title: Calcium-induced intracellular cross-linking of lipocortin I by tissue transglutaminase in A431 cells. Augmentation by membrane phospholipids.
abstract: 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.
authors.affiliation: Department of Dermatology, Faculty of Medicine, Kyoto University, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Ando
authors.forename: S
authors.initials: S
authors.lastname: Imamura
authors.forename: M K
authors.initials: MK
authors.lastname: Owada
authors.forename: R
authors.initials: R
authors.lastname: Kannagi
mesh_terms: D017302:Annexins; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002154:Calpain; D002402:Cathepsin D; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006801:Humans; D008563:Membrane Lipids / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D017302:Annexins; D002135:Calcium-Binding Proteins; D003432:Cross-Linking Reagents; D008563:Membrane Lipids; D010743:Phospholipids; D000001:Calcimycin; D004815:Epidermal Growth Factor; D011503:Transglutaminases; D002154:Calpain; D002402:Cathepsin D; D002118:Calcium