Polymerization of normal and intact β2-microglobulin as the amyloidogenic protein in dialysis-amyloidosis

Abstract

The primary structure of β2 microglobulin (β2m), the major constituent protein of β2-microglobulin amyloidosis (Aβ2m) or dialysis-amyloidosis, was initially shown to be identical to serum β2m, thereby strongly suggesting the polymerization of intact β2m in tissues. Recent biochemical data have been controversial, showing β2m acidic isoforms, fragmentalion and amino acid sequence alteration of deposited β2m. The aim of this study was to reinvestigate p,m amyloid deposits for the presence of β2m fragments andior amino acid sequence alteration. Fouramyloid-laden tissues (3 femoral bone amyloid cysts and 1 heart tissue) from dialysis patients were used to isolate amyloidogenic β2m. Amyloid fibrils were isolated using the classic water extraction method, and purified in 6 M guanidine on a gel-filtration column. The protein was further purified on 17% SDS-PAGE gel, and transferred to a nitrocellulose membrane for immunostaining with antihuman β2m. β2m-PAGE samples were microsequenced using the standard 03RPTH program on. 470A gas-phase sequencer, and HPLC was performed after digestion with trypsin. Two peaks were obtained with the gel filtration column, the second corresponding by molecular weight to β2m. SDS-PAGE analysis ofthis peak under reducing conditions, demonstrated qne major band at 12,000 Da and a minor band at 25,000 Da (monomer and dimer), and no lower molecular weight bands were observed. The 12 kDa band was micro-sequencedand the amino acid sequence corresponded to that of normal β2m through the 40th residue. Amino acid sequence analysis showed no difference from normal β2m in any of the p,m proteins contained in the amyloid deposits isolated from the four studied tissues. Als, the HPLC profile of the four protein samples were strictly normal and identical to a commercial preparation of β2m. The present study demonstrates that β2m molecules polymerized in amyloid fibrils and deposits are intact and have a normal amino acid sequence, and produced by a specific and unique fibrillogenetic mechanism, which does not require proteolytic processing from the precursor protein to the amyloid fibrils.