Studies on the human intestinal di-/tripeptide transporter HPT-1 as a potential carrier for orally administered drugs
Abstract
The purpose of the present study was to isolate and characterize the human intestinal di-/tripeptide transporter HPT-1 and to establish functional assays using isolated HPT-1. In addition, molecular recognition between the immobilized artificial membranes and solute molecules was also investigated in this study. HPT-1 was isolated for the first time from Caco-2 cell membranes and human intestinal brush border membranes. An efficient two-step procedure has been developed to purify sufficient quantity of HPT-1 for functional assays. In the first step of purification, a lectin was selected to remove over 95% of contaminating proteins. The second step uses either IAM (immobilized artificial membrane) chromatography or immobilized monoclonal antibodies that block the transport function of HPT-1. Amino acid sequencing confirmed that the isolated protein was HPT-1. A model of the membrane topology of HPT-1 was proposed based on amino acid sequence analysis. The purified HPT-1 was functional after being reconstituted in liposomes. Moreover, HPT-1 isoforms have been identified in different human intestinal segments. This study establishes the foundation for future studies on substrate structural requirements of HPT-1. Molecular dynamics (MD) simulations have been used in this study to provide molecular insights into the interactions involved in solute partitioning into the IAM surface. It was observed during the MD simulations that IAM surfaces not only model all interactions involved in solute partitioning into phospholipid membranes, but are also flexible enough to form solute cavities. Inside the solute cavities in IAM surfaces, the solute molecules adopt specific orientations which are determined by interactions between the solutes and IAM phospholipids. Consequently, different solutes have different solvent accessible surface areas and contact with different parts of phospholipids. All these factors profoundly affect the overall interaction energy between the solutes and their boundary phospholipids, and regulate solute partitioning in fluid and immobilized membranes. This is the first study to demonstrate the importance of the unique microstructure of IAM surfaces, in addition to their bulk interfacial properties, in predicting drug-membrane interaction and drug transport across biological membranes.
Degree
Ph.D.
Advisors
Pidgeon, Purdue University.
Subject Area
Biochemistry|Pharmacology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.