Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ.
Short amino acid sequences that interact with the Ca2+ binding protein S-100b were identified by screening a bacteriophage random peptide display library. S-100b binding bacteriophages were selected by Ca(2+)-dependent affinity chromatography, and the sequence of the random peptide insert contained in 51 clones was determined. Alignment of the sequence of 44 unique S-100b binding peptides identified a common motif of eight amino acids. A subgroup of peptides that contained sequences with the highest degree of similarity had the consensus motif (K/R)(L/I)XWXXIL, in which predominantly P, S, and N were found in position 3, and S and D were found in position 5. Analysis of sequence databanks identified a similar sequence in the COOH-terminal region of the alpha-subunit of actin capping proteins. The peptide TRTKIDWNKILS (TRTK-12), corresponding to the region of greatest homology within this region of the subunit of actin capping proteins (e.g. amino acids 265-276 in CapZ alpha 1 and CapZ alpha 2), was synthesized and shown by fluorescence spectrophotometry to bind S-100b in a Ca(2+)-dependent manner. Gel overlay and cross-linking experiments demonstrated the interaction of S-100b with CapZ to be Ca2+ dependent. Moreover, this interaction was blocked by addition of TRTK-12 peptide. These results identify Ca(2+)-dependent S-100b target sequence epitopes and designate the carboxyl terminus of the alpha-subunit of actin capping proteins, like CapZ, to be a target of S-100b activity. The high level of conservation within this region of actin capping proteins and the apparent high affinity of this interaction strongly suggest that the interaction between S-100b and the alpha-subunit of actin capping proteins is biologically significant.
Ivanenkov
V V
VV
Department of Emergency Medicine, University of Cincinnati College of Medicine, Ohio 45267, USA.
Jamieson
G A
GA
Gruenstein
E
E
Dimlich
R V
RV
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051347:CapZ Actin Capping Protein; D002417:Cattle; D003432:Cross-Linking Reagents; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism*; D005454:Fluorescence Polarization; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism*; D009414:Nerve Growth Factors; D009457:Neuroglia / Q000378:metabolism; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D051381:Rats; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D002135:Calcium-Binding Proteins; D051347:CapZ Actin Capping Protein; D003432:Cross-Linking Reagents; D000939:Epitopes; D008840:Microfilament Proteins; D009124:Muscle Proteins; D009414:Nerve Growth Factors; D009842:Oligopeptides; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins