title: The human colonic thiamine pyrophosphate transporter (hTPPT) is a glycoprotein and N-linked glycosylation is important for its function.
abstract: The recently identified human thiamine pyrophosphate transporter (hTPPT; product of the SLC44A4 gene) is responsible for absorption of the microbiota-generated TPP in the large intestine. The hTPPT is highly expressed in the colon, but not in other regions of the intestinal tract and is localized exclusively at the apical membrane domain of epithelia. The hTPPT protein is predicted to have multiple TM domains with a number of putative N-glycosylation sites, but it is not known if the protein is actually glycosylated, and if so at which site, and their role in the functionality of the transporter. Using several approaches including inhibiting de novo N-glycosylation in human colonic epithelial NCM460 cells with tunicamycin as well as enzymatic de-glycosylation, we show that the hTPPT protein is, indeed, a glycoprotein. Glycosylation of hTPPT was shown, by mean of site-directed mutagenesis, to occur at Asn(69), Asn(155), Asn(197), Asn(393), and Asn(416). However, only N-glycosylation at Asn(69), Asn(155), and Asn(393) appeared to be important for transporter functionality possibly through an effect on protein conformation and/or interaction with its ligand (but not through changes in expression at the cell membrane as determined by live cell confocal imaging). Results of this study showed, for the first time, that the hTPPT is glycosylated and that N-linked glycosylation occurs at multiple sites with some of them being important for function. The results also provide an indirect support for a membrane topology for hTPPT with 10 transmembrane domains as predicted by the TMHMM transmembrane helixes prediction program.
authors.affiliation: Department of Medicine, University of California, Irvine, CA 92697, United States; Department of Physiology/Biophysics, University of California, Irvine, CA 92697, United States; Department of Veterans Affairs Medical Center, Long Beach, CA 90822, United States.
authors.forename: Svetlana M
authors.initials: SM
authors.lastname: Nabokina
authors.affiliation: Department of Medicine, University of California, Irvine, CA 92697, United States; Department of Physiology/Biophysics, University of California, Irvine, CA 92697, United States; Department of Veterans Affairs Medical Center, Long Beach, CA 90822, United States.
authors.forename: Veedamali S
authors.initials: VS
authors.lastname: Subramanian
authors.affiliation: Department of Medicine, University of California, Irvine, CA 92697, United States; Department of Physiology/Biophysics, University of California, Irvine, CA 92697, United States; Department of Veterans Affairs Medical Center, Long Beach, CA 90822, United States. Electronic address: hmsaid@uci.edu.
authors.forename: Hamid M
authors.initials: HM
authors.lastname: Said
mesh_terms: D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003106:Colon / Q000737:chemistry* / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary
chemical_list: D006023:Glycoproteins; D026901:Membrane Transport Proteins; C583905:SLC44A4 protein, human
keywords: Glycosylation; Mutation; Thiamine pyrophosphate; Transport; Uptake
references.citation: Gastroenterology. 2010 May;138(5):1802-9
references.pmid: 19879271
references.citation: PLoS One. 2010;5(10):e13616
references.pmid: 21049048
references.citation: Nature. 2011 May 12;473(7346):174-80
references.pmid: 21508958
references.citation: Biochim Biophys Acta. 2011 Aug;1808(8):2073-80
references.pmid: 21570947
references.citation: Biochem J. 2011 Aug 1;437(3):357-72
references.pmid: 21749321
references.citation: Cent Nerv Syst Agents Med Chem. 2012 Jun;12(2):70-81
references.pmid: 22483272
references.citation: Am J Physiol Gastrointest Liver Physiol. 2012 Aug 1;303(3):G389-95
references.pmid: 22628036
references.citation: Mol Aspects Med. 2013 Apr-Jun;34(2-3):646-54
references.pmid: 23506897
references.citation: Am J Physiol Gastrointest Liver Physiol. 2013 Nov;305(9):G601-10
references.pmid: 23989008
references.citation: J Biol Chem. 2014 Feb 14;289(7):4405-16
references.pmid: 24379411
references.citation: Metab Brain Dis. 2014 Dec;29(4):1069-82
references.pmid: 24590690
references.citation: World Rev Nutr Diet. 2015;111:30-7
references.pmid: 25418386
references.citation: Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1835-40
references.pmid: 10677542
references.citation: Proc Soc Exp Biol Med. 2000 Sep;224(4):246-55
references.pmid: 10964259
references.citation: J Biol Chem. 2001 Jan 19;276(3):2168-73
references.pmid: 11036075
references.citation: Am J Physiol Cell Physiol. 2001 Sep;281(3):C786-92
references.pmid: 11502555
references.citation: Curr Mol Med. 2001 May;1(2):197-207
references.pmid: 11899071
references.citation: J Biol Chem. 2003 Feb 7;278(6):3976-84
references.pmid: 12454006
references.citation: Nat Med. 2003 Mar;9(3):294-9
references.pmid: 12592403
references.citation: Diabetes. 2003 Aug;52(8):2110-20
references.pmid: 12882930
references.citation: Am J Physiol Gastrointest Liver Physiol. 2004 Mar;286(3):G491-8
references.pmid: 14615284
references.citation: Anal Biochem. 1989 Aug 1;180(2):195-204
references.pmid: 2510544
references.citation: J Neurochem. 1995 May;64(5):2013-21
references.pmid: 7722487
references.citation: J Neuropathol Exp Neurol. 1999 Sep;58(9):946-58
references.pmid: 10499437
references.citation: Am J Physiol. 1999 Oct;277(4 Pt 1):C645-51
references.pmid: 10516094
references.citation: Mol Pharmacol. 2005 Mar;67(3):868-76
references.pmid: 15576633
references.citation: Am J Physiol Cell Physiol. 2006 Jan;290(1):C1-C10
references.pmid: 16338974
references.citation: Biochim Biophys Acta. 2008 Jun;1778(6):1407-14
references.pmid: 18405659
references.citation: Biochem Biophys Res Commun. 2008 Sep 12;374(1):123-7
references.pmid: 18619416
references.citation: Basic Clin Pharmacol Toxicol. 2008 Nov;103(5):482-6
references.pmid: 18715237
references.citation: Am J Physiol Renal Physiol. 2009 Mar;296(3):F459-69
references.pmid: 18971212
references.citation: Alcohol Alcohol. 2009 Mar-Apr;44(2):141-7
references.pmid: 19151161
references.citation: FEBS J. 2009 Jun;276(11):2917-25
references.pmid: 19490098