Apolipoprotein A-I amyloidogenic variant L174S, expressed and isolated from stably transfected mammalian cells, is associated with fatty acids

Abstract

Sixteen variants of apolipoprotein A-I (ApoA-I) are associated with hereditary systemic amyloidoses, characterized by amyloid deposition in peripheral organs of patients. As these are heterozygous for the amyloidogenic variants, their isolation from plasma is impracticable and recombinant expression systems are needed. Here we report the expression of recombinant ApoA-I amyloidogenic variant Leu174 with Ser (L174S) in stably transfected Chinese hamster ovary-K1 cells. ApoA-I variant L174S was found to be efficiently secreted in the culture medium, from which it was isolated following a one-step purification procedure. Mass spectrometry analyses allowed the qualitative and quantitative definition of the amyloidogenic variant lipid content, which was found to consist of two saturated and two monounsaturated fatty acids. Interestingly, the same lipid species were found to be associated with the wild-type ApoA-I, expressed and isolated using the same cell system, with lower values of the lipid to protein molar ratios with respect to the amyloidogenic variant. A possible role of fatty acids in trafficking and secretion of apolipoproteins may be hypothesized.

Keywords::

• Amyloidosis
• ApoA-I variants
• apolipoprotein A-I
• mass spectrometry
• recombinant amyloidogenic proteins

Abbreviations:	=
ApoA-I,	=	apolipoprotein A-I;
AApoA-I(L174S),	=	ApoA-I variant carrying the mutation L174S;
CAD,	=	coronary artery disease;
CHO,	=	Chinese ovary hamster;
ES-MS,	=	electrospray mass spectrometry;
GC-MS,	=	gas chromatography mass spectrometry;
HDL,	=	high-density lipoproteins;
RCT,	=	reverse cholesterol transport

Abbreviations:	=
ApoA-I,	=	apolipoprotein A-I;
AApoA-I(L174S),	=	ApoA-I variant carrying the mutation L174S;
CAD,	=	coronary artery disease;
CHO,	=	Chinese ovary hamster;
ES-MS,	=	electrospray mass spectrometry;
GC-MS,	=	gas chromatography mass spectrometry;
HDL,	=	high-density lipoproteins;
RCT,	=	reverse cholesterol transport

Declaration of interest: This work was partially supported by Ministero della Istruzione, Università e Ricerca (MIUR), Italy, with PRIN 2009. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.