Adenoviral expression of murine serum amyloid A proteins to study amyloid fibrillogenesis

Serum amyloid A (SAA) proteins are one of the most inducible acute-phase reactants and are precursors of secondary amyloidosis. In the mouse, SAA1 and SAA2 are induced in approximately equal quantities in response to amyloid induction models. These two isotypes differ in only 9 of 103 amino acid residues; however, only SAA2 is selectively deposited into amyloid fibrils. SAA expression in the CE/J mouse species is an exception in that gene duplication did not occur and the CE/J variant is a hybrid molecule sharing features of SAA1 and SAA2. However, even though it is more closely related to SAA2 it is not deposited as amyloid fibrils. We have developed an adenoviral vector system to overexpress SAA proteins in cell culture to determine the ability of these proteins to form amyloid fibrils, and to study the structural features in relation to amyloid formation. Both the SAA2 and CE/J SAA proteins were synthesized in large quantities and purified to homogeneity. Electron microscopic analysis of the SAA proteins revealed that the SAA2 protein was capable of forming amyloid fibrils, whereas the CE/J SAA was incapable. Radiolabelled SAAs were associated with normal or acute-phase high-density lipoproteins (HDLs); we examined them for their clearance from the circulation. In normal mice, SAA2 had a half-life of 70 min and CE/J SAA had a half-life of 120 min; however, in amyloid mice 50% of the SAA2 cleared in 55 min, compared with 135 min for the CE/J protein. When the SAA proteins were associated with acute-phase HDLs, SAA2 clearance was decreased to 60 min in normal mice compared with 30 min in amyloidogenic mice. Both normal and acute-phase HDLs were capable of depositing SAA2 into preformed amyloid fibrils, whereas the CE/J protein did not become associated with amyloid fibrils. This established approach opens the doors for large-scale SAA production and for the examination of specific amino acids involved in the fibrillogenic capability of the SAA2 molecule in vitro and in vivo.

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Serum amyloid A is a chemoattractant: induction of migration, adhesion, and tissue infiltration of monocytes and polymorphonuclear leukocytes.

Journal of Experimental Medicine ◽

10.1084/jem.180.1.203 ◽

1994 ◽

Vol 180 (1) ◽

pp. 203-209 ◽

Cited By ~ 331

Author(s):

R Badolato ◽

J M Wang ◽

W J Murphy ◽

A R Lloyd ◽

D F Michiel ◽

...

Keyword(s):

Acute Phase ◽

Polymorphonuclear Leukocytes ◽

Serum Amyloid A ◽

High Density ◽

Serum Amyloid ◽

High Density Lipoproteins ◽

Secondary Amyloidosis ◽

Polymorphonuclear Cells ◽

Amyloid A ◽

Cell Adhesion Molecule 1

Serum amyloid A (SAA) is an acute phase protein that in the blood is bound to high density lipoproteins; SAA is secreted mainly by hepatocytes, and its concentration increases in the blood up to 1000 times during an inflammatory response. At present, its biological function is unclear. Since some forms of secondary amyloidosis are caused by deposition in tissues of peptides derived from the SAA and leukocytes seem to be involved in this process, we investigated the effect of human SAA on human monocytes and polymorphonuclear cells (PMN). When recombinant human SAA (rSAA) was used at concentrations corresponding to those found during the acute phase (> 0.8 microM), it induced directional migration of monocytes and polymorphonuclear leukocytes. Preincubation of rSAA with high density lipoproteins blocked this chemoattractant activity for both monocytes and PMN. rSAA also regulated the expression of the adhesion proteins CD11b and leukocyte cell adhesion molecule 1 and induced the adhesion of PMN and monocytes to umbilical cord vein endothelial cell monolayers. When subcutaneously injected into mice, rSAA recruited PMN and monocytes at the injection site. On the basis of these data, we suggest that SAA may participate in enhancing the migration of monocytes and PMN to inflamed tissues during an acute phase response.

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Serum amyloid A and high density lipoproteins during the acute phase response

European Journal of Clinical Investigation ◽

10.1111/j.1365-2362.1988.tb01277.x ◽

1988 ◽

Vol 18 (6) ◽

pp. 619-626 ◽

Cited By ~ 30

Author(s):

LINDA L. BAUSSERMAN ◽

D. N. BERNIER ◽

K. P. W. J. McADAM ◽

P. N. HERBERT

Keyword(s):

Acute Phase ◽

Acute Phase Response ◽

Serum Amyloid A ◽

High Density ◽

Phase Response ◽

Serum Amyloid ◽

High Density Lipoproteins ◽

Amyloid A

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Heterogeneity of human serum amyloid A proteins.

