Topological assignment of the N-terminal extension of plasma gelsolin to the gelsolin surface.
The actin-binding protein gelsolin is highly conserved in vertebrates and exists in two isoforms, a cytoplasmic and an extracellular variant, generated by alternative splicing. In mammals, these isoforms differ only by an N-terminal extension in plasma gelsolin, a short sequence of up to 25 amino acids. Cells and tissues may contain both variants, as plasma gelsolin is secreted by many cell types. The tertiary structure of equine plasma gelsolin has been elucidated, but without any information on the N-terminal extension. In this paper, we present topographical data on the N-terminal extension, derived using a biochemical and immunological approach. For this purpose, a monoclonal antibody was generated that exclusively recognizes cytoplasmic gelsolin but not the extracellular variant and thus allows isoform-specific immunodetection and quantification of cytoplasmic gelsolin in the presence of plasma gelsolin. Using limited proteolysis and pepscan analysis, we mapped the binding epitope and localized it within two regions in segment 1 of the cytoplasmic gelsolin sequence: Tyr34-Ile45 and Leu64-Ile78. In the tertiary structure of the cytoplasmic variant, these sequences are mutually adjacent and located in the proximity of the N-terminus. We therefore conclude that the binding site of the antibody is covered by the N-terminal extension in plasma gelsolin and thus sterically hinders antibody binding. Our results allow for a topological model of the N-terminal extension on the surface of the gelsolin molecule, which was unknown previously.
Fock
Ulrike
U
Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38106 Braunschweig, Germany.
Jockusch
Brigitte M
BM
Schubert
Wolf-Dieter
WD
Hinssen
Horst
H
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001665:Binding Sites; D003593:Cytoplasm / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D018260:Gelsolin / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012680:Sensitivity and Specificity; D013552:Swine
D000911:Antibodies, Monoclonal; D000939:Epitopes; D018260:Gelsolin