Galactan (Potato)

Microfold cells (M cells) in the gut serve as a key gateway to enhance the potency of oral vaccines. Although natural polysaccharides show promise as carriers, their development is limited by inherent drawbacks such as scarce availability and lack of intrinsic targeting capacity. Therefore, it is essential to identify suitable materials from well-defined, commercially available polysaccharide standards. In this study, a library of well-defined, high-purity commercial polysaccharides was screened, and identified Xylan as a lead candidate with superior M cell transport efficiency in an in vitro human M-like cell model. This transport was confirmed in vivo, where mass spectrometry quantified a significant delivery (2.45-9.97 μg) of Xylan into murine Peyer's patches. Structural confirmation via monosaccharide composition, enzymatic oligosaccharide profiling, methylation, and NMR analyses identified this active polymer as a β-1, 4-Xylan branched with α-1, 2-glucuronic acid bearing methyl substitution. Mechanistically, molecular docking predicted the M cell surface protein Uromodulin (Umod) as a potential receptor, a hypothesis subsequently validated by isothermal titration calorimetry and surface plasmon resonance. In conclusion, our findings reveal that a specific, commercial Xylan is selectively transported by M cells, a process potentially mediated by Umod, highlighting its great promise as a novel and well-defined carrier for oral vaccine development.

The blood-brain barrier (BBB) experiences dysfunction during physiological aging, potentially leading to cognitive decline and the onset of neurodegenerative diseases. Recent research highlights brain endothelial cell (BEC) senescence as a key factor contributing to BBB impairment. In this study, polymeric nanoparticles (NPs) loaded with a senolytic agent - Navitoclax - were designed to selectively eliminate senescent cells more effectively. These NPs are taken up by both proliferative and senescent BECs, but are only degraded in the latter due to the presence of senescence-associated beta-galactosidase (SA-β-gal), releasing the drug to induce cell death. NPs effectively and selectively eliminate senescent BECs at lower doses, without affecting proliferative cells, outperforming the soluble senolytic. NPs were then evaluated in a chronologically aged rat model exhibiting BEC senescence, increased BBB permeability, and diminished exploratory behavior. In comparison to high-dose soluble senolytic, the NPs successfully restored BBB integrity by reducing permeability, diminishing the burden of senescent BECs, and partially improving rat exploratory performance. Notably, only the NPs were able to reduce the presence of p21⁺ and p16⁺ BECs in vivo and enhance exploratory behavior. These findings suggest that NPs containing senolytics represent a promising intervention for decreasing BECs senescence, reducing BBB permeability, and enhancing exploratory behavior in naturally aged rats.

Pectins are a major class of plant cell wall polysaccharide crucial for plant survival and adaptation. Despite their importance, the biosynthesis of pectin remains poorly understood due to its exceptional structural and functional diversity. Functional interrogation of pectin biosynthetic enzymes in planta is often limited by severe developmental or difficult to interpret carbohydrate phenotypes resulting from genetic disruption. Using a direct biochemical approach, we report the identification and functional characterization of a Galactan Initiation (GalTi) enzyme from the aquatic monocot Spirodela polyrhiza, a member of CAZy glycosyltransferase family 47, that initiates galactan side chain biosynthesis on rhamnogalacturonan I. We further show that orthologs from Arabidopsis thaliana exhibit identical catalytic activity, revealing conservation of this biosynthetic initiation mechanism across monocots and dicots. Comprehensive carbohydrate structural analyses demonstrate that GalTi enzymes specifically catalyze the transfer of single β D galactopyranosyl residues to the O 4 position of backbone rhamnose residues, establishing their direct role in galactan side chain biosynthesis. Co-synthesis experiments show 3-O-acetylation of backbone rhamnose residues inhibits SpGalTi mediated galactosylation, suggesting a potential regulatory mechanism by which plants use to modulate side chain patterning in vivo. Together, these findings provide a new framework for RG I galactan assembly, advancing our mechanistic understanding of plant cell wall biosynthesis.

Bacteria and fungi are well known for efficient degradation of plant polysaccharides thanks to various enzymes involved in plant cell wall decomposition. However, little is known about the role of archaea in this process or the repertoire and features of their polysaccharide-degrading enzymes. In our previous work, we discovered an archaeal multidomain glycosidase (MDG) composed of three catalytic domains (GH5 and two GH12) and two cellulose-binding modules (CBM2). The recombinant MDG and individual GH5 catalytic domain were active against cellulose and a number of other polysaccharides at a wide range of temperatures, with optimum temperatures (T_opt) of 60°C and 80°C, respectively. The present study was focused on the characterization of two GH12 domains of the MDG. Purified recombinant TMDG_GH12-1 and TMDG_GH12-2 proteins were active as individual enzymes but exhibited distinct catalytic properties. Both enzymes were thermostable and active at extremely high temperatures: TMDG_GH12-1 was active at 40-130°C (T_opt 100°C), and its half-life (t_½) at 100°C was 42 h, which makes it one of the most thermostable glycosidases known so far, whereas TMDG_GH12-2 was active at 50-100°C (T_opt 90°C) with t_½ at 100°C being 30 min. Phylogenetic and structural analysis of both TMDG_GH12 proteins together with molecular docking and site-directed mutagenesis suggested that the presence of two disulfide bridges and the W → Q mutation in the active site contribute to the exceptional thermostability of TMDG_GH12-1. Further structural and mutational studies of the TMDG_GH12-1 domain will help to gain a better understanding of the molecular mechanisms of its extraordinary thermostability and substrate specificity.

There is growing interest in members of the genus Segatella (family Prevotellaceae) as members of a well-balanced human gut microbiota (HGM). Segatella are particularly associated with the consumption of a diet rich in plant polysaccharides comprising dietary fiber. However, understanding of the molecular basis of complex carbohydrate utilization in Segatella species is currently incomplete. Here, we used RNA sequencing (RNA-seq) of the type strain Segatella copri DSM 18205 (previously Prevotella copri CB7) to define precisely individual polysaccharide utilization loci (PULs) and associated carbohydrate-active enzymes (CAZymes) that are implicated in the catabolism of common fruit, vegetable, and grain polysaccharides (viz. mixed-linkage β-glucans, xyloglucans, xylans, pectins, and inulin). Although many commonalities were observed, several of these systems exhibited significant compositional and organizational differences vis-à-vis homologs in the better-studied Bacteroides (sister family Bacteroidaceae), which predominate in post-industrial HGM. Growth on β-mannans, β(1, 3)-galactans, and microbial β(1, 3)-glucans was not observed, due to an apparent lack of cognate PULs. Most notably, S. copri is unable to grow on starch, due to an incomplete starch utilization system (Sus). Subsequent transcriptional profiling of bellwether Ton-B-dependent transporter-encoding genes revealed that PUL upregulation is rapid and general upon transfer from glucose to plant polysaccharides, reflective of de-repression enabling substrate sensing. Distinct from previous observations of Bacteroides species, we were unable to observe clearly delineated substrate prioritization on a polysaccharide mixture designed to mimic in vitro diverse plant cell wall digesta. Finally, co-culture experiments generally indicated stable co-existence and lack of exclusive competition between S. copri and representative HGM Bacteroides species (Bacteroides thetaiotaomicron and Bacteroides ovatus) on individual polysaccharides, except in cases where corresponding PULs were obviously lacking.