α₁‐Microglobulin chromophores are located to three lysine residues semiburied in the lipocalin pocket and associated with a novel lipophilic compound

Abstract

Alpha1-microglobulin (alpha1m) is an electrophoretically heterogeneous plasma protein. It belongs to the lipocalin superfamily, a group of proteins with a three-dimensional (3D) structure that forms an internal hydrophobic ligand-binding pocket. Alpha1m carries a covalently linked unidentified chromophore that gives the protein a characteristic brown color and extremely heterogeneous optical properties. Twenty-one different colored tryptic peptides corresponding to residues 88-94, 118-121, and 122-134 of human alpha1m were purified. In these peptides, the side chains of Lys92, Lys118, and Lys130 carried size heterogeneous, covalently attached, unidentified chromophores with molecular masses between 122 and 282 atomic mass units (amu). In addition, a previously unknown uncolored lipophilic 282 amu compound was found strongly, but noncovalently associated with the colored peptides. Uncolored tryptic peptides containing the same Lys residues were also purified. These peptides did not carry any additional mass (i.e., chromophore) suggesting that only a fraction of the Lys92, Lys118, and Lys130 are modified. The results can explain the size, charge, and optical heterogeneity of alpha1m. A 3D model of alpha1m, based on the structure of rat epididymal retinoic acid-binding protein (ERABP), suggests that Lys92, Lys118, and Lys130 are semiburied near the entrance of the lipocalin pocket. This was supported by the fluorescence spectra of alpha1m under native and denatured conditions, which indicated that the chromophores are buried, or semiburied, in the interior of the protein. In human plasma, approximately 50% of alpha1m is complex bound to IgA. Only the free alpha1m carried colored groups, whereas alpha1m linked to IgA was uncolored.

A1M, an extravascular tissue cleaning and housekeeping protein

“…(50) mg albumin and A1M were purified from human urine as described [5], dialyzed against water and freeze-dried. Brown-colored heterogeneous covalent modifications of the human urine A1M were identified on Lys69, Lys92, Lys118 and Lys130 [3,47]. Albumin is shown as a control protein, localized in blood plasma and purified from urine, but unmodified, illustrating the specificity of the radical trapping mechanism of A1M.…”

“…absolute requirement for the reductase and cell-protection activities [38,40], is involved in radical scavenging [10], and was suggested to participate in heme-binding [8]. Lys69, Lys92, Lys118, and Lys130 (blue) are involved in reductase activity [38] and are covalently modified in vivo [3]. Tyr22 and Tyr132 (black) are covalently modified in radical scavenging [10].…”

“…Besides these two tyrosyl-residues, several lysyl side-chains were shown to be modified by radical trapping. Thus, Lys69 [47], 92, 118 and 130 [3] are covalently modified in human A1M isolated from urine. Furthermore, the tryptophan metabolite 3-OH-kynurenin, which has a propensity to form free radicals [48], was identified as a lysyl modification on urinary A1M from hemodialysis patients [49].…”

“…The yellow-brown coloration was demonstrated to consist of very heterogeneous covalent modifications of several amino acid side-groups [3]. The last ten years, a series of reports have revealed A1M:s physiological role as a cell-and tissue protective antioxidant, operating 4 by clearing extravascular fluids of free radicals and heme-groups, and transporting them to the kidneys for degradation [4] (Figure 1).…”

Nitrophorins and nitrobindins: structure and function

“…The Lys118 and Lys92 residues pave the top region of the Hs-α 1 -m central cavity, which is wider than in most lipocalins, while Lys130 residue is positioned halfway down towards the bottom of the cavity. Cys34, Met62, Met99, Lys118 and Lys130 residues are involved in ligand recognition (104,106,107), although none of them are conserved among orthologous Hs-α 1 -m species (104). Residues Arg43, Arg66, Arg68, Lys69, Lys92, Lys94, Lys118 and Lys130 line the upper part of the pocket, whereas the hydrophobic side chains of the Tyr79, Phe88, Phe114 and Tyr132 residues form the lower cavity of the pocket.…”