Journal of Experimental Medicine ◽

10.1084/jem.152.3.641 ◽

1980 ◽

Vol 152 (3) ◽

pp. 641-656 ◽

Cited By ~ 100

Author(s):

L L Bausserman ◽

P N Herbert ◽

K P McAdam

Keyword(s):

Amino Acid ◽

Serum Amyloid A ◽

Polyacrylamide Gel ◽

Serum Amyloid ◽

High Density Lipoproteins ◽

Systemic Lupus Erythematosis ◽

Secondary Amyloidosis ◽

Single Chain ◽

Amino Acid Compositions ◽

Amyloid A

Serum amyloid A proteins (SAA), presumed precursors of the tissue amyloid A proteins (AA) characteristic of secondary amyloidosis, have been isolated from the plasma high-density lipoproteins (HDL) of normals after etiocholanolone-induced inflammation and from patients with Wegener's granulomatosis, systemic lupus erythematosis, juvenile rheumatoid arthritis, Waldenström's macroglobulinemia, and Goodpasture's syndrome. At least six polymorphic forms of SAA wer identified among the low molecular weight proteins of HDL, and these comprosed up to 27% of the total HDL protein. Gel and ion-exchange chromatography permitted isolation of the SAA polymorphs in homogeneous form. Their amino acid compositions were very similar, they were indistinguishable in cationic and sodium dodecyl sulfate-polyacrylamide gel electrophoresis systems, and each had the terminal sequency COOH-Tyr-Lys-Phe-. Charge heterogeneity in anionic-urea polyacrylamide gel electropherograms was unaffected by neuaminidase treatment, and none of the SAA protein bands stained with the periodate-Schiff reagent. The two major SAA polymorphs, designated SAA4 and SAA5 according to their order of elution from DEAE-cellulose, had different NH2-terminal sequences. Manual Edman degradation demonstrated NH2-arg-ser-phe-phe- for SAA4 and NH2-ser-phe-phe- for SAA5. This NH2-terminal heterogeneity corresponds to that most frequently reported for AA and suggests that microheterogeneity in SAA may underlie that already documented in AA. Sufficient quantitites of the other SAA polymorphs were not available for similar analyses, but the amino acid compositions do not indicate that NH2-terminal heterogeneity accounts for all of the observed polymorphism. Artifactual polymorphism also appears unlikely, and the heterogeneiy of SAA may reflect origin from more than one cell type with or without posttranslational modificaton. We calculate from quantitative COOH-terminal analyses that SAA is of 11,000-11,900 mol wt. Primary structure studies have shown AA t be a single chain protein of 76 residues, and SAA, therefore, appears to contain a peptide of 33 amino acids that is missing from AA.

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Differential plasma clearance of murine acute-phase serum amyloid A proteins SAA1 and SAA2

Biochemical Journal ◽

10.1042/bj3220663 ◽

1997 ◽

Vol 322 (2) ◽

pp. 663-669 ◽

Cited By ~ 38

Author(s):

Barbara KLUVE-BECKERMAN ◽

Toshiyuki YAMADA ◽

Joyce HARDWICK ◽

Juris J. LIEPNIEKS ◽

Merrill D. BENSON

Keyword(s):

