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

J Biol Chem. 1991 Jan 15;266(2):1101-8.

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.

MeSH terms

  • Annexins
  • Blotting, Western
  • Calcimycin / pharmacology
  • Calcium / metabolism*
  • Calcium-Binding Proteins / metabolism*
  • Calpain
  • Cathepsin D
  • Cross-Linking Reagents*
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme-Linked Immunosorbent Assay
  • Epidermal Growth Factor / pharmacology
  • Fluorescent Antibody Technique
  • Humans
  • Membrane Lipids / metabolism*
  • Phospholipids / metabolism*
  • Transglutaminases / metabolism*

Substances

  • Annexins
  • Calcium-Binding Proteins
  • Cross-Linking Reagents
  • Membrane Lipids
  • Phospholipids
  • Calcimycin
  • Epidermal Growth Factor
  • Transglutaminases
  • Calpain
  • Cathepsin D
  • Calcium