Characterization of heme binding to recombinant α1-microglobulin.

Alpha-1-microglobulin (A1M), a small lipocalin protein found in plasma and tissues, has been identified as a heme and radical scavenger that may participate in the mitigation of toxicities caused by degradation of hemoglobin. The objective of this work was to investigate heme interactions with A1M in vitro using various analytical techniques and to optimize analytical methodology suitable for rapid evaluation of the ligand binding properties of recombinant A1M versions

The cysteine 34 residue of A1M/α1-microglobulin is essential for protection of irradiated cell cultures and reduction of carbonyl groups.

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

A1M: a heme and radical binding protein. A study on structure, function and mechanisms

This thesis describes functional and structural studies of the human protein α1-microglobulin (A1M), an evolutionarily well-conserved 26 kDa plasma and tissue protein mainly synthesized in the liver. A1M belongs to the lipocalin protein family. Lipocalins share a very similar structur, an 8-stranded β-barrel forming a hydrophobic pocket. A1M has reductase and radical scavenging properties. A1M also binds the iron-containing heme-group, a lipophilic small compound found in many proteins and enzymes, but toxic in free form. A truncated form of A1M (t-A1M) catalyzes degradation of the heme-group. These properties give A1M antioxidative properties in human tissues. The research focus in this thesis has been to study the protection mechanisms of A1M with a special focus on the structure, and to investigate its protection properties in vivo. The results show that radiation-induced cell damage induces reactive oxygen species (ROS) and that ROS mediates a so-called bystander effect (a spreading of damage to cells not directly hit by the radiation). A1M inhibited the bystander effect and by using site-directed mutagenesis it was shown that this protection is dependent on a cysteine side-chain in position 34, and regulated by three lysine residues in position 92, 118 and 130. Furthermore, when A1M binds heme, the cysteine in position 34, a histidine in position 123, and the three lysine-residues in position 92, 118 and 130 are involved in co-ordination of the iron-atom in heme. The results support a model in which two heme-groups are bound simultaneously. Molecular simulation suggests binding of the first heme group inside the lipocalin pocket, followed by a structural shift that allows binding of a second heme group, with lower affinity, near the opening of pocket. Finally, the in vivo therapeutic effects of A1M were investigated using a sheep model of the pregnancy disease preeclampsia. Starvation induced preeclampsia-like symptoms in the pregnant ewes manifesting in hemolysis and subsequent damage to the placenta and kidneys. A1M was well tolerated and displayed positive therapeutic effects

Bystander cell death and stress response is inhibited by the radical scavenger α(1)-microglobulin in irradiated cell cultures.

Alpha-particle irradiation of cells damages not only the irradiated cells but also nontargeted bystander cells. It has been proposed that the bystander effect is caused by oxidants and free radicals generated by the radiation. Recent studies have shown that α(1)-microglobulin protects against cell damage caused by oxidants and free radicals. Using a novel experimental system that allows irradiation of 0.02% of a human hepatoma monolayer, leaving 99.98% as bystander cells, we investigated the influence of oxidative stress and the cell-protective effects of α(1)-microglobulin during α-particle irradiation. The results showed an increase in cell death in both irradiated cells and bystander cells. A significant increase in apoptosis, oxidation markers and expression of the stress response genes heme oxygenase 1, superoxide dismutase, catalase, glutathione peroxidase 1, p21 and p53 were observed. Addition of α(1)-microglobulin reduced the amount of dead cells and inhibited apoptosis, formation of oxidation markers, and up-regulation of stress response genes. The results emphasize the role of oxidative stress in promoting bystander effects. Furthermore, the results suggest that α(1)-microglobulin protects nonirradiated cells by eliminating oxidants and free radicals generated by radiation and imply that α(1)-microglobulin can be used in radiation therapy of tumors to minimize damage to surrounding tissues

M1 protein from Streptococcus pyogenes induces nitric oxidemediated vascular hyporesponsiveness to phenylephrine: Involvement of toll-like receptor activation.

Streptococcus pyogenes carrying M1 protein causes the severe and increasingly prevalent streptococcal toxic shock syndrome and necrotizing fasciitis. M1 protein is an important virulence factor of Streptococcus pyogenes and induces an inflammatory response in human monocytes. We wanted to investigate if purified M1 protein in solution could induce vascular nitric oxide (NO) production leading to vasopressor hyporesponsiveness. Rat aorta segments were incubated with M1 protein or lipopolysaccharide (LPS) in vitro. M1 protein (10 mug ml) and LPS (1 ng ml) to a similar extent induced NO production and hyporesponsiveness to the vasoconstrictor phenylephrine. Immuno-gold electron microscopy demonstrated that M1 protein binds to toll-like receptor (TLR) 2 as well as TLR4 in mouse aorta but only to TLR2 in human omental artery. Incubation with M1 protein caused a reduction in the contractile response to phenylephrine in aorta segments from wild type and TLR2 knockout but not from TLR4 knockout mice. In conclusion, M1 protein causes vascular NO production leading to hyporesponsiveness to vasopressors via a mechanism involving TLR but the subtypes may be species-dependent. M1 protein could contribute to the circulatory disturbances accompanying severe invasive streptococcal infections

Fetal hemoglobin and alpha(1)-microglobulin as first- and early second-trimester predictive biomarkers for preeclampsia

