Introduction
A progressive neurological disease, which mainly affects the senile population, Alzheimer’s disease (AD) is one of the most prominent form of dementia [1]. The advances in molecular biology, structural biology and neurobiology in the recent past have enhanced our understanding of the pathological process which underlies this progressive neurodegenerative disease, but still we do not have a complete understanding of the molecular mechanisms associated with the disease. An estimated 55 million people across the globe suffer from Alzheimer’s and other related dementia, with no disease modifying drugs or therapy still available [2]. The current strategy of treatments revolves around providing symptomatic relief with cognition enhancement drugs and early diagnosis with biomarkers [3]. Central to the neuropathology of AD is the loss of synapse and synaptic plasticity, which reduce the ability of individuals to make and store new memories [4]. The two most prominent hypotheses which help explain this cognitive decline are the amyloid cascade hypothesis and the tau hyperphosphorylation hypothesis [5, 6].
Tau protein also called microtubule associated protein tau, is an intrinsically distorted protein (IDP), which does not have a complete secondary or tertiary structure when it’s in the unbound form. Encoded by the MAPT gene located on chromosome 17, with an mRNA of 16 exons, tau protein occurs in different isoforms in the human brain [7, 8]. Alternate splicing of the exons 2, 3 & 10 leads to the formation of six different isoforms with chain lengths ranging from 352-441 amino acids [9, 10]. The major role of tau protein in neurons is that they bind and stabilize the cytoskeletal fragments called microtubules. This in turn helps in the proper intracellular trafficking [11, 12]. Once bound to the microtubules, the tau protein will have residue specific secondary structure which stabilizes the binding [13, 14]. Hyperphosphorylation of tau protein have been attributed as one of the main causes of AD because of its ability to destabilize the tau-microtubule binding and induce the formation of neurofibrillary tangles [15, 16].
The structure of the longest tau isomer with 441 amino acids can be divided into three different domains, the projection domain, proline rich domain and microtubule binding domain. The projection domain is the region that is close to the N terminal and has two sub regions called the N1 & N2 (1-165), which are not attached but projected away from the microtubules. The proline rich domain extends from amino acid 166 to 242 having a high density of proline residues. The microtubule binding region consists of four repeat regions (R1, R2, R3 & R4) extending from residues 243 to 367, which binds to the microtubules and have certain residue specific secondary structures, followed by the C terminal region [17, 18].
Post translational modifications (PTMs) have been associated with the pathophysiology of many intrinsically distorted proteins like tau [19]. Alquezer and coworkers have summarized various PTMs on tau protein where they have given a detailed review of all modifications along with the probable modification sites [20]. The most widely studied PTM associated with AD is tau hyperphosphorylation. Glycosylation is another important PTM on tau, which has been shown to be correlated with AD [21, 22]. Tau undergoes O-GlcNAcylation, which involves an attachment of the β-N-acetylglucosamine (GlcNAc) monosaccharide to either Serine or Threonine residues, and it has been found that the O-GlcNAcylation levels have been reduced in AD brains. Since both phosphorylation and O-GlcNAcylation takes place on serine/threonine residues, the presence of one is thought to hinder the other [23, 24]. Another interesting PTM is N-glycosylation of the tau protein. It was found that tau protein obtained from AD brains have been N-glycosylated but not those from the healthy brains [25]. N-glycosylation of tau was an unusual observation as the machinery needed for tau N-glycosylation, i.e., endoplasmic reticulum and Golgi are located on pathways for secreted proteins and usually not accessible to tau protein, which is an intracellular protein [26-28]. It was also observed that N-glycosylation takes place on asparagine residues having sequence N–X–S/T (where X can be any amino acid except proline) [29] and the three potential N-glycosylation sites of human tau protein are Asn167, Asn359, and Asn410; numbering based on longest tau isoform [26].
The fact that tau is N-glycosylated in AD brains and not in healthy brains, suggested the possible involvement of this PTM in AD pathology. It was also found that paired helical filament (PHF) tau when deglycosylated, changed the structure from the folded state to straight filaments [30]. A recent in vitro study conducted by Losev and coworkers using site specific mutation of putative N-glycosylation sites in tau protein expressed in Drosophila and SH-SY5Y cells, suggested the involvement of the three potential N-glycosylation sites in tau aggregation. SH-SY5Y cells and transgenic Drosophila expressing tau protein with asparagine to glutamine mutants at the putative N-glycosylation sites were generated, and it was found that N-glycosylation at Asn167 & Asn359 enhances the tau aggregation, while Asn410 reduces tau aggregation. They also found that expression of the N359Q hTau mutant leads to amelioration of AD-like symptoms, reduced neurodegeneration and importantly, N359Q is the only mutant that improved the lifespan of the flies [31].
Even though these in vitro results suggest that N glycosylation of tau protein at specific residues (Asn359) leads to tau aggregation, the exact mechanism by which it occurs is still not fully understood. Understanding the N-glycosylation induced effects on protein dynamics of tau can help us gain some insights into misfolding patterns and some of the important residue interactions. This in turn can help the scientific community to further explore the pathogenic role played by N-glycosylation in AD and identify new targets in the pathogenic N-glycosylation pathway. Here, in this study we used molecular dynamics (MD) simulations coupled with essential dynamics, free energy landscape analysis (FEL) & residue interaction network analysis to evaluate the effects caused by the N-glycosylation on Asn-359 residue, which forms part of the microtubule binding region of the tau protein. Through this study, we aimed to study whether N-glycosylation can induce tau protein folding and what are the important residue specific interactions which helps in the process. We hope that the information that we gain from this study can be used in future research to build new anti-tau aggregation compounds as disease modifying therapeutics against AD and other related tauopathies.