Human α-1-Microglobulin Is Covalently Bound to Kynurenine-derived Chromophores

Abstract

␣-1-Microglobulin carries a set of covalently linked chromophores that give it a peculiar yellow-brown color, fluorescence properties, and both charge and size heterogeneity. In this report it is shown that these features are due to the adducts with the tryptophan metabolite, 3-hydroxykynurenine, and its autoxidation products and that the modification is more pronounced in the protein isolated from urine of hemodialyzed patients. The light yellow amniotic fluid ␣-1-microglobulin acquires the optical properties and charge heterogeneity of the urinary counterpart following incubation with kynurenines. The colored amino acid adducts of urinary and amniotic fluid ␣-1-microglobulins were separated by chromatography after acid hydrolysis and analyzed by mass spectrometry. Human serum albumin samples, native and treated with 3-hydroxykynurenine in the presence of oxygen, were used as a control. The retention times and mass fragmentation products were compared, and a lysyl adduct with hydroxantommathin was identified in the urinary ␣-1-microglobulin and in the modified albumin samples. The more extensive modification of the urinary protein appears to be correlated with uremia, a condition in which the catabolism of tryptophan via the kynurenine pathway is increased, and the consequent rise in the concentration of its derivatives is accompanied by the oxidative processes due to the hemodialysis treatment. The oxidative derivatives of 3-hydroxykynurenine, which are known to act as protein crosslinking agents, are the likely cause of the propensity of urinary ␣-1-microglobulin to form dimers and oligomers. This process, as well as the redox properties of these metabolites, may contribute to the toxic effects of the kynurenine species.1 also known as protein HC, is a widely distributed glycosylated protein that on the basis of sequence homology has been included in the lipocalin family. This large group of predominantly extracellular molecules shares a common ␤-barrel fold but has been assigned different functional roles (1). ␣-1-m is present in many tissues and, although its function is not known, several reports hint at a role as an immunomodulator (2, 3). Its single polypeptide chain consists of 183 residues (4) and contains one O-linked and two N-linked oligosaccharide moieties (5). The synthesis takes place mainly in the liver and, in humans, the ␣-1-m gene has been mapped to chromosome 9 in a region where other lipocalin genes are clustered (4, 6). As for all other species, it encodes also bikunin, the light chain of a plasma proteinase inhibitor family (7), and the precursor polypeptide chain contains an internal basic tripeptide recognized by a specific endoprotease. The cleavage occurs in the Golgi apparatus before secretion and, so far, no functional correlation between the lipocalin and bikunin has been found. ␣-1-m is then catabolized in the proximal tubule cells of the kidneys, and its concentration increases in urines of patients with renal failure (8). The protein has thus been recognized as a very sensitive ...

Coelution of Other Proteins with Albumin during Size-Exclusion HPLC: Implications for Analysis of Urinary Albumin

“…The aggregate concentration in plasma of globulins with a molecular size similar to albumin, proteins such as transferrin, ␣ 1 -acid glycoprotein, ␣ 1 -proteinase inhibitor, ␣ 1 -antichymotrypsin, ␣ 2 -HS glycoprotein, hemopexin, Gc-globulin, and many others (31 ), in healthy individuals is ϳ25% that of albumin; this aggregate concentration is even higher during acutephase reactions (32 ). Because the passage of plasma proteins through the glomerular barrier into urine is primarily a size-dependent process with a size-exclusion limit approximately the size of albumin (33 ), it is not surprising that all of the albumin-sized plasma components are also found in urine (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(33)(34)(35)(36)(37)(38)(39). There is considerable variation in the reported proportions of individual globulins relative to albumin in urine (Table 3).…”

RT-PCR- and MALDI-TOF Mass Spectrometry-Based Identification and Discrimination of Isoforms Homologous to Pufferfish Saxitoxin- and Tetrodotoxin-Binding Protein in the Plasma of Non-Toxic Cultured Pufferfish (Takifugu rubripes)

“…1, potential N-glycosylation sites). Tr1 might form an SDS-resistant dimer even under reducing conditions, as observed for other lipocalins, e.g., dimerization of -1-microglobulin via in vivo covalent cross-linking with oxidized metabolites, 17) and that of -lactoglobulin via ex vivo denaturation/aggregation. 18) The possibility that one of the 120-kDa proteins is the product of an unidentified gene that encodes tandemrepeated Tr1-like domains, however, cannot be ruled out.…”

A1M, an extravascular tissue cleaning and housekeeping protein

“…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 side-chains of Lys92, 118 and 130 may therefore serve a dual role both in activation of the Cys34 thiol by lowering its pK a [38] and as electron-donors in the radical-trapping mechanism.…”