrophoresis and immunoblot analysis with mouse anti-recombinant German cockroach tropomyosin serum was performed to investigate the isoforms at the protein level. Reverse transcriptase PCR (RT-PCR) was applied to examine the sequence diversity. Eleven different variants of the deduced amino acid sequences were identified by RT-PCR. German cockroach tropomyosin has only minor sequence variations that did not seem to affect its allergenicity significantly. These results support the molecular basis underlying the cross-reactivities of arthropod tropomyosins. Recombinant fragments were also generated by PCR, and IgE-binding epitopes were assessed by enzyme-linked immunosorbent assay. Sera from seven patients revealed heterogeneous IgE-binding responses. This study demonstrates multiple IgE-binding epitope regions in a single molecule, suggesting that full-length tropomyosin should be used for the development of diagnostic and therapeutic reagents.
Jeong
Kyoung Yong
KY
Department of Parasitology and Institute of Tropical Medicine, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.
Lee
Jongweon
J
Lee
In-Yong
IY
Ree
Han-Il
HI
Hong
Chein-Soo
CS
Yong
Tai-Soon
TS
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D000937:Antigen-Antibody Reactions; D003058:Cockroaches / Q000276:immunology*; D003429:Cross Reactions / Q000235:genetics / Q000276:immunology; D018076:DNA, Complementary; D016056:Immunodominant Epitopes / Q000235:genetics*; D007073:Immunoglobulin E / Q000276:immunology*; D020033:Protein Isoforms; D020539:Sequence Analysis, Protein; D014335:Tropomyosin / Q000235:genetics / Q000276:immunology*
D018076:DNA, Complementary; D016056:Immunodominant Epitopes; D020033:Protein Isoforms; D014335:Tropomyosin; D007073:Immunoglobulin E