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Siglec-6 mediates the uptake of extracellular vesicles through a noncanonical glycolipid binding pocket.

ABSTRACT: Immunomodulatory Siglecs are controlled by their glycoprotein and glycolipid ligands. Siglec-glycolipid interactions are often studied outside the context of a lipid bilayer, missing the complex behaviors of glycolipids in a membrane. Through optimizing a liposomal formulation to dissect Siglec-glycolipid interactions, it is shown that Siglec-6 can recognize glycolipids independent of its canonical binding pocket, suggesting that Siglec-6 possesses a secondary binding pocket tailored for recognizing glycolipids in a bilayer. A panel of synthetic neoglycolipids is used to probe the specificity of this glycolipid binding pocket on Siglec-6, leading to the development of a neoglycolipid with higher avidity for Siglec-6 compared to natural glycolipids. This neoglycolipid facilitates the delivery of liposomes to Siglec-6 on human mast cells, memory B-cells and placental syncytiotrophoblasts. A physiological relevance for glycolipid recognition by Siglec-6 is revealed for the binding and internalization of extracellular vesicles. These results demonstrate a unique and physiologically relevant ability of Siglec-6 to recognize glycolipids in a membrane.

SUBMITTER: Schmidt EN

PROVIDER: S-EPMC10122656 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Siglec-6 mediates the uptake of extracellular vesicles through a noncanonical glycolipid binding pocket.

Schmidt Edward N EN Lamprinaki Dimitra D McCord Kelli A KA Joe Maju M Sojitra Mirat M Waldow Ayk A Nguyen Jasmine J Monyror John J Kitova Elena N EN Mozaneh Fahima F Guo Xue Yan XY Jung Jaesoo J Enterina Jhon R JR Daskhan Gour C GC Han Ling L Krysler Amanda R AR Cromwell Christopher R CR Hubbard Basil P BP West Lori J LJ Kulka Marianne M Sipione Simonetta S Klassen John S JS Derda Ratmir R Lowary Todd L TL Mahal Lara K LK Riddell Meghan R MR Macauley Matthew S MS

Nature communications 20230422 1

Immunomodulatory Siglecs are controlled by their glycoprotein and glycolipid ligands. Siglec-glycolipid interactions are often studied outside the context of a lipid bilayer, missing the complex behaviors of glycolipids in a membrane. Through optimizing a liposomal formulation to dissect Siglec-glycolipid interactions, it is shown that Siglec-6 can recognize glycolipids independent of its canonical binding pocket, suggesting that Siglec-6 possesses a secondary binding pocket tailored for recogni ...[more]

PMID: 37087495

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Project description:BackgroundExtracellular vesicles (EVs) are a heterogeneous group of cell-derived membranous structures that are important mediators of intercellular communication. Arthropods transport nutrients, signaling molecules, waste and immune factors to all areas of the body via the hemolymph. Little is known about tick hemolymph EVs.MethodsHemolymph was collected from partially fed Rhipicephalus haemaphysaloides and Hyalomma asiaticum ticks by making an incision with a sterile scalpel in the middle (between the femur and metatarsus) of the first pair of legs, which is known as leg amputation. EVs were isolated from hemolymph by differential centrifugation and characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Proteins extracted from the hemolymph EVs were analyzed by 4D label-free proteomics. The EVs were also examined by western blot and immuno-electron microscopy analysis. Intracellular incorporation of PHK26-labeled EVs was tested by adding labeled EVs to tick salivary glands and ovaries, followed by fluorescence microscopy.ResultsIn this study, 149 and 273 proteins were identified by 4D label-free proteomics in R. haemaphysaloides and H. asiaticum hemolymph EVs, respectively. TEM and NTA revealed that the sizes of the hemolymph EVs from R. haemaphysaloides and H. asiaticum were 133 and 138 nm, respectively. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analyses of identified proteins revealed pathways related to binding, catalytic and transporter activity, translation, transport and catabolism, signal transduction and cellular community. The key EV marker proteins RhCD9, RhTSG101, Rh14-3-3 and RhGAPDH were identified using proteomics and western blot. The presence of RhFerritin-2 in tick hemolymph EVs was confirmed by western blot and immuno-electron microscopy. We demonstrated that PKH26-labeled hemolymph EVs are internalized by tick salivary glands and ovary cells in vitro.ConclusionsThe results suggest that tick EVs are secreted into, and circulated by, the hemolymph. EVs may play roles in the regulation of tick development, metabolism and reproduction.

Dataset Information

Siglec-6 mediates the uptake of extracellular vesicles through a noncanonical glycolipid binding pocket.

Publications

Siglec-6 mediates the uptake of extracellular vesicles through a noncanonical glycolipid binding pocket.

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