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Locating Apolipoprotein A2 Protein in the Central Nervous System
Author(s) -
Azzam Sausan,
Gamble Jenniffer,
Decker Michael,
Li Xiaolin,
Hernandez Yeritza,
Strohl Kingman P
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.653.7
Subject(s) - peptide , hypothalamus , apelin , medicine , proteomics , chemistry , microbiology and biotechnology , endocrinology , gene , biology , biochemistry , receptor
Objectives Apolipoprotein A2 (Apoa2) gene expression in the brain is deterministic of apnea in the C57BL/6J sleep apnea mouse model, with both alleles needed for brain (but not liver) mRNA expression, and post‐hypoxic pauses (Gillombardo et al. 2016). Functional Apoa2 gene products, well described in the liver, are not reported as present in CNS. The purpose was to determine if Apoa2 protein was in the brain, and to validate two customized Apoa2 antibodies. Methods 12–16 week old B6‐WT (+/+) and Apoa2 KO (−/−) mice were sacrificed for the tissue used in this study. Two Apoa2 monoclonal antibodies were customized using two specific epitopes‐ THEQLTPLVRC (TH Rabbit mAb) and NLEEKPAPAAK (NL Rabbit mAb). Both antibodies were tested by Western immunoblotting. Liver and brain tissue (hypothalamus, hippocampus, and brainstem) samples were lysed, FASPed, quant, digested, and analyzed by SRM quantitative proteomics as previously described (Azzam et al 2016). Stable‐isotope labeled peptide was synthesized for the Apoa2 target peptide (THEQLTPLVRC). All samples were analyzed by LC/MS using a Waters Nano‐ACQUITY Ultra performance liquid chromatography system and a TSQ Quantum Ultra mass spectrometry system. The abundance of each peptide was calculated on the basis of the peak area intensity, and the peak areas for the target peptide was normalized to the internal standard for accurate quantification of the target peptide. Statistical analysis was performed using one‐way ANOVA. Results Proteomics data detected no Apoa2 in the KO mouse; however, protein was present in WT brainstem, hypothalamus, and hypothalamus, but in lesser abundance than either WT liver or plasma (P=0.0001). Apoa2 was present in the brain in dimeric and tetramer formations. Further, Western blots from brain, using either customized Apoa2 antibody, showed polymeric bands, but also a band at 33K previously ascribed in plasma samples as representing bonding with Apolipoprotein D. Conclusions Apoa2 protein is present in the B6 mouse CNS, indicating links to lipid‐cholesterol pathways operating not only for breathing and possibly for stress and memory functions as well. Support or Funding Information Supported by VA Merit Award and the NIH