Yusuke Masuo1, Yuri Ohba1, Kohei Yamada1, Aya Hasan Al-Shammari1, Natsumi Seba1, Noritaka Nakamichi1, Takuo Ogihara2, Munetaka Kunishima1, Yukio Kato3. 1. Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan. 2. Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan. 3. Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan. ykato@p.kanazawa-u.ac.jp.
Abstract
PURPOSE: Solute carrier SLC22A4 encodes the carnitine/organic cation transporter OCTN1 and is associated with inflammatory bowel disease, although little is known about how this gene is linked to pathogenesis. The aim of the present study was to identify endogenous substrates that are associated with gastrointestinal inflammation. METHODS: HEK293/OCTN1 and mock cells were incubated with colon extracts isolated from dextran sodium sulfate-induced colitis mice; the subsequent cell lysates were mixed with the amino group selective reagent 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS), to selectively label OCTN1 substrates. Precursor ion scanning against the fragment ion of APDS was then used to identify candidate OCTN1 substrates. RESULTS: Over 10,000 peaks were detected by precursor ion scanning; m/z 342 had a higher signal in HEK293/OCTN1 compared to mock cells. This peak was detected as a divalent ion that contained four APDS-derived fragments and was identified as spermine. Spermine concentration in peripheral blood mononuclear cells from octn1 gene knockout mice (octn1-/-) was significantly lower than in wild-type mice. Lipopolysaccharide-induced gene expression of inflammatory cytokines in peritoneal macrophages from octn1-/- mice was lower than in wild-type mice. CONCLUSIONS: The combination metabolomics approach can provide a novel tool to identify endogenous substrates of OCTN1.
PURPOSE: Solute carrier SLC22A4 encodes the carnitine/organic cation transporter OCTN1 and is associated with inflammatory bowel disease, although little is known about how this gene is linked to pathogenesis. The aim of the present study was to identify endogenous substrates that are associated with gastrointestinal inflammation. METHODS: HEK293/OCTN1 and mock cells were incubated with colon extracts isolated from dextran sodium sulfate-induced colitismice; the subsequent cell lysates were mixed with the amino group selective reagent 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS), to selectively label OCTN1 substrates. Precursor ion scanning against the fragment ion of APDS was then used to identify candidate OCTN1 substrates. RESULTS: Over 10,000 peaks were detected by precursor ion scanning; m/z 342 had a higher signal in HEK293/OCTN1 compared to mock cells. This peak was detected as a divalent ion that contained four APDS-derived fragments and was identified as spermine. Spermine concentration in peripheral blood mononuclear cells from octn1 gene knockout mice (octn1-/-) was significantly lower than in wild-type mice. Lipopolysaccharide-induced gene expression of inflammatory cytokines in peritoneal macrophages from octn1-/- mice was lower than in wild-type mice. CONCLUSIONS: The combination metabolomics approach can provide a novel tool to identify endogenous substrates of OCTN1.
Authors: Petra Krumpochova; Sunny Sapthu; Jos F Brouwers; Marcel de Haas; Ric de Vos; Piet Borst; Koen van de Wetering Journal: FASEB J Date: 2011-10-27 Impact factor: 5.191
Authors: Sook Wah Yee; Adrian Stecula; Huan-Chieh Chien; Ling Zou; Elena V Feofanova; Marjolein van Borselen; Kit Wun Kathy Cheung; Noha A Yousri; Karsten Suhre; Jason M Kinchen; Eric Boerwinkle; Roshanak Irannejad; Bing Yu; Kathleen M Giacomini Journal: PLoS Genet Date: 2019-09-25 Impact factor: 5.917