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Nature CommunicationsPharmacology & ToxicologyPeer-reviewed journal article

1,3- and 1,4-linked Polysaccharides Uptake in Intestinal Cells Relies on Clathrin/Dynamin 1/Rab5-dependent Endocytosis

Wenfeng Liao, Dianxiu Cao, Ying Wang, Zhenyun Du, Yao Jx, Pengfei Dou, Yuandong Zheng, Zhiming WangiD, Xia Chen, Peipei WangiD, Chungwah Ma, Hao CheniD, Xingxing DiaoiD, Kaiping WangiD, Kan DingiD

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Abstract

Natural polysaccharides are considered to be a promising feedstock for manufacturing sustainable materials. However, polysaccharides are believed by most biologists that they could not be absorbed by intestinal mucosa to enter circulation system owing to their high hydrophilicity and large molecule size. Herein, {beta}-1,3-linked glucan (GFPBW1) and -1,4-linked glucan (WGE) is used as the model polysaccharide and the transport experiments show that different linked and charged polysaccharides can permeate Caco-2 cell monolayers. The in vivo experiments demonstrate that GFPBW1 and WGE are detectable in rodent plasma and liver after oral administration the isotope (99mTc, 3H) and fluorescein labeled polysaccharides. Further study show that polysaccharide uptake requires clathrin heavy chain (CLTC) and its associated factors Rab5 and dynamin1 in intestinal cells using gene-knockdown strategies and inhibitors. Strikingly, polysaccharide absorption is attenuated in both CLTC intestine deficient mice and Rab5, dynamin1 conventional-knockout mice. Importantly, membrane receptors bone morphogenetic protein receptor type IA (BMPRIA), Dectin-1 and epidermal growth factor receptor (EGFR) are also critical for specific structural polysaccharide internalization. These findings provide novel insight to understand polysaccharide absorption mechanism and lay foundation for oral polysaccharide- based new drugs development.

In-depth summary

Claude Opus 5.5 · xhigh effort · 5 Nov 2026 · from the full text of version 1 (3 Jan 2025)

Orally administered β-1,3- and α-1,4-linked polysaccharides cross intestinal epithelium into rodent plasma and liver as macromolecules via clathrin-, dynamin 1-, and Rab5-dependent endocytosis mediated by EGFR, Dectin-1, and BMPRIA.

Natural polysaccharides are widely assumed to be unabsorbable by the gastrointestinal mucosa after oral administration because of their high molecular weight and hydrophilicity, leaving the pharmacokinetics and cellular uptake mechanisms of oral polysaccharide drugs unresolved. Tracing unlabeled, fluorescent, and radiolabeled glucans across cultured intestinal cells and knockout mice establishes macromolecular intestinal absorption via clathrin-mediated endocytosis.

Key results

  1. ShownPolysaccharides with diverse glycosidic linkages and surface charges permeated Caco-2 monolayers intact and entered cultured intestinal and cancer cells in a concentration-dependent manner.

    Model glucans GFPBW1 (β-1,3-linked, 300 kDa) and WGE (α-1,4-linked, 700 kDa), along with lentinan, MDG-1, dextran, and -NH3/-SO4 derivatives, crossed Caco-2 monolayers with apparent permeability coefficients (Papp) > 1 × 10^-6 cm/s and unchanged HPLC retention times, internalizing into HIEC-6, Caco-2, IEC-6, and T24 cells within 5 minutes at concentrations of 1.25 mg/mL (GFPBW1) and 2.5 mg/mL (WGE). · 2.1 Different linked type and charged…; 2.9 Polysaccharide could be internalized in T24…; 2.9 The internalized polysaccharides might be…

  2. ShownOrally administered GFPBW1 and WGE were absorbed across the rodent intestinal lining and detected as intact macromolecules in plasma and liver.

    Unlabeled, fluorescein/Cy5.5-labeled, and radiolabeled (^3H, ^99mTc) GFPBW1 and WGE retained canonical HPLC and Radio-HPGPC peaks in rat and C57BL/6J mouse plasma and liver lysates after oral gavage, with BALB/c mouse intestinal enterocyte uptake detected at 20 and 15 minutes and peaking at 80 and 30 minutes for GFPBW1 and WGE, respectively. · 2.2 Polysaccharides were absorbed in rat and…; 2.9 The internalized polysaccharides might be…

  3. ShownPolysaccharide internalization depends on clathrin heavy chain (CLTC), dynamin 1 (DNM1), and Rab5 (RAB5A) both in cultured cells and in knockout mice, rather than lipid-raft, caveolar, or macropinocytic pathways.

