Binding site of annexin XI on the calcyclin molecule.
We purified rabbit calcyclin of S100 family protein and a calcyclin associated protein which has proved to be a novel annexin, annexin XI. Using a co-precipitation assay of annexin XI with phospholipid, the binding site of annexin XI on calcyclin was examined. The peptide fragment of calcyclin, CNBr-3 (residues 1-57), digested with cyanogen bromide completely inhibited the interaction of native calcyclin with annexin XI, while CNBr-1 (residues 83-90) and CNBr-2 (residues 58-82) did not affect the binding. We then constructed and expressed recombinant cDNAs for wild type and four different deletion mutants lacking N-terminal portions. The wild type (wt) and mt1 mutant lacking three amino acids from N-terminal bound to annexin XI with phosphatidylserine and Ca2+, whereas mt2, mt3 and mt4 with seven, twelve and eighteen amino acids deleted, respectively, did not bind to annexin XI. Moreover, the truncated mutant from residues 4 to 7 (mt5) decreased the binding capacity. These observations suggest that four amino acids (residues 4-7) at the N-terminal portion of calcyclin play an important role in the interaction of calcyclin with annexin XI.
Watanabe
M
M
Department of Pharmacology, Nagoya University School of Medicine, Japan.
Ando
Y
Y
Tokumitsu
H
H
Hidaka
H
H
D000595:Amino Acid Sequence; D000818:Animals; D017302:Annexins / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000378:metabolism; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000096:biosynthesis; D008168:Lung / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D009418:S100 Proteins
D017302:Annexins; D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D009418:S100 Proteins; D004815:Epidermal Growth Factor; D005982:Glutathione Transferase