Structure-Function Analysis of Lactoferrin

California State University Long Beach, Long Beach CA

Investigators

Abstract

DESCRIPTION (provided by applicant): The goal of this project is to understand the in vivo functions of lactoferrin (Lf). Lf is an important Fe-binding protein widely distributed in exocrine fluids that helps regulate inflammation, hematopoiesis, and Fe-transport. A full explanation of its structure-function relationships requires an understanding of how it binds receptors and other target molecules. This study focuses on determining how Lf interacts with the asialoglycoprotein receptor (ASGP-R) and with specfic serum Lf-binding proteins. Specific Aim 1 is the analysis of serum Lf-binding proteins. A Lf-binding protein from human serum (p165) will be identified and its interaction with Lf examined. Lf-binding proteins from non-human sera will be identified. Specific Aim 2 is the analysis of Lf glycosylation mutants. The hypothesis that Lf interacts with ASGP-R in a glycan-independent manner will be tested. Bovine Lf cDNAs mutated to remove the consensus N-linked glycosylation sites in the C-lobe will be expressed in the yeast Pichia pastoris. Recombinant Lfs will be examined for Fe-binding and ASGP-R binding activity. Specific Aim 3 is the analysis of the ASGP-R binding activity of recombinant Lf-transferrin (Tf) hybrid proteins. A series of human Lf-Tf hybrid cDNAs have been constructed in which various sub-domains of the two proteins have been swapped. These hybrid constructs will be expressed in insect Sf9 cells using a baculovirus expression system. Purified recombinant Lf-Tf hybrids will be examined for Fe-binding, carbohydrate content, secondary structure alterations, and ASGP-R binding activity. These studies will test the hypothesis that the ASGP-R recognition elements are located in the C2 sub-domain of Lf. Lt sub-domains, which when removed from Lf-Tf hybrids elicit a loss-of-binding phenotype, will be placed into a Tf background to determine if they confer a gain-of-binding phenotype on Tf.