The molecular characterization of tau glycoprotein/glycan interactions and phosphorylation in alzheimer’s disease

Abstract

Alzheimer’s disease (AD) is the 7th leading cause of death in the United States as of 2021, with poorly understood etiology and limited therapeutic measures. Of the various hypotheses for AD pathogenesis, herpes virus has grown as an attractive contributing agent due to a myriad of correlative evidence. Herpes virus shares an intriguing connection to one of the pathological hallmarks of AD, hyperphosphorylated Tau; both are internalized by neurons via a 3-O-sulfated heparan sulfate (3-O-S HS) chain on heparan sulfate proteoglycans. These are two of the only eight known proteins to specifically recognize the 3-O-S moiety on HS. However, to investigate the potential ternary interaction of AD-related Tau, herpes virus, and heparan sulfate, we must first define each binary interactions of our biomolecular triad, as two of these interfaces, herpes viral glycoprotein D and heparan sulfate, and phosphorylated Tau and heparan sulfate, are not fully understood. Herein, we present here the results of investigations into gD-HS interface, the ternary interaction between gD-HS-Tau, and the impact of phosphorylation on the Tau-HS interface. For the gD-HS interface, we utilized various biophysical assays to clarify the protein regions involved in HS binding and to investigate the glycan preferences of gD. We found that the first 22 amino acids of gD are disposable for HS binding, contrary to previous literature. We also show that gD has a strong preference for 6-O-sulfation, a variable requirement for 2-O-sulfation, and recognizes 3-O-sulfation using SPR competition assays, SV-AUC, and glycan microarray. Furthermore, a de-glycosylated mutant of gD revealed a different dissociation mode when interacting with heparin, pointing to the involvement of glycosylation in the gD-HS interaction. SPR, affinity chromatography, and SV-AUC provide indirect and direct evidence of a ternary complex between full-length Tau, gD285, and an HS analog heparin. Phosphorylation is the most important post-translational modification of Tau in Alzheimer’s disease. We accomplished phosphorylation of Tau by co-expressing a kinase, glycogen synthase kinase 3β (GSK3β) and Tau in E. coli. Phosphorylation by GSK3β did not impact Tau’s affinity for heparin, a sulfated analog of heparan sulfate, as shown by SPR kinetic assays and heparin affinity chromatography. However, phosphorylation did change the conformational dynamics of Tau and its complex with heparin as shown by SV-AUC. Furthermore, SPR competition assays and glycan microarray revealed that phosphorylation shifted the glycan preferences of Tau away from 3-O-S. This may have implications for the formation of ternary complexes of HS, pTau, and other proteins in AD pathogenesis. This dissertation provides detailed characterization on two important protein-glycan interactions, glycoprotein D/heparan sulfate and phosphorylated Tau/heparan sulfate, and investigates a ternary complex between glycoprotein D/heparan sulfate/Tau which has implications for Alzheimer’s disease, herpes viral entry, and drug development.