Calcyclin-binding site located on the NH2-terminal domain of rabbit CAP-50 (annexin XI): functional expression of CAP-50 in Escherichia coli.
CAP-50 (annexin XI) is a member of annexin family proteins originally identified and characterized as a target protein for calcyclin (H. Tokumitsu et al. (1992) J. Biol. Chem. 267, 8919-8924). In the present work, the calcyclin-binding site of CAP-50 was determined by proteolytic study and by using various deletion mutants expressed in Escherichia coli. The 43-kDa fragment of CAP-50 digested with Staphylococcus aureus V8 protease did not bind to calcyclin in the presence of Ca2+. CAP-50 fusion proteins, including various NH2-terminal deletion mutants were expressed in E. coli using rabbit CAP-50 cDNA and the calcyclin-binding potential was examined using the 125I-calcyclin gel overlay method and coprecipitation with phosphatidylserine-containing vesicles in the presence of Ca2+. All recombinant protein carried the potential for Ca(2+)-dependent phospholipid binding, due to the presence of the COOH-terminal domain (core domain). Calcyclin-binding experiments showed that CAP-50 molecules lacking the NH2-terminal 26 residues retain binding potential for calcyclin; however, deletion of an additional 26 amino acids from the NH2 terminus abolishes specific calcyclin binding. These observations suggest that the calcyclin-binding site is located on the NH2-terminal region of CAP-50, probably adjacent to or within the region from Tyr27 to Leu52.
Department of Pharmacology, Nagoya University School of Medicine, Japan.
H
H
Tokumitsu
A
A
Mizutani
H
H
Hidaka
D000818:Animals; D017302:Annexins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D017353:Gene Deletion; D015870:Gene Expression; D015151:Immunoblotting; D008168:Lung / Q000737:chemistry; D016296:Mutagenesis; D010446:Peptide Fragments / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D009418:S100 Proteins; D012697:Serine Endopeptidases / Q000378:metabolism
D017302:Annexins; D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D009418:S100 Proteins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase