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 Analysis of the Secondary Structure of β-Amyloid (Aβ42) Fibrils by Systematic Proline Replacement

2004 ◽  
Vol 279 (50) ◽  
pp. 52781-52788 ◽  
Author(s):  
Akira Morimoto ◽  
Kazuhiro Irie ◽  
Kazuma Murakami ◽  
Yuichi Masuda ◽  
Hajime Ohigashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Amyloid Fibrils ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Aβ Peptides ◽  
Amyloid Peptides ◽  
Aggregation Inhibitors ◽  
Β Amyloid ◽  
Β Sheet

Amyloid fibrils in Alzheimer's disease mainly consist of 40- and 42-mer β-amyloid peptides (Aβ40 and Aβ42) that exhibit aggregative ability and neurotoxicity. Although the aggregates of Aβ peptides are rich in intermolecular β-sheet, the precise secondary structure of Aβ in the aggregates remains unclear. To identify the amino acid residues involved in the β-sheet formation, 34 proline-substituted mutants of Aβ42 were synthesized and their aggregative ability and neurotoxicity on PC12 cells were examined. Prolines are rarely present in β-sheet, whereas they are easily accommodated in β-turn as a Pro-Xcorner. Among the mutants at positions 15-32, only E22P-Aβ42 extensively aggregated with stronger neurotoxicity than wild-type Aβ42, suggesting that the residues at positions 15-21 and 24-32 are involved in the β-sheet and that the turn at positions 22 and 23 plays a crucial role in the aggregation and neurotoxicity of Aβ42. The C-terminal proline mutants (A42P-, I41P-, and V40P-Aβ42) hardly aggregated with extremely weak cytotoxicity, whereas the C-terminal threonine mutants (A42T- and I41T-Aβ42) aggregated potently with significant cytotoxicity. These results indicate that the hydrophobicity of the C-terminal two residues of Aβ42 is not related to its aggregative ability and neurotoxicity, rather the C-terminal three residues adopt the β-sheet. These results demonstrate well the large difference in aggregative ability and neurotoxicity between Aβ42 and Aβ40. In contrast, the proline mutants at the N-terminal 13 residues showed potent aggregative ability and neurotoxicity similar to those of wild-type Aβ42. The identification of the β-sheet region of Aβ42 is a basis for designing new aggregation inhibitors of Aβ peptides.

Interaction of serum amyloid A with human cystatin C-assessment of amino acid residues crucial for hCC-SAA formation (part II)

2013 ◽  
Vol 26 (9) ◽  
pp. 415-425 ◽  
Author(s):  
Marta Spodzieja ◽  
Monika Rafalik ◽  
Aneta Szymańska ◽  
Aleksandra S. Kołodziejczyk ◽  
Paulina Czaplewska
Keyword(s):  
Amino Acid ◽  
Cystatin C ◽  
Serum Amyloid A ◽  
Serum Amyloid ◽  
Amino Acid Residues ◽  
Amyloid A ◽  
Human Cystatin C ◽  
Human Cystatin

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 Molecular and Functional Analyses of Kunjin Virus Infectious cDNA Clones Demonstrate the Essential Roles for NS2A in Virus Assembly and for a Nonconservative Residue in NS3 in RNA Replication

2003 ◽  
Vol 77 (14) ◽  
pp. 7804-7813 ◽  
Author(s):  
Wen Jun Liu ◽  
Hua Bo Chen ◽  
Alexander A. Khromykh
Keyword(s):  
Amino Acid ◽  
Virus Assembly ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Common Amino Acid

ABSTRACT A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by ∼100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an ∼5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type Ile residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in trans by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in trans by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.

scholarly journals Serum Amyloid a Low-Density Lipoprotein Levels and Smoking Status in Obese Japanese Patients

2011 ◽  
Vol 39 (5) ◽  
pp. 1917-1922 ◽  
Author(s):  
K Kotani ◽  
N Satoh-Asahara ◽  
Y Kato ◽  
R Araki ◽  
A Himeno ◽  
...  
Keyword(s):  
Serum Amyloid A ◽  
Smoking Status ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Japanese Patients ◽  
Serum Amyloid ◽  
Low Density ◽  
Amyloid A

scholarly journals Effects of Specific Amino Acid Substitutions on Activities of Dinitrogenase Reductase-Activating Glycohydrolase fromRhodospirillum rubrum

2001 ◽  
Vol 183 (19) ◽  
pp. 5743-5746 ◽  
Author(s):  
Babu S. Antharavally ◽  
Russell R. Poyner ◽  
Yaoping Zhang ◽  
Gary P. Roberts ◽  
Paul W. Ludden
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Paramagnetic Resonance ◽  
Specific Amino Acid ◽  
Dinitrogenase Reductase ◽  
Electron Paramagnetic ◽  
Hydrolysis Of

ABSTRACT Site-directed mutagenesis of the draG gene was used to generate altered forms of dinitrogenase reductase-activating glycohydrolase (DRAG) with D123A, H142L, H158N, D243G, and E279R substitutions. The amino acid residues H142 and E279 are not required either for the coordination to the metal center or for catalysis since the variants H142L and E279R retained both catalytic and electron paramagnetic resonance spectral properties similar to those of the wild-type enzyme. Since DRAG-H158N and DRAG-D243G variants lost their ability to bind Mn(II) and to catalyze the hydrolysis of the substrate, H158 and D243 residues could be involved in the coordination of the binuclear Mn(II) center in DRAG.

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