Abstract
Tetraspan proteins are significantly enriched in the membranes of exosomal vesicles (EVs) and their extracellular domains are attractive targets for engineering towards specific antigen recognition units. To enhance the tolerance of a tetraspanin fold to modification, we achieved significant thermal stabilization of the human CD81 large extracellular loop (hCD81 LEL) via de novo disulfide bonds. The best mutants were shown to exhibit a positive shift in the melting temperature (Tm) of up to 25 °C. The combination of two most potent disulfide bonds connecting different strands of the protein resulted in a mutant with a Tm of 109 °C, 43 °C over the Tm of the wild-type hCD81 LEL. A peptide sequence binding to the human transferrin receptor (hTfr) was engrafted into the D-segment of the hCD81 LEL, resulting in a mutant that still exhibited a compact fold. Grafting of the same peptide sequence between helices A and B resulted in a molecule with an aberrant profile in size exclusion chromatography (SEC), which could be improved by a de novo cysteine bond connecting both helices. Both peptide-grafted proteins showed an enhanced internalization into the cell line SK-BR3, which strongly overexpresses hTfr. In summary, the tetraspan LEL fold could be stabilized to enhance its amenability for engineering into a more versatile protein scaffold.
Highlights
In the last decade, the research into exosomes has intensified as they have been recognized as significant mediators of cell-to-cell communication [1]
This motif is conserved among the protein members of the tetraspanin family [14] and the oxidation of cysteine bonds is a prerequisite for high-affinity binding of the E2 envelope protein of the hepatitis C virus (HCV), the natural ligand of CD81 [15]
After blocking with 5% bovine serum albumin (BSA)-PBS for 1 h at room temperature (RT), supernatants of HEK293-6E cells transfected with human CD81 (hCD81) LEL variants or purified variants of hCD81 LEL diluted in 2.5% BSA-PBS were allowed to bind for 1 h at RT
Summary
The research into exosomes has intensified as they have been recognized as significant mediators of cell-to-cell communication [1]. The large extracellular loop of CD81, topologically located between transmembrane domains 3 and 4, is characterized by five helical elements forming a mushroom-like structure [12,13] stabilized by two pairs of cysteines This motif is conserved among the protein members of the tetraspanin family [14] and the oxidation of cysteine bonds is a prerequisite for high-affinity binding of the E2 envelope protein of the hepatitis C virus (HCV), the natural ligand of CD81 [15]. Segment D of CD81 should be able to guide specific homomeric clustering [22] To enhance their potential as the next-generation therapeutic carriers, exosome-mediated delivery systems need to be further developed, especially to improve their inherently low efficiency of cellular uptake, which can be achieved by the engineering of exosomal membrane proteins. A stabilizing disulfide bond connecting helices A and B was shown to enhance the biophysical properties of the protein modified in this way
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