Structural characterization of a biologically active human lipocortin 1 expressed in Escherichia coli

Abstract

Lipocortin or annexin 1 is a calcium-dependent phospholipid-binding protein which probably acts as a glucocorticoid-regulated anti-inflammatory factor. cDNA for human lipocortin 1 was cloned in the pT7.7 expression plasmid under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl thio-p-D-galactoside, large amounts of the protein were produced and accumulated in Escherichia coli in a soluble form. The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps. The final yield was about 30 mg/l bacterial culture. Electrospray mass spectrometric analysis of the purified protein demonstrated that the recombinant product corresponds to the native human lipocortin 1, without the initial methionine and with a free N-terminal alanine; tryptic peptide mapping by fast-atom-bombardment mass spectrometry showed that the recombinant protein contains cysteine residues at positions 263 and 324 with free thiol groups, whereas Cys270 and Cys343 are probably involved in an intrachain disulfide bridge. Recombinant human lipocortin 1 reduces the carrageenininduced paw oedema in rat in vivo and inhibits porcine pancreatic phospholipase Az activity in vitro; in both cases, a dose-related response is observed.The lipocortin (LC) or annexin family includes structurally and immunologically related calcium and phospholipid-binding proteins, initially described for their anti-inflammatory properties [l, 21. These proteins are widely distributed in nature between and within species and are involved in several different calcium-mediated physiological and biochemical processes [3]. Up to now, 11 different annexin members have been identified [4, 51; LC-1 was initially purified from rat [6] and the corresponding human cDNA was cloned [7].Human LC-1 is a 38-kDa protein with a 33-kDa core, responsible of Ca2 + and phospholipid binding, linked to an Nterminal region consisting of 43 amino acids [2] which includes tyrosine and serine residues which are substrates for epidermal growth factor, tyrosine kinase receptor [8] and protein kinase C [9]. This suggests the involvement of LC-1 also as a mediator for intracellular signal transduction. Interestingly, phosphorylation occurring in the LC-1 N-terminal region have been found to alter Ca2+-dependent phospholipid-binding affinity of the core region [lo]. Other biological functions such as inhibition of synthesis and release of prolactin from human decidual cells [l 11, macrophage-mediated suppression in Correspondence to R. Arcone, CEINGE, Dipartimento di Biochimica e Biotecnologie Mediche, Universita di Napoli Federico 11, Via S. Pansini 5,I-80131 Napoli, ItalyAbbreviations. ES/MS, electrospray mass spectrometry ; FAB-MS, fast-atom-bombardment mass spectrometry; IPTG, isopropyl thio-jl-D-galactoside; LC-1, lipocortin 1 ; rhLC-1, recombinant human lipocortin 1 ; PLA2, phospholipase A,; NbsZ, 5,5'-dithiobis(2-nitrobenzoic acid).tumor-bearing mice have been described [12]. Besides, a new cerebral anti...

ANXA1 as a Prognostic and Immune Microenvironmental Marker for Gliomas Based on Transcriptomic Analysis and Experimental Validation

“…ANXA1 is a membrane-localized protein that binds phospholipids and has an anti-inflammatory activity. It plays important roles in the innate immune response as an effector of glucocorticoid-mediated responses and a regulator of the inflammatory processes ( Arcone et al, 1993 ; Leoni et al, 2015 ). It contributes to the adaptive immune response by enhancing the signaling cascades that are triggered by T-cell activation, regulates differentiation and proliferation of activated T-cells, promotes the differentiation of T-cells into Th1 cells, and negatively regulates their differentiation into Th2 cells ( D’Acquisto et al, 2007 ).…”

Annexin‐A1: a pivotal regulator of the innate and adaptive immune systems

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Targeting ANXA1 abrogates Treg-mediated immune suppression in triple-negative breast cancer

“…12 It plays important roles in the innate immune response as effector of glucocorticoid-mediated responses and regulator of the inflammatory process, and has anti-inflammatory activity. 13 In resting conditions, cells contain high levels of ANXA1 in cytoplasm; after being activated, ANXA1 is mobilized to cell surface and secreted. 14 ANXA1 signals through a seven-membranespanning G-protein-coupled receptor, known as formyl peptide receptor 2 (FPR2; also known as ALXR in humans).…”