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Review
. 2021 May 31;26(11):3299.
doi: 10.3390/molecules26113299.

The Cellular and Chemical Biology of Endocytic Trafficking and Intracellular Delivery-The GL-Lect Hypothesis

Ludger Johannes  1
Affiliations
Review

The Cellular and Chemical Biology of Endocytic Trafficking and Intracellular Delivery-The GL-Lect Hypothesis

Ludger Johannes. Molecules. .

Abstract

Lipid membranes are common to all forms of life. While being stable barriers that delimitate the cell as the fundamental organismal unit, biological membranes are highly dynamic by allowing for lateral diffusion, transbilayer passage via selective channels, and in eukaryotic cells for endocytic uptake through the formation of membrane bound vesicular or tubular carriers. Two of the most abundant fundamental fabrics of membranes-lipids and complex sugars-are produced through elaborate chains of biosynthetic enzymes, which makes it difficult to study them by conventional reverse genetics. This review illustrates how organic synthesis provides access to uncharted areas of membrane glycobiology research and its application to biomedicine. For this Special Issue on Chemical Biology Research in France, focus will be placed on synthetic approaches (i) to study endocytic functions of glycosylated proteins and lipids according to the GlycoLipid-Lectin (GL-Lect) hypothesis, notably that of Shiga toxin; (ii) to mechanistically dissect its endocytosis and intracellular trafficking with small molecule; and (iii) to devise intracellular delivery strategies for immunotherapy and tumor targeting. It will be pointed out how the chemical biologist's view on lipids, sugars, and proteins synergizes with biophysics and modeling to "look" into the membrane for atomistic scale insights on molecular rearrangements that drive the biogenesis of endocytic carriers in processes of clathrin-independent endocytosis.

Keywords: Casimir force; endocytosis; galectin; glycosphingolipid; glycosylation; immunotherapy; raft; retrograde trafficking; small molecule; tumor targeting.

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Conflict of interest statement

The author declares no conflict of interest.

Figures

Figure 1
Figure 1
Endocytic pit construction according to the GL–Lect hypothesis. The sugar binding protein galectin-3 (Gal3) is monomeric in solution. It binds to complex carbohydrates of plasma membrane glycoproteins (glycosylated cargo). This leads to Gal3 oligomerization, thereby enabling its capacity to interact with glycosphingolipids (GSLs) in a way such as to induce narrow membrane bending and the clathrin-independent formation of endocytic pits into which the glycosylated cargoes are recruited. From these pit, endocytic carriers are then generated for the cellular uptake of the glycosylated cargoes (not shown). Reproduced with permission from Ref. [16].
Figure 2
Figure 2
GSL biosynthesis pathways. Left panel: ceramide (Cer) can be acylated (acyl-Cer), phosphorylated (Cer-1-phosphate), or conveyed to the TGN for the synthesis of sphingomyelin (SM). Alternatively, Cer is glycosylated for the synthesis of the GSL precursors, glucosylceramide (GlcCer) or galactosylceramide (GalCer) along the secretory pathway. GalCer is then processed for the production of sulfatides. Right panel: GlcCer is galactosylated to lactosylceramide (LacCer), which serves as a common precursor for the different GSL series: globo (red), ganglio (green), asialo (blue) and lacto (purple). Glycosphingolipid-synthetizing enzymes catalyzing the major synthetic reactions are shown in dark orange. Reproduced with permission from The Journal of Cell Science, Ref. [41].
Figure 3
Figure 3
Hypothesis on fluctuation force-driven clustering. Thermally excited fluctuations are suppressed by membrane inclusions (yellow) of nanometric size like STxB, not only at the sites of binding to the membrane, but also for the membrane patch between the two nanoparticles, if they are separated only by a distance of their own size. This perturbation results in an attractive force whose strength is expected to be similar to that of shielded electrostatics or van der Waals interactions. Reproduced with permission from Toxins, Ref. [24].
Figure 4
Figure 4
Hypothesis on asymmetric compressive stress leading to negative membrane curvature. Membrane compression of the exoplasmic leaflet to which STxB is bound will force the double-layered plasma membrane to curve in the direction of the toxin. From Reference [20]; courtesy of the Cold Spring Harbor Laboratory Archives.
Figure 5
Figure 5
Negative curvature induction based on the geometry of GSL binding sites on lectins. (a) All atom molecular dynamics simulation of the STxB homopentamer (gray ribbon structure, side view) on a membrane patch. Gb3 molecules in blue. Note the increment of spontaneous membrane curvature that is observed in silico due to the presence of STxB. (b) Representation (extracted from (a)) of STxB (side view) with three carbohydrates (blue, green and red). For simplicity, only the three Gb3 binding sites on one monomer are shown. Note that sites 1 (red) and 2 (green) are at the periphery of the protein, while site 3 (blue) faces down under the protein. (c) Schematic representation of the situation in (b) which illustrates that the membrane (black line) needs to bend up at the edges of STxB to plug the sugars of Gb3 receptor molecules (red and green) into the binding pockets of sites 1 and 2. This would then lead to the generation of negative membrane curvature. (d) Overlay of the structures of the following proteins in interaction with GSL receptor analogues: STxB in green with Gb3 (Reference [53]), CTxB in red with GM1 (Reference [54]), the capsid protein VP1 from SV40 in blue (Reference [55]). Note that despite the fact that these proteins do not have any sequence similarity, they fold such that the conserved GSL binding site 2 is presented with the same geometry in space (the sugars of the three structures overlay). It therefore seems likely that this fold was selected by convergent evolution for the same function: to generate negative membrane curvature for building endocytic sites for clathrin-independent endocytosis. Reproduced with permission from Toxins, Ref. [24].
Figure 6
Figure 6
Induction of tubular membrane invaginations by fluorescently labeled STxB on Gb3-containing GUVs. Note that less than 1 min after addition of STxB, tubular membrane invaginations (arrows) were observed on GUVs that contained a C22 Gb3 species with a single unsaturated acyl chain (top panel, C22:1)). In contrast, no tubules were observed on GUVs that contained a C22 Gb3 species with fully saturated acyl chain (bottom panel, C22:0), even after dozens of minutes of incubation with STxB. Bars = 5 µm. Reproduced with permission from Ref. [14].
Figure 7
Figure 7
Model of how Retro-2 induces a block of Shiga toxin trafficking at the level of early endosomes. In untreated conditions (left), Shiga toxin trafficking from early endosomes to the TGN requires the interaction of GPP130 (blue) with syntaxin-5 (Syn5, yellow). From the TGN, Shiga toxin then moves on to the ER from where the catalytic A-subunit is translocated to the cytosol to inactivate ribosomes. Under Retro-2 treatment conditions (right), the anterograde transport of syntaxin-5 between ER and Golgi is slowed down by the interaction of Retro-2 with Sec16A, leading to the partial depletion of syntaxin-5 in the Golgi, a loss of its interaction with GPP130, and thereby the blockage of STxB in early endosomes. Reproduced with permission from Ref. [70].
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