Polymerization of normal and intact beta(2)-microglobulin as the amyloidogenic protein in dialysis-amyloidosis

The primary structure of beta(2)-microglobulin (beta(2)m), the major constituent protein of beta(2)-microglobulin amyloidosis (A beta(2)m) or dialysis-amyloidosis, was initially shown to be identical to serum beta(2)m, thereby strongly suggesting the polymerization of intact beta(2)m in tissues. Recent biochemical data have been controversial, showing beta(2)m acidic isoforms: fragmentation and amino acid sequence alteration of deposited beta(2)m. The aim of this study was to reinvestigate beta(2)m amyloid deposits for the presence of beta(2)m fragments and/or amino acid sequence alteration. Four amyloid-laden tissues (3 femoral bone amyloid cysts and 1 heart tissue) from dialysis patients were used to isolate amyloidogenic beta(2)m. 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 beta(2)m. beta(2)m samples were microsequenced using the standard O3RPTH program on a 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 beta(2)m. SDS-PAGE analysis of this peak under reducing conditions, demonstrated one 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-sequenced and the amino acid sequence corresponded to that of normal beta(2)m through the 40th residue, Amino acid sequence analysis showed no difference from normal beta(2)m in any of the beta(2)m proteins contained in the amyloid deposits isolated from the four studied tissues. Also, the HPLC profile of the four protein samples were strictly normal and identical to a commercial preparation of beta(2)m. The present study demonstrates that beta(2)m molecules polymrrized 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.