Campistol J M, Bernard D, Papastoitsis G, Solé M, Kasirsky J, Skinner M
Arthritis Center, Thorndike Memorial Laboratories, Boston City Hospital, Boston University School of Medicine, Massachusetts, USA.
Kidney Int. 1996 Oct;50(4):1262-7. doi: 10.1038/ki.1996.436.
The primary structure of beta 2-microglobulin (beta 2m), the major constituent protein of beta 2-microglobulin amyloidosis (A beta 2m) or dialysis-amyloidosis, was initially shown to be identical to serum beta 2m, thereby strongly suggesting the polymerization of intact beta 2m in tissues. Recent biochemical data have been controversial, showing beta 2m acidic isoforms, fragmentation and amino acid sequence alteration of deposited beta 2m. The aim of this study was to reinvestigate beta 2m amyloid deposits for the presence of beta 2m 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 2m. 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 2m. beta 2m samples were microsequenced using the standard 03RPTH 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 2m. 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 2m through the 40th residue. Amino acid sequence analysis showed no difference from normal beta 2m in any of the beta 2m 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 2m. The present study demonstrates that beta 2m 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.
β2微球蛋白淀粉样变性（Aβ2m）或透析淀粉样变性的主要组成蛋白β2微球蛋白（β2m）的一级结构最初被证明与血清β2m相同，从而有力地表明完整的β2m在组织中发生了聚合。最近的生化数据存在争议，显示出β2m酸性异构体、沉积的β2m的片段化和氨基酸序列改变。本研究的目的是重新研究β2m淀粉样沉积物中是否存在β2m片段和/或氨基酸序列改变。使用来自透析患者的四个富含淀粉样蛋白的组织（3个股骨骨淀粉样囊肿和1个心脏组织）来分离淀粉样生成性β2m。使用经典的水提取方法分离淀粉样纤维，并在凝胶过滤柱上于6M胍中纯化。蛋白质在17%SDS-PAGE凝胶上进一步纯化，并转移至硝酸纤维素膜上用抗人β2m进行免疫染色。使用470A气相测序仪上的标准03RPTH程序对β2m样品进行微量测序，并在用胰蛋白酶消化后进行HPLC分析。凝胶过滤柱得到两个峰，第二个峰的分子量与β2m相对应。在还原条件下对该峰进行SDS-PAGE分析，显示在12,000Da处有一条主要条带，在25,000Da处有一条次要条带（单体和二聚体），未观察到分子量更低的条带。对12kDa条带进行微量测序，其氨基酸序列与正常β2m直至第40个残基的序列一致。氨基酸序列分析表明，从四个研究组织中分离出的淀粉样沉积物中所含的任何β2m蛋白与正常β2m均无差异。此外，四个蛋白质样品的HPLC图谱完全正常，且与市售β2m制剂相同。本研究表明，在淀粉样纤维和沉积物中聚合的β2m分子是完整的，具有正常的氨基酸序列，并且是由一种特定且独特的纤维生成机制产生的，该机制不需要从前体蛋白到淀粉样纤维的蛋白水解加工。
