title: Stable Expression and Characterization of an Optimized Mannose Receptor.
abstract: The mannose receptor (MR) is a macrophage surface receptor that recognizes pathogen associated molecular patterns (PAMPs) from a diverse array of bacterial, fungal and viral pathogens. Functional studies of the MR are hampered by the scarcity of human cell lines that express the receptor. Current model systems available for the study of MR biology often demonstrate low levels of expression and do not retain many of the classical MR properties. Although several laboratories have reported transient and stable expression of MR from plasmids, preliminary data from our laboratory suggests that these plasmids produce a protein that lacks critical domains and is often not stable over time. In this current report we describe the generation and characterization of a novel human codon-optimized system for transient and stable MR expression. Rare codons and sequences that contribute to mRNA instability were modified to produce mRNA that is qualitatively and quantitatively improved. Confocal imaging of the transient and stably expressed optimized receptor demonstrates a distribution consistent with previous reports. To demonstrate the functional characteristics of the optimized receptor, we further show that the introduction of codon-optimized MR plasmid can confer MR-associated phagocytosis ofS. aureusto non-phagocytic HeLa cells. We show that three molecules participate in the engagement and internalization ofS. aureus. MR was found to colocalize with Toll-like receptor 2 (TLR2) and Rab5 following exposure to pHrodo-stainedS. aureus, suggesting cooperation among the three molecules to engage and internalize the bacterial particle. This study describes a transfection capable, optimized MR receptor with functional characteristics similar to the wild type receptor and further demonstrates a new system for the continued study of MR biology and function.
authors.affiliation: Department of Veterans Affairs Medical Center, USA ; Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville TN 37212, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Vigerust
authors.affiliation: Department of Veterans Affairs Medical Center, USA.
authors.forename: Sherell
authors.initials: S
authors.lastname: Vick
authors.affiliation: Department of Veterans Affairs Medical Center, USA ; Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville TN 37212, USA.
authors.forename: Virginia L
authors.initials: VL
authors.lastname: Shepherd
keywords: Macrophages; Mannose receptor