title: The cysteine 34 residue of A1M/α1-microglobulin is essential for protection of irradiated cell cultures and reduction of carbonyl groups.
abstract: α1-microglobulin (A1M) is a 26 kDa plasma and a tissue protein belonging to the lipocalin family. The reductase and free radical scavenger A1M has been shown to protect cells and extracellular matrix against oxidative and irradiation-induced damage. The reductase activity was previously shown to depend upon an unpaired cysteinyl side-chain, C34, and three lysyl side-chains, K92, 118, and 130, located around the open end of the lipocalin pocket. The aim of this work was to investigate whether the cell and matrix protection by A1M is a result of its reductase activity by using A1M-variants with site-directed mutations of the C34, K92, K118, and K130 positions. The results show that the C34 side-chain is an absolute requirement for protection of HepG2 cell cultures against alpha-particle irradiation-induced cell death, upregulation of stress response and cell cycle regulation genes. Mutation of C34 also resulted in loss of the reduction capacity toward heme- and hydrogen peroxide-oxidized collagen, and the radical species 2,2´-azino-bis (3-ethyl-benzo-thiazoline-6-sulphonic acid) (ABTS). Furthermore, mutation of C34 significantly suppressed the cell-uptake of A1M. The K92, K118, and K130 side-chains were of minor importance in cell protection and reduction of oxidized collagen but strongly influenced the reduction of the ABTS-radical. It is concluded that antioxidative protection of cells and collagen by A1M is totally dependent on its C34 amino acid residue. A model of the cell protection mechanism of A1M should be based on the redox activity of the free thiolyl group of the C34 side-chain and a regulatory role of the K92, K118, and K130 residues.
authors: Division of Infection Medicine, Lund University, Lund, Sweden.
authors: S
authors: S
authors: Rutardottir
authors: E J C
authors: EJ
authors: Nilsson
authors: J
authors: J
authors: Pallon
authors: M
authors: M
authors: Gram
authors: B
authors: B
authors: Åkerström
mesh_terms: D000512:Alpha Particles / Q000009:adverse effects*; D000510:Alpha-Globulins / Q000737:chemistry* / Q000378:metabolism*; D000975:Antioxidants / Q000378:metabolism*; D018929:Cell Culture Techniques; D002453:Cell Cycle / Q000235:genetics / Q000528:radiation effects; D016923:Cell Death / Q000528:radiation effects; D003545:Cysteine / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D008958:Models, Molecular; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000510:Alpha-Globulins; D000975:Antioxidants; D013438:Sulfhydryl Compounds; C016001:alpha-1-microglobulin; D003545:Cysteine