Calcium-induced intracellular cross-linking of lipocortin I by tissue transglutaminase in A431 cells. Augmentation by membrane phospholipids

• Published in: The Journal of biological chemistry 1991-01, Vol.266 (2), p.1101-1108
• Main Authors: Ando, Y, Imamura, S, Owada, M K, Kannagi, R
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Annexins; Biological and medical sciences; Blotting, Western; Calcimycin - pharmacology; calcium; Calcium - metabolism; Calcium-Binding Proteins - metabolism; Calpain; Cathepsin D; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Enzymes and enzyme inhibitors; Epidermal Growth Factor - pharmacology; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Humans; Membrane Lipids - metabolism; phospholipids; Phospholipids - metabolism; protein-glutamine gamma -glutamyltransferase; Transferases; Transglutaminases - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)35288-2

Covalently cross-linked multimers of lipocortin I are shown to be present in human epidermoid carcinoma A431 cells treated with epidermal growth factor or the calcium ionophore A23187. This intracellular cross-linking of lipocortin I is suggested to be mediated by the action of tissue transglutaminase, a Ca2(+)-dependent protein cross-linking enzyme. Cross-linking of lipocortin I competes with proteolytic digestion of the protein, and pretreatment of the cells with inhibitors for calpain (Ca2(+)-dependent intracellular protease) markedly enhanced the cross-linking of lipocortin I. Cross-linked lipocortin I is shown to be present in the soluble fraction of A431 cells as well as in the particulate fraction; a 34-kDa fragment of lipocortin I was solubilized successfully by plasmin digestion of the latter fraction. Immunofluorescence microscopy using specific antilipocortin-I antibody showed that cross-linked lipocortin I forms an envelope-like structure, which is not extracted with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) or Triton X-100. In vitro incubation of purified lipocortin I with tissue transglutaminase resulted in the formation of covalently cross-linked lipocortin I dimer, tetramer, and so on. Amine incorporation and cross-linking studies using lipocortin I and its N-terminal truncated derivatives indicated that the cross-linking site is localized within the plasmin-susceptible N-terminal 29 amino acids of lipocortin I. The cross-linking of lipocortin I is shown to be accelerated more than 10 times by the addition of phosphatidylserine vesicles, on which lipocortin I molecules are most likely aligned in a conformation suitable for cross-linking. Collectively, these findings suggest that an increase of intracellular calcium concentration results in the attachment of lipocortin I onto the plasma membrane phospholipids through the C-terminal domain of the molecule where the membrane-bound lipocortin I is cross-linked by the action of tissue transglutaminase through the N-terminal domain.