• Home
  • Search Results
  • Uncleaved ApoM signal peptide is required for formation of large ApoM/sphingosine 1-phosphate (S1P)-enriched HDL particles.

Uncleaved ApoM signal peptide is required for formation of large ApoM/sphingosine 1-phosphate (S1P)-enriched HDL particles.

The Journal of biological chemistry (2015-01-30)
Mingxia Liu, Jeremy Allegood, Xuewei Zhu, Jeongmin Seo, Abraham K Gebre, Elena Boudyguina, Dongmei Cheng, Chia-Chi Chuang, Gregory S Shelness, Sarah Spiegel, John S Parks
ABSTRACT

Apolipoprotein M (apoM), a plasma sphingosine 1-phosphate (S1P) carrier, associates with plasma HDL via its uncleaved signal peptide. Hepatocyte-specific apoM overexpression in mice stimulates formation of both larger nascent HDL in hepatocytes and larger mature apoM/S1P-enriched HDL particles in plasma by enhancing hepatic S1P synthesis and secretion. Mutagenesis of apoM glutamine 22 to alanine (apoM(Q22A)) introduces a functional signal peptidase cleavage site. Expression of apoM(Q22A) in ABCA1-expressing HEK293 cells resulted in the formation of smaller nascent HDL particles compared with wild type apoM (apoM(WT)). When apoM(Q22A) was expressed in vivo, using recombinant adenoviruses, smaller plasma HDL particles and decreased plasma S1P and apoM were observed relative to expression of apoM(WT). Hepatocytes isolated from both apoM(WT)- and apoM(Q22A)-expressing mice displayed an equivalent increase in cellular levels of S1P, relative to LacZ controls; however, relative to apoM(WT), apoM(Q22A) hepatocytes displayed more rapid apoM and S1P secretion but minimal apoM(Q22A) bound to nascent lipoproteins. Pharmacologic inhibition of ceramide synthesis increased cellular sphingosine and S1P but not medium S1P in both apoM(WT) and apoM(Q22A) hepatocytes. We conclude that apoM secretion is rate-limiting for hepatocyte S1P secretion and that its uncleaved signal peptide delays apoM trafficking out of the cell, promoting formation of larger nascent apoM- and S1P-enriched HDL particles that are probably precursors of larger apoM/S1P-enriched plasma HDL.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Methanol, HPLC Plus, ≥99.9%, poly-coated bottles
Supelco
Sodium dodecyl sulfate, dust-free pellets, suitable for electrophoresis, for molecular biology, ≥99.0% (GC)
Supelco
Methanol, Pharmaceutical Secondary Standard; Certified Reference Material
SAFC
Cholesterol, from sheep wool, Controlled origin, meets USP/NF testing specifications
USP
Dehydrated Alcohol, United States Pharmacopeia (USP) Reference Standard
Supelco
Dehydrated Alcohol, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Sodium dodecyl sulfate, 92.5-100.5% based on total alkyl sulfate content basis
Sigma-Aldrich
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, clone M2, purified immunoglobulin (Purified IgG1 subclass), buffered aqueous solution (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.02% sodium azide)
Supelco
Ethanol standards 10% (v/v), 10 % (v/v) in H2O, analytical standard
Sigma-Aldrich
Ethanol Fixative 80% v/v, suitable for fixing solution (blood films)
Sigma-Aldrich
Cholesterol, from sheep wool, ≥92.5% (GC), powder
Sigma-Aldrich
Cholesterol, Sigma Grade, ≥99%
Sigma-Aldrich
Cholesterol, powder, BioReagent, suitable for cell culture, ≥99%
Sigma-Aldrich
Sodium dodecyl sulfate, BioReagent, suitable for electrophoresis, for molecular biology, ≥98.5% (GC)
Sigma-Aldrich
Bicinchoninic acid disodium salt hydrate, ≥98% (HPLC)
Sigma-Aldrich
Sodium dodecyl sulfate, ReagentPlus®, ≥98.5% (GC)
Sigma-Aldrich
Sodium dodecyl sulfate, BioXtra, ≥99.0% (GC)
Supelco
Fumonisin B1 solution, ~50 μg/mL in acetonitrile: water, analytical standard
Supelco
Cholesterol solution, certified reference material, 10 mg/mL in chloroform
Sigma-Aldrich
SyntheChol® NS0 Supplement, 500 ×, synthetic cholesterol, animal component-free, aqueous solution, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Methanol, anhydrous, 99.8%
Supelco
Ethanol solution, certified reference material, 2000 μg/mL in methanol
Sigma-Aldrich
Sodium dodecyl sulfate, ACS reagent, ≥99.0%
Sigma-Aldrich
Methanol, suitable for HPLC, gradient grade, 99.93%
Sigma-Aldrich
Sodium trichloroacetate, 97%
Sigma-Aldrich
Sodium dodecyl sulfate, ≥90%
Sigma-Aldrich
Sodium dodecyl sulfate, tested according to NF, mixture of sodium alkyl sulfates consisting mainly of sodium dodecyl sulfate
Supelco
Methanol, analytical standard
Sigma-Aldrich
Sodium dodecyl sulfate, ≥98.0% (GC)
Sigma-Aldrich
Sodium dodecyl sulfate, BioUltra, for molecular biology, ≥99.0% (GC)