(489.13) Synthesis and Conformational Analysis of APP O-Glycopeptides Bearing the Tn Antigen

Poster Board Number: A83

Mare Cudic (Florida Atlantic University), YashoNandini Singh (Florida Atlantic University)

Objective:  To perform a conformational analysis of APP glycopeptides by CD spectroscopy and evaluate the impact of site specific mucin-type O-glycosylation and/or unique patterns of O-glycan clusters proximal to secretase cleavage sites on the APP’s secondary structure.

Alzheimer’s disease (AD) is characterized by the accumulation of the neurotoxic deposits of β-amyloid peptide (Aβ) within the brain along with hyperphosphorylated and cleaved forms of the microtubule-associated tau protein. Genetic, biochemical, and behavioral research suggest that physiologic generation of the Aβ forming fibrils stems from the proteolytic processing of the amyloid precursor protein (APP), a type 1 transmembrane glycoprotein, by β- and γ-secretases. A small percent of AD is also caused by single genetic mutations that are passed down through families known as familial AD (FAD). The Swedish double mutation (Lys670Asn/Met671Leu), located near the N-terminus region of Aβ results in a major increase in the total output of Aβ40 and Aβ42 by providing a better substrate for the β-secretase enzyme. Recent findings suggest possible role of O‐glycosylation in APP’s proteolytic processing. However, due to the inherited complexity of protein glycosylation, the effect of O-glycans on APP function, and its subsequent role in AD pathogenesis, remains challenging. Our preliminary data suggest that a unique type of O-glycosylation, on the phenolic hydroxyl group of the tyrosine (Tyr681) can induce conformational change in APP and affect its proteolytic processing fate towards the amyloidogenic pathway. Intrigued by the number of mucin-type O-glycan sites that have been reported for APP in the proximity of β-secretase site, we focused on three sites in this study, Thr663, Ser667 and Tyr681, modified with the simplest mucin-type structure, O-α-GalNAc (Tn), to obtain an initial account of its direct impact on protein’s secondary structure. The APP glycopeptide models incorporating β- and α-secretases sites, along with their Swedish-double mutated versions, and the non-glycosylated counterparts were synthesized by Fmoc solid phase peptide synthesis approach. Circular dichroism (CD) was used to analyze the secondary structure of peptides and evaluate the role of peptide environment on inducing conformational changes. The spectra were further analysed for secondary structure estimations by Beta Structure Selection (BeStSel) method, specifically designed for the analysis of beta sheet-rich proteins. Our results show that conformation is strongly dependent on external conditions in the peptide environment such as buffer ions and solvent polarity as well as its internal modifications such as sequence length, O-glycosylation, and Swedish mutation.