scholarly journals Accumulation of pro-apolipoprotein A-II in mouse senile amyloid fibrils

1997 ◽  
Vol 325 (3) ◽  
pp. 653-659 ◽  
Author(s):  
Keiichi HIGUCHI ◽  
Kumiko KOGISHI ◽  
Jing WANG ◽  
Chen XIA ◽  
Takuya CHIBA ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Density Lipoprotein ◽  
Relative Abundance ◽  
Mouse Liver ◽  
Amyloid Fibrils ◽  
Amyloid Fibril ◽  
Density Lipoprotein ◽  
Amyloid Protein ◽  
Amino Acid Residues ◽  
Apolipoprotein A

Apolipoprotein A-II (apoA-II), the major apoprotein of serum high-density lipoprotein, is deposited as amyloid fibrils (AApoAII) in murine senile amyloidosis. We have identified and purified a more basic amyloid protein from old-mouse liver. N-terminal sequencing of the protein revealed that the pro-segment of five amino acid residues (Ala-Leu-Val-Lys-Arg) extended from the N-terminal glutamine residue of mature apoA-II protein. MS analysis revealed the deposit of intact pro-apoA-II protein (molecular mass 9319 Da). Antiserum was prepared for staining of the AApoAII amyloid deposition. The relative abundance of pro-apoA-II to mature apoA-II in the amyloid-fibril fraction isolated from livers of mice with severe amyloidosis was 14.1%. The similar abundance of pro-apoA-II in the amyloid fibril fraction from the spleen (16.3%) suggested that deposited pro-apoA-II originated from the blood. The concentration of pro-apoA-II was much lower in the serum (1.5% of mature apoA-II) than in the amyloid-fibril fraction. There was no difference in the content of pro-apoA-II between the amyloidogenetic R1.P1-Apoa2c and amyloid-resistant SAMR1 strains at the age of 3 months. The abundance of pro-apoA-II in the amyloid-fibril fraction compared with the serum suggested that it plays a key role in the initialization of mouse senile amyloidosis.

scholarly journals 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

1996 ◽  
Vol 318 (3) ◽  
pp. 1041-1049 ◽  
Author(s):  
Himakshi PATEL ◽  
Jo BRAMALL ◽  
Helen WATERS ◽  
Maria C. DE BEER ◽  
Patricia WOO
Keyword(s):  
Amino Acid ◽  
Serum Amyloid A ◽  
Amyloid Fibrils ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Amyloid A ◽  
Amyloid Fibril Formation

Site-directed mutagenesis of the acute-phase human serum amyloid A (SAA1α) protein was used to evaluate the importance of the N-terminal amino acid residues, namely RSFFSFLGEAF. The full-length cDNA clone of SAA1α (pA1.mod.) was used to create two mutations, namely Gly-8 to Asp-8 and an 11 amino acid truncation between Arg-1 and Phe-11 respectively. Wild-type 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 SAA1α and SAA1α desArg found in acute-phase plasma. N-terminal sequencing of 56 residues confirmed its identity with human SAA1α. 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 HDL3 [Turnell, Sarra, Glover, Baum, Caspi, Baltz and Pepys (1986) Mol. 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 mutSAAΔ1–11 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.

scholarly journals The amino acid sequence of a carbohydrate-containing immunoglobulin-light-chain-type amyloid-fibril protein

1985 ◽  
Vol 232 (1) ◽  
pp. 183-190 ◽  
Author(s):  
T Tveteraas ◽  
K Sletten ◽  
P Westermark
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Light Chain ◽  
Amyloid Fibrils ◽  
Amyloid Fibril ◽  
Amino Acid Residues ◽  
Hypervariable Regions ◽  
Glycosylation Sites ◽  
Amyloid Fibril Protein ◽  
Chain Type

The amino acid sequence of an amyloid-fibril protein Es492 of immunoglobulin-lambda-light-chain origin (AL) was elucidated. The amyloid fibrils were obtained from the spleen of a patient who died from systemic amyloidosis. The amino acid sequence was elucidated from structural studies of peptides derived from digestion of the protein with trypsin, thermolysin, chymotrypsin and Staphylococcus aureus V8 proteinase and from cleavage of the protein with CNBr and BNPS-skatole. A heterogeneity in the length of the polypeptide was seen in the C-terminal region. The protein was by sequence homology to other lambda-chains shown to be of the V lambda II subgroup. Although an extensive homology was seen, some amino acid residues in positions 26, 31, 32, 40, 44, 93, 97, 98 and 99 have not previously been reported in these positions of V lambda II proteins. The significance of these residues in the fibril formation is unclear. The protein was found to contain carbohydrate, with glycosylation sites in two of the hypervariable regions.

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