Sakanovic, A. | bioRxiv Science

bioRxiv2020

Insight into glycosphingolipid crypticity: Crystal structure of the anti-tumor antibody 14F7 and recognition of NeuGc GM3 ganglioside

Tumor-associated glycolipids such as NeuGc GM3 are auspicious molecular targets in antineoplastic therapies and vaccine strategies. 14F7 is an anti-tumor antibody with high clinical potential, which has extraordinary specificity for NeuGc GM3, but does not recognize the very similar, ubiquitous NeuAc GM3. Here we present the 2.3 [A] crystal structure of the 14F7 binding domain (14F7 scFv) in complex with the NeuGc GM3 trisaccharide. Intriguingly, a water molecule appears to shape the specificity of 14F7. Using model membrane systems, we show that 14F7 recognizes NeuGc GM3 only above lipid concentrations that are likely to form glycolipid-rich domains. This "all-or-nothing" effect was exacerbated in giant unilamellar vesicles and multilamellar vesicles, whereas no binding was observed to 100 nm liposomes, emphasizing that the 14F7-NeuGc GM3 interaction is additionally modulated by membrane curvature. Unexpectedly, adding NeuAc GM3 strongly increased binding affinity to NeuGc GM3-containing liposomes. This effect may be important for tumor recognition, where the ubiquitous NeuAc GM3 may enhance 14F7 binding to NeuGc GM3-expressing cancer cells.

biochemistry↗

bioRxiv2020

Redistribution of cholesterol from vesicle to plasmalemma controls fusion pore geometry

Eukaryotic vesicles fuse with the plasmalemma to form the fusion pore, previously considered to be unstable with widening of the pore diameter. Recent studies established that the pore diameter is stable, reflecting balanced forces of widening and closure. Proteins are considered key regulators of the fusion pore, whereas the role of membrane lipids remains unclear. Super-resolution microscopy revealed that lactotroph secretory vesicles discharge cholesterol after stimulation of exocytosis; subsequently, vesicle cholesterol redistributes to the outer leaflet of the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes evokes release of vesicle hormone, indicating that cholesterol constricts the fusion pore. A new model of cholesterol-dependent fusion pore diameter regulation is proposed. High-resolution measurements of fusion pore conductance confirmed that the fusion pore widens with cholesterol depletion and constricts with cholesterol enrichment. In fibroblasts lacking the Npc1 protein, in which cholesterol accumulates in vesicles, the fusion pore is narrower than in controls, showing that cholesterol regulates fusion pore geometry. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/027169v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@73168borg.highwire.dtl.DTLVardef@1cd2afdorg.highwire.dtl.DTLVardef@c170f9org.highwire.dtl.DTLVardef@1dfff4f_HPS_FORMAT_FIGEXP M_FIG C_FIG Top: stages through which a vesicle interacts with the plasmalemma. Stage A denotes hemifusion, which proceeds to stage B, with a narrow fusion pore, which can then reversibly open (stage C), before widening fully (stage D). Bottom: redistribution of cholesterol from the vesicle to the outer leaflet of the plasmalemma controls fusion pore constriction. In BriefA membrane pore is formed when the vesicle membrane fuses with the plasmalemma. Proteins were considered key regulators of the opening and closing of this fusion pore. Here, evidence is provided to show that cholesterol, a membrane constituent, determines a radial force constricting the fusion pore, revealing that the fusion pore functions as a proteolipidic structure. HighlightsO_LIIntravesicular cholesterol redistributes to the outer leaflet of the plasmalemma. C_LIO_LICholesterol depletion widens the fusion pore, whereas cholesterol enrichment constricts the fusion pore. C_LIO_LIA model of cholesterol-dependent force preventing fusion pore widening is developed. C_LIO_LIDisease-related increase in vesicle cholesterol constricts the fusion pore. C_LI

cell biology↗