Expression of recombinant human serum amyloid A in mammalian cells and demonstration of the region necessary for high-density lipoprotein binding and amyloid fibril formation by site-directed mutagenesis
Expression of recombinant human serum amyloid A in mammalian cells and demonstration of the region necessary for high-density lipoprotein binding and amyloid fibril formation by site-directed mutagenesis
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DOI：
10.1042/bj3181041
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发表时间：
1996-09-15
影响因子：
4.1
通讯作者：
Woo, P
中科院分区：
文献类型：
--
作者：
Patel, H;Bramall, J;Woo, P
用人血清淀粉样蛋白A(SAA1α)蛋白的定点突变来评价N-末端氨基酸残基RSFFSFLGEAF的重要性。SAA1α(pA1.mod.)被用来产生两个突变，即Gly-8到Asp-8，以及在Ag-L和Phe-11之间截断11个氨基酸。在人巨细胞病毒启动子的控制下，野生型和突变型cDNAs在中国仓鼠卵巢(CHO)细胞中表达，使处理后的蛋白分泌到培养基中。野生型重组人SAA(RSAA)蛋白的pI分别为6.0和6.4，与急性期血浆中发现的人SAA亚型SAA1α和SAA1αdesArg相似。对56个残基的N末端测序证实了其与人SAA1α的同源性。用ELISA法测得野生型RSAA的总产量在3.50~30 mg/L之间，两个突变导致突变型SAA蛋白的表达水平降低(3~10 mg/L)。进一步测定浓度为1.21g/ml(高密度脂蛋白)和1.063~1.18g/ml(极低密度脂蛋白/极低密度脂蛋白)的培养液中脂质部分的RSAA浓度，发现76%的野生型蛋白存在于高密度脂蛋白部分，其余24%存在于非脂蛋白部分。相反，突变的RSAA在高密度脂蛋白和非高密度脂蛋白中的相对浓度是相反的。这与先前提出的11个氨基酸多肽参与将SAA蛋白锚定在高密度脂蛋白(8)上是一致的[Turnell，Sarra，Coverer，Baum，Caspi，Baltz和Pepys(1986)Mel。比奥尔。地中海医院。3,387-407]。电子显微镜显示，野生型RSAA蛋白在体外酸性条件下形成淀粉样纤维，刚果红染色阳性，偏振光下呈现苹果绿双折射。在相同条件下，MutSAA(G8D)和MutSAA Delta 1-11不形成淀粉样纤维。总之，用Asp-8替换Gly-8或删除RSAA N-末端的前11个氨基酸残基会降低其与高密度脂蛋白的结合能力，并减少淀粉样纤维的形成。
Site-directed mutagenesis of the acute-phase human serum amyloid A (SAA1 alpha) protein was used to evaluate the importance of the N-terminal amino acid residues, namely RSFFSFLGEAF. The full-length cDNA clone of SAA1 alpha (pA1.mod.) was used to create two mutations, namely Gly-8 to Asp-8 and an 11 amino acid truncation between Ag-l and Phe-ll respectively. Wildtype and mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells under the control of the human cytomegalovirus promoter, which resulted in the secretion of the processed proteins into the culture media. Wild-type recombinant human SAA (rSAA) protein was shown to have pi values of 6.0 and 6.4, similar to the human SAA isoform SAA1 alpha and SAA1 alpha desArg found in acute-phase plasma. N-terminal sequencing of 56 residues confirmed its identity with human SAA1 alpha. The total yield of wild-type rSAA measured by ELISA was between 3.5 and 30 mg/l. The two mutations resulted in reduced expression levels of the mutant SAA proteins (3-10 mg/l). Further measurements of rSAA concentration in lipid fractions of culture medium collected at a density of 1.21 g/ml (high-density lipoprotein; HDL) and 1.063-1.18 g/ml (very-low-density lipoprotein/low-density lipoprotein; VLDL/LDL) showed that 76 % of the wild-type protein was found in the HDL fraction and the remaining 24 % in the infranatant non-lipid fraction. In contrast the relative concentration of mutant rSAA in HDL and infranatant fractions was reversed. This is consistent with the previously proposed involvement of the 11 amino acid peptide in anchoring SAA protein on to HDL(8) [Turnell, Sarra, Clover, Baum, Caspi, Baltz and Pepys (1986) Mel. Biol. Med. 3, 387-407]. Wild-type rSAA protein was shown to form amyloid fibrils in vitro under acidic conditions as shown by electron microscopy, and stained positive with Congo Red and exhibited apple-green birefringence when viewed under polarized light. Under the same conditions mutSAA(G8D) and mutSAA Delta 1-11 did not form amyloid fibrils. In conclusion, replacement of Gly-8 by Asp-8 or deletion of the first 11 amino acid residues at the N-terminus of rSAA diminishes its capacity to bind to HDL and decreases amyloid fibril formation.