Investigations of tetraspanin functions using large extracellular loops

Abstract

This thesis describes our characterization of a specific tetraspanin domain: the large extracellular loop (LEL). Tetraspanins are involved in cellular migration, adhesion, and metastasis, sperm-egg fusion, and viral infectivity. The large extracellular loop domain is the major extracellular domain of tetraspanins and the binding of a monoclonal antibody against the tetraspanin CD9 serves to inhibit fertilization, consistent with the CD9-null mouse model. The first area of focus in this thesis is the characterization of the murine CD9-LEL domain. We present a methodology to express and purify the mCD9-LEL to homogeneity. Biophysical characterization of the mCD9-LEL protein reveals that it is an autonomously folding, [alpha]-helical dimer. Mutagenesis over much of the mCD9-LEL protein reveals that it is composed of two subdomains: a dimerization subdomain and a variable subdomain proposed to mediate heterotypic interactions. These results suggest both a means for exploring endogenous tetraspanins functions and a mechanism by which tetraspanins may oligomerize. Surprisingly, we were not able to detect oligomerization of the intact CD9 molecule, in discordance with our biophysical data on the mCD9-LEL.

(cont.) In the latter part of this thesis, we expand our methodology to purify and characterize three different tetraspanins-LELs, the hCD9-LEL, the hCD63-LEL, and the hCD8 1-LEL. These tetraspanins-LELs all exhibit similar characteristics to the mCD9-LEL, consistent with a published crystal structure of the hCD81-LEL. Lastly, we demonstrate the ability of our tetraspanin-LEL proteins to bind integrins, to inhibit sperm-egg fusion, and to inhibit hepatitis C viral infectivity. Taken as a whole, these studies present novel, biophysically validated tetraspanins-LELs that lend insight into endogenous tetraspanins functions.