Cloning, Sequencing and Expression of a Porcine Intestinal Peptide Transporter in a Mammalian Cell Line

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dc.contributor.authorKlang, Judith Elisaen
dc.contributor.committeechairWong, Eric A.en
dc.contributor.committeememberWebb, Kenneth E. Jr.en
dc.contributor.committeememberJiang, Honglinen
dc.contributor.departmentAnimal and Poultry Sciencesen
dc.date.accessioned2014-03-14T20:30:21Zen
dc.date.adate2003-05-30en
dc.date.available2014-03-14T20:30:21Zen
dc.date.issued2002-12-10en
dc.date.rdate2004-05-30en
dc.date.sdate2003-01-10en
dc.description.abstractAbsorption of dietary proteins can be met through the uptake of free amino acids or as small peptides. A peptide transport protein, PepT1, is responsible for the absorption of intact peptides arising from digestion of dietary proteins. PepT1 is driven by a H+-coupled transport system that allows for the absorption of small peptides through the intestinal brush border membrane. Screening of a porcine intestinal cDNA library with a sheep PepT1 cDNA probe resulted in the identification of three porcine PepT1 (pPepT1) cDNAs of varying sizes and sequences. Each variant cDNA isolated was cloned into a mammalian expression vector, sequenced, and expressed in Chinese hamster ovary (CHO) cells. Peptide transport was assessed by uptake studies using the radiolabeled dipeptide [3H]-Gly-Sar. Only one of the three cDNAs encoding for a protein of 708 amino acids induced H+-dependent peptide transport activity. Through computer analysis, a putative protein structure for pPepT1 was developed. The transporter has an unusual 13 transmembrane structure with the N-terminus located extracellularly and the C-terminus located intracellularly. Seven glycosylation sites and three protein kinase C phosphorylation sites are located throughout the protein. Expression of pPepT1 activity in CHO cells had a optimal peptide uptake at 18-24 hours. The transporter showed optimal uptake at a pH of 5.5-6.0. Eighteen different unlabeled dipeptides and tripeptides were found to inhibit the uptake of [3H] -Gly-Sar in competition studies. The IC50 of 13 of the dipeptides and two tripeptides ranged between 0.015 to 0.4 mmol/L. The exceptions were Lys-Lys, Arg-Lys, and Lys-Trp-Lys, which showed IC50 values greater than 1.37 mmol/L and appear to be poor substrates for pPepT1. All three of the tetrapeptides examined showed very high IC50 values and inhibition of the uptake of Gly-Sar was too small to measure even at a 10mM concentration. Dipeptides and tripeptides appear to be substrates for the porcine intestinal peptide transporter while tetrapeptides do not appear to be transported.en
dc.description.degreeMaster of Scienceen
dc.identifier.otheretd-01102003-135233en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-01102003-135233/en
dc.identifier.urihttp://hdl.handle.net/10919/30880en
dc.publisherVirginia Techen
dc.relation.haspartpPepT1thesis.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectPeptide Transporten
dc.subjectPigen
dc.subjectPepT1en
dc.titleCloning, Sequencing and Expression of a Porcine Intestinal Peptide Transporter in a Mammalian Cell Lineen
dc.typeThesisen
thesis.degree.disciplineAnimal and Poultry Sciencesen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

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