Discovery of conserved motifs in MARCO through evolutionary analyses and molecular biology

Abstract

<p>Of the pattern recognition receptors involved in innate immunity, the class A Scavenger receptors are involved in the recognition and clearance of bacteria, yeast, and senescent molecules. Previous research has implicated the intracellular region of these receptors as essential for the clearance of these substances via endocytosis. In this work, I used a bioinformatic approach to define the evolutionary history of the class A Scavenger receptor family while elucidating areas of conservation within the cytoplasmic domains of these proteins. With this information, in addition to further predictions of post translational modifications and potential docking motifs for interacting proteins, I conducted molecular biology experiments to study the in vitro functionality of the macrophage receptor with collagenous domain (MARCO), a member of the class A Scavenger receptors.</p> <p>Evolutionary analyses of the 5 class A Scavenger receptors identified a shared ancestry between these proteins and allowed me to postulate that 4 distinct gene du- plication events in addition to subsequent domain fusions, internal repeats, and dele- tions are responsible for the diverse protein structures and functions of this family. Despite some variation in domain structure, I found highly conserved regions across all 5 members, including a negatively charged region in the cytoplasmic domain. Further analyses of MARCO across organisms identified other conserved regions, in- cluding 2 residues predicted to be ubiquitinated, sumoylated, or phosphorylated by in silico predictive methods. However, molecular biology experiments demonstrated that these post translational modifications to not occur in the steady state. Addi- tional in vitro experiments, including isolations of MARCO and an artifical construct containing only the intracellular regions of the protein, were unable to identify any candidate adaptor binding proteins. Further research is needed to determine whether modifications in this region occur in the presence of bound ligands and/or known co-receptors.</p>