Carbohydrates are often utilized to provide hydrophilicity and hydroxyl-based hydrogen bonds in self-assembling glycopeptides, affording versatile scaffolds with wide applicability in biomedical research. However, how stereochemistry of carbohydrates impacts the self-assembly process remains unclear.Here we have established a dimeric tyrosine-rich glycopeptide system for probing the corresponding hydrogelating behavior under the influence of site-and stereospecific glycosylations. Comparison of 18 glycoforms bearing monosaccharides at Tyr 4 and Tyr 4′ shows that the glycopeptides with either αor β-anomers exhibit contrary gelating abilities, when the glycan moieties contain axial hydroxyl groups. A high-resolution X-ray crystallographic structure of the β-galactose-containing gelator, along with other results from spectroscopic, microscopic, and rheological experiments, indicate an unusual carbohydrate−aromatic CH−π bonding that promotes glycopeptide self-assembly. These mechanistic findings, particularly evidence obtained at the angstrom scale, illuminate an unconventional role that carbohydrates can play in building supramolecules. Potential biomaterials exploiting the CH−π bond-based stabilization, as exemplified by an enzymeresistant hydrogel, may thus be developed.

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Brain accumulation of amyloid-β (Aβ) peptides (resulting from a disrupted balance between biosynthesis and clearance) occurs during the progression of Alzheimer’s disease (AD). Aβ peptides have diverse posttranslational modifications (PTMs) that variously modulate Aβ aggregation into fibrils, but understanding the mechanistic roles of PTMs in these processes remains a challenge. Here, we chemically synthesized three homogeneously modified isoforms of Aβ (1–42) peptides bearing Tyr10 O-glycosylation, an unusual PTM initially identified from the cerebrospinal fluid samples of AD patients. We discovered that O-glycans significantly affect both the aggregation and degradation of Aβ42. By combining cryo-EM and various biochemical assays, we demonstrate that a Galβ1-3GalNAc modification redirects Aβ42 to form a new fibril polymorphic structure that is less stable and more vulnerable to Aβ-degrading enzymes (e.g., insulin-degrading enzyme). Thus, beyond showing how particular O-glycosylation modifications affect Aβ42 aggregation at the molecular level, our study provides powerful experimental tools to support further investigations about how PTMs affect Aβ42 fibril aggregation and AD-related neurotoxicity.

D-半乳糖胺(GalNAc)与丝氨酸或苏氨酸的羟基连接;后者则通常以N-乙酰-α-D-半乳糖胺(GlcNAc)与特定 序列Asn-Xaa-Ser/Thr中的天冬酰胺残基侧链连接 [7] .从结构上看, 氮连接聚糖(N-linked glycan)普遍具有共 同的核心五糖Man 3 GlcNAc 2 , 并在此基础上根据远端 位置糖型的差异可进一步衍生出3个亚型(图1): 高甘 露糖型(high-mannose type)、杂化型(hybrid type)和复 杂型(complex type). 每种类型可包含两个或两个以上 的分支(antenna), 每个分支可能含有数目不等的单糖, 这些特点进一步增加了氮连接聚糖结构的复杂性 [8] .

The intracellular liquid-liquid phase separation (LLPS) of biomolecules gives rise to condensates that carry vital functions as membrane-less organelles. The RNA-binding protein FUS is a model system for LLPS and the often disease-linked liquid-to-solid transition of biomolecular condensates in aging. However, the mechanism of such maturation processes, as well as the structural and physical properties of the system, remain unclear, partly attributable to difficulties in scrutinizing the internal structures of the micrometer-sized condensates with diffraction-limited optical microscopy. Harnessing a set of multidimensional super-resolution fluorescence microscopy tools, here we uncover nanoscale heterogeneities in the aging process of FUS condensates. Through spectrally resolved single-molecule localization microscopy (SR-SMLM) with a solvatochromic dye, we unveil distinct hydrophobic nanodomains at the condensate surface. Through SMLM with a fluorogenic amyloid probe, we identify these nanodomains as amyloid aggregates. Through single-molecule displacement/diffusivity mapping (SMdM), we show that such nanoaggregates drastically impede local diffusion. Notably, upon aging or mechanical shears, these nanoaggregates progressively expand on the condensate surface, thus leading to a growing low-diffusivity shell while leaving the condensate interior diffusion-permitting. Together, beyond uncovering fascinating nanoscale spatial heterogeneities in the single-component FUS condensates, the demonstrated synergy of multidimensional super-resolution approaches in this study opens new paths for understanding LLPS systems.