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 c onstituent protein of beta(2)-microglobulin amyloidosis (A beta(2)m) o r dialysis-amyloidosis, was initially shown to be identical to serum b eta(2)m, thereby strongly suggesting the polymerization of intact beta (2)m in tissues. Recent biochemical data have been controversial, show ing beta(2)m acidic isoforms: fragmentation and amino acid sequence al teration of deposited beta(2)m. The aim of this study was to reinvesti gate beta(2)m amyloid deposits for the presence of beta(2)m fragments and/or amino acid sequence alteration. Four amyloid-laden tissues (3 f emoral bone amyloid cysts and 1 heart tissue) from dialysis patients w ere used to isolate amyloidogenic beta(2)m. Amyloid fibrils were isola ted using the classic water extraction method, and purified in 6 M gua nidine on a gel-filtration column. The protein was further purified on 17% SDS-PAGE gel, and transferred to a nitrocellulose membrane for im munostaining with antihuman beta(2)m. beta(2)m samples were microseque nced using the standard O3RPTH program on a 470A gas-phase sequencer. and HPLC was performed after digestion with trypsin. Two peaks were ob tained with the gel filtration column, the second corresponding by mol ecular weight to beta(2)m. SDS-PAGE analysis of this peak under reduci ng conditions, demonstrated one major band at 12,000 Da and a minor ba nd at 25,000 Da (monomer and dimer), and no lower molecular weight ban ds were observed. The 12 kDa band was micro-sequenced and the amino ac id sequence corresponded to that of normal beta(2)m through the 40th r esidue, Amino acid sequence analysis showed no difference from normal beta(2)m in any of the beta(2)m proteins contained in the amyloid depo sits isolated from the four studied tissues. Also, the HPLC profile of the four protein samples were strictly normal and identical to a comm ercial preparation of beta(2)m. The present study demonstrates that be ta(2)m molecules polymrrized in amyloid fibrils and deposits are intac t and have a normal amino acid sequence, and produced by a specific an d unique fibrillogenetic mechanism, which does not require proteolytic processing from the precursor protein to the amyloid fibrils.