Amyloid-enhancing factor mediates amyloid formation on fibroblasts via a nidus/template mechanism.
OBJECTIVE
To determine the mechanism by which amyloid-enhancing factor (AEF) promotes amyloid deposition, and to test whether AEF seeds deposition of serum amyloid A (SAA) and facilitates conversion to beta-sheet structure.


METHODS
Fibroblasts were cultured with mouse recombinant SAA1.1 and AEF, SAA1.1, or AEF. AEF was prepared as a glycerol extract of spleen from amyloidotic mice. Amyloid was identified by staining with Congo red and examining for green birefringence under polarized light. SAA was localized immunohistochemically. Texas Red-labeled SAA was visualized in living cultures by fluorescence confocal microscopy. AEF was characterized by Western blot analysis using anti-SAA antiserum and N-terminal sequence analysis. Subunits comprising amyloid in fibroblast cultures were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.


RESULTS
Amyloid was produced in fibroblast cultures by an AEF-dependent mechanism. AEF, added to culture medium as insoluble protein precipitates, adhered to fibroblast monolayers. SAA bound preferentially to the adherent precipitates. Coincident with SAA binding, precipitates developed an affinity for Congo red. Over time, as more SAA was added, networks of Congo red-positive material producing bright green birefringence also developed outward from AEF precipitates. Amyloid built upon AEF in this manner was composed of full-length SAA. No amyloid was produced in cultures treated with either SAA or AEF alone. SAA and SAA peptides processed in the C-terminal region were the most prominent proteins in the glycerol-extracted AEF preparation.


CONCLUSIONS
AEF binds to fibroblast monolayers and acts as a sink for SAA. SAA that collects on AEF assembles into an amyloid structure. Thus, it is concluded that AEF serves as both a nidus and a template for amyloid formation.
Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Nadine
N
Magy
Juris J
JJ
Liepnieks
Merrill D
MD
Benson
Barbara
B
Kluve-Beckerman
D000818:Animals; D001053:Apolipoproteins / Q000096:biosynthesis* / Q000235:genetics; D001704:Biopolymers; D002478:Cells, Cultured; D004396:Coloring Agents; D003224:Congo Red; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006023:Glycoproteins / Q000494:pharmacology*; D006801:Humans; D007124:Immunoenzyme Techniques; D051379:Mice; D018613:Microscopy, Confocal; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D000685:Serum Amyloid A Protein / Q000096:biosynthesis* / Q000235:genetics; D012867:Skin / Q000187:drug effects / Q000378:metabolism; D013698:Templates, Genetic
D001053:Apolipoproteins; D001704:Biopolymers; D004396:Coloring Agents; D006023:Glycoproteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D000685:Serum Amyloid A Protein; C046748:amyloid enhancing factor; D003224:Congo Red