    Chlorpromazine and dynasore (but not nystatin or cytochalasin D) inhibited uptake in HIEC-6, IEC-6, and T24 cells, polysaccharides still entered caveolin-free Huh-7 cells, siRNA knockdown of CLTC, DNM1 (but not DNM2), or RAB5A blocked cellular uptake, and intestinal uptake was sharply reduced in intestinal-specific CLTC CKO mice, heterozygous DNM1 KO mice, and RAB5A^-/- KO mice. · 2.3 Clathrin mediated endocytosis is critical…; 2.4 Polysaccharides uptake was impaired in CLTC…; 2.5 Dynamin1 and Rab5 were required for…; 2.9 Polysaccharide could be internalized in T24…

  4. ShownInternalized polysaccharides traffic through early endosomes, lysosomes, and the Golgi apparatus, and require membrane receptors EGFR, Dectin-1, and BMPRIA as well as Wnt/β-catenin and NF-κB signaling.

    GFPBW1 and WGE colocalized with EEA1, LAMP1, and RCAS1 in HIEC-6 and T24 cells (and ER marker PDI in HIEC-6 but not T24, with minimal mitochondrial AIF colocalization); uptake of both glucans was suppressed by EGFR knockdown/Gefitinib, XAV939 (Wnt/β-catenin inhibitor), and TPCA-1 (NF-κB inhibitor), while Dectin-1 depletion specifically blocked GFPBW1 and BMPRIA depletion blocked WGE. · 2.6 Internalized polysaccharides were detectable…; 2.7 EGFR, Dectin-1 and BMPRIA were implicated in…; 2.8 Wnt/β-catenin and NF-κB signaling pathways…; 2.9 The internalized polysaccharides might be…

Methods, data and reproducibility

Evaluated trans-epithelial transport and uptake of unlabeled, fluorescent, and ^3H/^99mTc-labeled beta-1,3-glucan (GFPBW1, 300 kDa), alpha-1,4-glucan (WGE, 700 kDa), and derivatives across 5 cell lines and orally/IV dosed rats and mice via HPLC, Radio-HPGPC, QCM, and confocal microscopy. Dissected endocytosis and receptors using inhibitors, siRNAs, and intestinal CLTC CKO, DNM1+/-, and RAB5A-/- mice.

  • In vitro monolayer permeability (Caco-2) and fluorescence/QCM uptake and colocalization assays in cultured intestinal and cancer cell lines · human (Caco-2, HIEC-6, Huh-7, T24) and rat (IEC-6) · n = 5 cell lines tested across multiple polysaccharide structures and derivatives
  • In vivo oral and intravenous pharmacokinetics, biodistribution, and intestinal uptake in wild-type and knockout rodents · mouse (BALB/c, C57BL/6J, CLTC CKO, DNM1 KO heterozygote, RAB5A KO) and rat · n = wild-type rats and mice plus 3 genetic knockout mouse models
Methods & tools
HPLC, Radio HPGPC, NMR, QCM, confocal microscopy, Western blotting, Fiji
Reagents & environment
Fiji
Reproducibility gaps
Experimental procedures are placed in Supporting Information and underlying datasets are only available from the corresponding author upon reasonable request.
Limitations (3)
  • Homozygous DNM1 knockout mice died postnatally, restricting in vivo dynamin 1 loss-of-function experiments to heterozygous DNM1 knockout mice. · stated by the authors
  • Overexpression of full-length CLTC cDNA plasmid in T24 cells did not significantly increase GFPBW1 or WGE internalization above control levels. · stated by the authors
  • The mechanism explaining why internalized polysaccharides localize to the endoplasmic reticulum (PDI) in HIEC-6 intestinal cells but not in T24 cancer cells remains undetermined. · stated by the authors

Read this if you study oral macromolecule drug delivery, glycobiology, or intestinal epithelial endocytosis mechanisms.

Citations
38
FWCI
104.71
Field percentile
top 1%

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