Alpha-1-microglobulin, heme-binding protein, reductase and antioxidant

Abstract: This thesis describes structural and functional studies of alpha-1-microglobulin (a1m), a 26 kDa plasma and tissue protein that is evolutionarily well-conserved and belongs to the lipocalin superfamily. The lipocalins are polypeptides of 160-190-amino acids that are folded into an 8-stranded beta-barrel forming a pocket with a hydrophobic interior. a1m carries heterogeneous yellow-brown chromophores covalently bound to one unpaired cysteinyl (C34) and three lysyl residues (K92, 118, 130) located at the entrance to the pocket. a1m is found both in free form and as a complex with IgA in blood and interstitial tissues. We show here that a1m is involved in heme metabolism. The protein binds to heme and, when exposed to hemoglobin or erythrocyte membranes, a processed form of a1m, t-a1m (t=truncated), with a free thiol group in position 34 and lacking the C-terminal tetrapeptide, is released from free a1m and the IgA-complex. The processed t-a1m has an intense yellow-brown colour and has properties suggestive of heme-degradation enzyme activity. The processed t-a1m-form is found in urine as well as in chronic ulcer fluids, where it is co-localized with heme. Chronic ulcers are characterized by long-standing inflammation and hemolysis with a continuous release of heme and iron, which are considered to be pathogenetic factors. These findings suggest that a1m is involved in the defence against heme-mediated oxidation. We also found that a1m has enzymatic reductase and dehydrogenase properties. Thus, a1m reduces cytochrome c, methemoglobin, and free iron to their non-oxidized forms, using the biological electron donors NADH and ascorbic acid as electron-supplying co-factors. Recombinant a1m-variants lacking C34 and/or the chromophore-carrying K92, 118 and 130 have less reducing activity, suggesting that the thiol group in position 34 as well as the nearby located lysyl groups are involved in the reaction. The protein has antioxidation properties, i.e. it inhibits the oxidation of biological targets as collagen and LDL, by the pro-oxidants heme, oxidized hemoglobin, hydrogen peroxide and oxidized iron. a1m can even remove some of the oxidation products on collagen and LDL after they have been formed. a1m has been found in LDL from human plasma, suggesting that a1m is a naturally occurring component of LDL. In vitro, a1m binds specifically to LDL-particles. a1m inhibits the hydroxyl radical-induced peroxidation of purified erythrocyte membranes. a1m can also inhibit the generation of intracellular reactive oxygen species in K562 cells after exposure of the cells to heme and hydrogen peroxide as well as in resting cells. In conclusion, a1m may be regarded as a heme-metabolising protein, a new regulator of oxidation and an extracellular antioxidant.