OBJECTIVE: The aim of this study was to evaluate fetal hemoglobin (HbF) and alpha(1)-microglobulin (A1M) in maternal serum as first-trimester biomarkers for preeclampsia (PE). STUDY DESIGN: The design was a case-control study. We included 96 patients in the first trimester of pregnancy (60 with PE and 36 controls). Venous serum samples were analyzed for HbF and total hemoglobin (Hb) by enzyme-linked immunosorbent assay and for A1M by radioimmunoassay. Sensitivity and specificity was calculated by logistic regression and receiver operating characteristic curve analysis. RESULTS: The HbF/Hb ratio and A1M concentration were significantly elevated in serum from women with subsequent development of PE (P < .0001). The optimal sensitivity and specificity was obtained using the biomarkers in combination; 69% sensitivity for a 5% screen positive rate and 90% sensitivity for a 23% screen positive rate. CONCLUSION: The study suggests that HbF/Hb ratio in combination with A1M is predictive biomarkers for PE

Structural and biochemical characterization of two heme binding sites on α1-microglobulin using site directed mutagenesis and molecular simulation.

α1-Microglobulin (A1M) is a reductase and radical scavenger involved in physiological protection against oxidative damage. These functions were previously shown to be dependent upon cysteinyl-, C34, and lysyl side-chains, K(92, 118,130). A1M binds heme and the crystal structure suggests that C34 and H123 participate in a heme binding site. We have investigated the involvement of these five residues in the interactions with heme

rA1M-035, a Physicochemically Improved Human Recombinant α-Microglobulin, Has Therapeutic Effects in Rhabdomyolysis-Induced Acute Kidney Injury

AIMS: Human α1-microglobulin (A1M) is an endogenous reductase and radical- and heme-binding protein with physiological antioxidant protective functions. Recombinant human A1M (rA1M) has been shown to have therapeutic properties in animal models of preeclampsia, a pregnancy disease associated with oxidative stress. Recombinant A1M, however, lacks glycosylation, and shows lower solubility and stability than A1M purified from human plasma. The aims of this work were to (i) use site-directed mutagenesis to improve the physicochemical properties of rA1M, (ii) demonstrate that the physicochemically improved rA1M displays full in vitro cell protective effects as recombinant wild-type A1M (rA1M-wt), and (iii) show its therapeutic potential in vivo against acute kidney injury (AKI), another disease associated with oxidative stress.RESULTS: A novel recombinant A1M-variant (rA1M-035) with three amino acid substitutions was constructed, successfully expressed, and purified. rA1M-035 had improved solubility and stability compared with rA1M-wt, and showed intact in vitro heme-binding, reductase, antioxidation, and cell protective activities. Both rA1M-035 and rA1M-wt showed, for the first time, potential in vivo protective effects on kidneys using a mouse rhabdomyolysis glycerol injection model of AKI.INNOVATION: A novel recombinant A1M-variant, rA1M-035, was engineered. This protein showed improved solubility and stability compared with rA1M-wt, full in vitro functional activity, and potential protection against AKI in an in vivo rhabdomyolysis mouse model.CONCLUSION: The new rA1M-035 is a better drug candidate than rA1M-wt for treatment of AKI and preeclampsia in human patients. Antioxid. Redox Signal. 00, 000-000

Increased levels of cell-free hemoglobin, oxidation markers, and the antioxidative heme scavenger alpha(1)-microglobulin in preeclampsia.

Preeclampsia is a major cause of morbidity and mortality during pregnancy. To date, the pathogenesis of the disease is not fully understood. Recent studies show that preeclampsia is associated with overexpression of the hemoglobin genes alpha2 and gamma and accumulation of the protein in the vascular lumen of the placenta. Hypothesizing that cell-free hemoglobin leaks from the placenta into the maternal circulation and contributes to the endothelial damage and symptoms by inducing oxidative stress, we analyzed fetal and adult hemoglobin (HbF, HbA), haptoglobin, oxidation markers, and the heme scavenger and antioxidant alpha(1)-microglobulin in plasma, urine, and placenta in preeclamptic women (n=28) and women with normal pregnancy (n=27). The mean plasma concentrations of HbF, HbA, protein carbonyl groups, membrane peroxidation capacity, and alpha(1)-microglobulin were significantly increased in preeclamptic women. The levels of total plasma Hb correlated strongly with the systolic blood pressure. The plasma haptoglobin concentrations of women with preeclampsia were significantly depressed. Increased amounts of alpha(1)-microglobulin mRNA and protein were found in placenta from preeclamptic women, and the levels of plasma and placenta alpha(1)-microglobulin correlated with the plasma Hb concentrations. The heme-degrading form t-alpha(1)-microglobulin was significantly increased in urine in preeclampsia. These results support the idea that hemoglobin-induced oxidative stress is a pathogenic factor in preeclampsia

Perfusion of human placenta with hemoglobin introduces preeclampsia-like injuries that are prevented by α(1)-microglobulin.

Preeclamptic women have increased plasma levels of free fetal hemoglobin (HbF), increased gene expression of placental HbF and accumulation of free HbF in the placental vascular lumen. Free hemoglobin (Hb) is pro-inflammatory, and causes oxidative stress and tissue damage