Glycation of Human β₂-Microglobulin in Patients with Hemodialysis-Associated Amyloidosis: Identification of the Glycated Sites

β₂-Microglobulin (β₂M) is a major component forming amyloid deposits in patients with hemodialysis-associated amyloidosis (HAA), a serious complication of long-term hemodialysis. Recently, we demonstrated that β₂M modified with the Maillard reaction is a definite constituent of amyloid deposits in patients with HAA. Our further study demonstrated that this modified β₂M induces not only chemotaxis of monocytes but also secretion of tumor necrosis factor-α, interleukin-1 β, and interleukin-6 from macrophages, suggesting the potential link of glycation of β₂M by the Maillard reaction to the pathogenesis of HAA. The present study was undertaken to identify the glycated site(s) of β₂M purified from long-term hemodialysis patients as well as β₂M incubated with glucose in vitro. Borotritide-treated β₂M was cleaved by endoproteinase Lys-C, and peptides were isolated by reverse-phase high-performance liquid chromatography, followed by amino acid sequence analysis and fast atom bombardment mass spectrometry to identify the glycated site. The glycated sites of β₂ formed in vivo were found to be almost the same as those of glycated β₂M in vitro. The primary glycated site was the α-amino group of the amino terminal isoleucine. Other minor sites were the ϵ-amino groups of Lys-19, −41, −48, −58, −91, and −94. Computer graphics of the three-dimensional structure of β₂M suggested that the high specificity for the glycated site at Ile-1 may be explained by its high solvent accessibility and the nearby imidazole group of His-31 as an acid-base catalyst of the Amadori rearrangement.