Acute Phase ◽

Half Life ◽

Plasma Clearance ◽

Serum Amyloid A ◽

Tissue Injury ◽

Density Lipoprotein ◽

Serum Amyloid ◽

Acute Phase Reactants ◽

Amyloid A ◽

Potential Factor

Serum amyloid A (SAA) proteins SAA1 and SAA2 are prominent acute-phase reactants which circulate in association with the high-density-lipoprotein (HDL) fraction of plasma. Plasma levels of SAA1 and SAA2 increase dramatically, by as much as 1000-fold, within 24 h of tissue injury and then rapidly decrease with cessation of the inflammatory stimulus, suggesting that SAA clearance and/or catabolism is important to the re-establishment of homoeostasis. In this context, aberrant SAA catabolism has long been considered a potential factor in the pathogenesis of reactive amyloidosis. To initiate studies aimed at understanding the differential regulation of SAA metabolism, we have produced 35S-labelled murine SAA1 and SAA2 in Escherichia coli, bound them individually to HDL, and then compared the plasma-clearance characteristics of SAA1 and SAA2 under normal and acute-phase conditions. When bound to normal HDL, SAA2 [half-life (t½) = 30 min] was cleared significantly faster than SAA1 (t½ = 75 min). Clearance of SAA1 and SAA2 was significantly slower when each was bound to acute-phase HDL as opposed to normal HDL, when clearance rates were determined in acute-phase mice versus normal mice, and when normal HDL was remodelled to contain both recombinant isotypes rather than just one of the isotypes. Thus it appears that an increased amount of SAA on HDL, or possibly the combined presence of both isotypes on HDL, is associated with a prolongation in the plasma half-life of SAA.

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Evaluation of goose serum amyloid a acute phase response by enzyme-linked immunosorbent assay

Acta Veterinaria Hungarica ◽

10.1556/avet.55.2007.3.9 ◽

2007 ◽

Vol 55 (3) ◽

pp. 349-357 ◽

Cited By ~ 7

Author(s):

Beáta Kovács ◽

Mathilda Toussaint ◽

E. Gruys ◽

Ibolya Fábián ◽

L. Szilágyi ◽

...

Keyword(s):

Acute Phase ◽

Pasteurella Multocida ◽

Serum Amyloid A ◽

Serum Amyloid ◽

Enzyme Linked Immunosorbent Assay ◽

Common Disease ◽

Post Inoculation ◽

Secondary Amyloidosis ◽

Phase Reaction ◽

Amyloid A

Serum amyloid A (SAA) is of interest as the circulating precursor of amyloid A protein, the fibrillar component of AA (secondary) amyloid deposits, and also as an extremely sensitive and rapid major acute phase protein. Serum concentrations of acute phase proteins (APPs) provide valuable information about the diagnosis and prognosis of various diseases, and thus the relevance of APPs for monitoring the health status of domestic animals is widely accepted. More importantly, the measurement of SAA concentration assists in assessing the prognosis in secondary amyloidosis, which is a common disease of geese, affecting an increasing number of animals. In the present study we introduce a highly sensitive goose-specific ELISA method for measuring SAA concentration in goose serum or plasma samples. Samples were taken from geese of the Landes Grey and Hungarian White breeds, which were stimulated for an acute phase reaction by administration of a commercially available fowl cholera vaccine containing inactivated Pasteurella multocida . Strong and characteristically rapid acute phase responses were measured in both breeds, peaking at approximately 24 h after inoculation. The maximum SAA concentration was 1200 μg/ml. At 72 h post-inoculation, the concentrations returned to pre-inoculation values. There was significantly (p = 0.004) less intense response in the control groups; however, a very mild increase of SAA levels was detected due to the stress inevitably caused by the sampling procedure.

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Acute-phase high-density lipoprotein in the rat does not contain serum amyloid A protein

Biochemical Journal ◽

10.1042/bj2420301 ◽

1987 ◽

Vol 242 (1) ◽

pp. 301-303 ◽

Cited By ~ 18

Author(s):

M L Baltz ◽

I F Rowe ◽

D Caspi ◽

W G Turnell ◽

M B Pepys

Keyword(s):

High Density Lipoprotein ◽

Acute Phase ◽

Serum Amyloid A ◽

Amyloid Fibrils ◽

Density Lipoprotein ◽

Lipid Binding ◽

High Density ◽

Serum Amyloid ◽

Amyloid A ◽

Serum Amyloid A Protein

Serum amyloid A protein (SAA) is an acute-phase apolipoprotein of high-density lipoprotein (HDL). Its N-terminal sequence is identical with that of amyloid A protein (AA), the subunit of AA amyloid fibrils. However, rats do not develop AA amyloidosis, and we report here that neither normal nor acute-phase rat HDL contains a protein corresponding to SAA of other species. mRNA coding for a sequence homologous with the C-terminal but not with the N-terminal part of human SAA is synthesized in greatly increased amounts in acute-phase rat liver. These observations indicate that the failure of rats to develop AA amyloid results from the absence of most of the AA-like part of their SAA-like protein, and that the N-terminal portion of SAA probably contains the lipid-binding sequences.

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