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

Absorption 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.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

masters

Discipline thesis:degree_discipline

Animal and Poultry Sciences

Department dc:contributor.department

Animal and Poultry Sciences

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

2002

Author dc:creator

• Klang, Judith Elisa

Chair dc:contributor.committeechair

• Wong, Eric A.

Committee members dc:contributor.committeemember

• Webb, Kenneth E. Jr.
• Jiang, Honglin

• Peptide Transport
• Pig
• PepT1

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Dc Identifier Other

etd-01102003-135233

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/30880