N-terminal sequence analysis of SAA-derivatives purified from murine inflammatory macrophages

Abstract

The pathogenesis of secondary amyloidosis in vivo is not well-understood. Experimental studies suggest that incomplete degradation of acute phase serum amyloid A (SAA), presumably endocytosed by activated monocytoid cells, may lead to intralysosomal formation of amyloid A (AA). To establish a possible link between these two events, we have carried out partial N-terminal sequence analysis of affinity purified SAA derivatives from peritoneal macrophages isolated at 4 weeks post-infection from alveolar hydatid cyst infected C57BL/6 mice. The macrophage lysates yielded five N-terminally intact SAA derivatives of- 5 to - 12 kDa which reacted with anti-mouse AA IgG, and contained a mixture of SAA₁ and SAA₂ isoforms. The SAA₂: SAA₁ ratio, evaluated from their proportion present in each M₁ SAA derivative, showed a decrease with the decreasing apparent mass of the N-terminally infected SAA material. These results not only confirm that both SAA₁ and SAA₂ are processed by activated monocytoid cells but, more importantly, establish a plausible link between N-terminally intact SAA derivatives and formation of AA within activated monocytoid cells.

Key Words:

• macrophage
• serum amyloid A derivatives
• N-terminal sequencing
• affinity chromatography
• immunoblotting