The amyloid-β precursor protein (APP) undergoes proteolytic cleavage by α-, β-, and γ-secretases, to determine its fate in Alzheimer's disease (AD) pathogenesis. Recent findings suggest a possible role of O-glycosylation in APP's proteolytic processing. Therefore, we synthesized native and Swedish-double-mutated APP (glyco)peptides with Tyr 681 -O-GalNAc. We studied conformational changes and proteolytic processing using circular dichroism (CD) spectroscopy and enzyme cleavage assay, respectively. CD analysis was carried out in four solvent systems to evaluate peptide environment and O-glycosylation induced conformational changes. The Swedish mutation and Tyr 681 -O-GalNAc were the key factors driving conformational changes. Furthermore, the level of αand β-secretase activity was increased by the presence of mutation and this effect was more pronounced for its glycosylated analogues. Our results suggest that O-glycosylation of Tyr 681 can induce a conformational change in APP and affect its proteolytic processing fate toward the amyloidogenic pathway.
The amyloid-β precursor protein (APP) undergoes proteolysis by β- and γ-secretases to form amyloid-β peptides (Aβ), which is a hallmark of Alzheimer’s disease (AD). Recent findings suggest a possible role of O-glycosylation on APP’s proteolytic processing and subsequent fate for AD-related pathology. We have previously reported that Tyr681-O-glycosylation and the Swedish mutation accelerate cleavage of APP model glycopeptides by β-secretase (amyloidogenic pathway) more than α-secretase (non-amyloidogenic pathway). Therefore, to further our studies, we have synthesized additional native and Swedish-mutated (glyco)peptides with O-GalNAc moiety on Thr663 and/or Ser667 to explore the role of glycosylation on conformation, secretase activity, and aggregation kinetics of Aβ40. Our results show that conformation is strongly dependent on external conditions such as buffer ions and solvent polarity as well as internal modifications of (glyco)peptides such as length, O-glycosylation, and Swedish mutation. Furthermore, the level of β-secretase activity significantly increases for the glycopeptides containing the Swedish mutation compared to their nonglycosylated and native counterparts. Lastly, the glycopeptides impact the kinetics of Aβ40 aggregation by significantly increasing the lag phase and delaying aggregation onset, however, this effect is less pronounced for its Swedish-mutated counterparts. In conclusion, our results confirm that the Swedish mutation and/or O-glycosylation can render APP model glycopeptides more susceptible to cleavage by β-secretase. In addition, this study sheds new light on the possible role of glycosylation and/or glycan density on the rate of Aβ40 aggregation.
Objective To synthesize carbohydrate building blocks bearing the sialyl‐Tn antigen, and incorporate them into a MUC1 peptide backbone for structural and functional studies with endogenous lectins. Mucin 1 (MUC1), a transmembrane mucin protein, is a well‐known biomarker of different cancer types. The aberrantly glycosylated forms of MUC1 display specific truncated tumor‐associated carbohydrate antigens (TACAs) on the surface of the cell such as: Tn (GalNAc), T (Galβ1,3GalNAc), sTn (NeuAcα2,6GalNAc), and sT (2,3‐sT and 2,6‐sT). These truncated glycan structures, often capped by sialic acid, play a key role in tumor initiation, progression, and metastasis, and accumulating evidence suggests their involvement in tumor escape from immune defenses through interaction with endogenous carbohydrate‐binding proteins (lectins). Macrophage galactose binding lectin (MGL) is a C‐type lectin receptor found on antigen‐presenting cells (APCs) which facilitates the uptake of carbohydrate antigens for antigen presentation, modulating the immune response homeostasis, autoimmunity, and cancer. The main goal of this research is to evaluate the interactions between Tn and sTn antigen of tumor‐associated MUC1 with MGL and to characterize the molecular origin of the high affinity and selectivity of this endogenous lectin for its natural MUC1 ligands. This interaction plays a crucial role in tumor immune evasion. Nonetheless, MGL targeting is an attractive approach to enhance cancer vaccine efficacy. It is of crucial importance to understand the fine‐intricate nature of immune cells to either augment the anti‐tumorigenic effects or minimize the pro‐tumorigenic effects. The aim of this research is to synthesize structurally well‐defined chemical probes, mono‐ and multiple‐glycosylated MUC1 peptide models carrying the Tn or sTn O‐glycans, that allow for the control of the complexity of the chemical space of the multivalent ligands. For this purpose, a concise scheme was developed for the synthesis of the sTn antigen building blocks to be used in the solid‐phase synthesis of glycopeptide models. We employed the use of orthogonally protected phenyl thioglycoside glycosyl donors in the presence TfOH/NIS or TMSOTf/NIS as promoter system. The glycosylated threonine and sialylated glycosylated threonine were synthesized in a relatively high yield, 85% and 60%, but with moderate stereoselectivity, α/β ratio 3.5:1 and 2:1, respectively. The subsequent reduction and N‐acetylation of an azido group resulted in targeted glycosylated amino acid building block that was used in the solid‐phase peptide synthesis of MUC1 sTn bearing glycopeptides. The secondary structure of synthesized peptides was determined by circular dichroism (CD) spectroscopy. The synthetic efforts will be followed by mechanistic studies including isothermal titration calorimetry (ITC) and AFM. Support or Funding Information This work was supported by the National Institutes of Health Grant CA242351 to M. Cudic.
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