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Cell surface photoengineering enables modeling of glycocalyx shedding dynamics.

ABSTRACT: The cellular glycocalyx, composed of membrane associated glycoproteins and glycolipids, is a complex and dynamic interface that facilitates interactions between cells and their environment. The glycocalyx composition is continuously changing through biosynthesis of new glycoconjugates and membrane turnover. Various glycocalyx components, such as mucins, can also be rapidly shed from the cell surface in response to acute events, such as pathogenic threat. Mucins, which are large extended glycoproteins, deliver important protective functions against infection by creating a physical barrier at the cell surface and by capturing and clearing pathogens through shedding. Evaluating these mucin functions may provide better understanding of early stages of pathogenesis; however, tools to tailor the composition and dynamics of the glycocalyx with precision are still limited. Here, we report a chemical cell surface engineering strategy to model the shedding behavior of mucins with spatial and temporal control. We generated synthetic mucin mimetic glycopolymers terminated with a photolabile membrane anchor, which could be introduced into the membranes of living cells and, subsequently, released upon exposure to UV light. By tuning the molecular density of the artificial glycocalyx we evaluated lectin crosslinking and its effect on shedding, showing that lectins can stabilize the glycocalyx and limit release of the mucin mimetics from the cell surface. Our findings indicate that endogenous and pathogen-associated lectins, which are known to interact with the host-cell glycocalyx, may alter mucin shedding dynamics and influence the protective properties of the mucosal barrier. More broadly, we present a method which enables photoengineering of the glycocalyx and can be used to facilitate the study of glycocalyx dynamics in other biological contexts.

SUBMITTER: Purcell SC

PROVIDER: S-EPMC9172368 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The invasive larvae (cercariae) of schistosomes penetrate the skin of their definitive hosts. During the invasion, they undergo dramatic ultrastructural and physiological transitions. These changes result in the development of the subsequent stage, schistosomulum, which migrates through host tissues in close contact with host's immune system. One of the striking changes in the transforming cercariae is the shedding of their thick tegumental glycocalyx, which represents an immunoattractive structure; therefore its removal helps cercariae to avoid immune attack. A set of commercial fluorescently labeled lectin probes, their saccharide inhibitors and monoclonal antibodies against the trisaccharide Lewis-X antigen (LeX, CD15) were used to characterize changes in the surface saccharide composition of the neuropathogenic avian schistosome Trichobilharzia regenti during the transformation of cercariae to schistosomula, both in vitro and in vivo. The effect of various lectins on glycocalyx shedding was evaluated microscopically. The involvement of peptidases and their inhibitors on the shedding of glycocalyx was investigated using T. regenti recombinant cathepsin B2 and a set of peptidase inhibitors. The surface glycocalyx of T. regenti cercariae was rich in fucose and mannose/glucose residues. After the transformation of cercariae in vitro or in vivo within their specific duck host, reduction and vanishing of these epitopes was observed, and galactose/N-acetylgalactosamine emerged. The presence of LeX was not observed on the cercariae, but the antigen was gradually expressed from the anterior part of the body in the developing schistosomula. Some lectins which bind to the cercarial surface also induced secretion from the acetabular penetration glands. Seven lectins induced the shedding of glycocalyx by cercariae, among which five bound strongly to cercarial surface; the effect could be blocked by saccharide inhibitors. Mannose-binding protein, part of the lectin pathway of the complement system, also bound to cercariae and schistosomula, but had little effect on glycocalyx shedding. Our study did not confirm the involvement of proteolysis in glycocalyx shedding.

Project description:BackgroundThe endothelial glycocalyx (EG) on the luminal surface of endothelial cells contributes to the permeability barrier of vessels and prevents activation of the coagulation cascade. Endothelial glycocalyx damage, which occurs in the shock state, results in endotheliopathy. Interleukin (IL)-22 is a cytokine with both proinflammatory and anti-inflammatory properties, and how IL-22 affects the EG has not been studied. We hypothesized that IL-22:Fc, a recombinant fusion protein with human IL-22 and the Fc portion of human immunoglobulin G1 (which extends the protein half-life), would not affect EG shedding in endothelium after injury.MethodsHuman umbilical vein endothelial cells (HUVECs) were exposed to 1 μg/mL lipopolysaccharide (LPS). Lipopolysaccharide-injured cells (n = 284) were compared with HUVECs with LPS injury plus 0.375 μg/mL of IL-22:Fc treatment (n = 293) for 12 hours. These two cohorts were compared with control HUVECs (n = 286) and HUVECs exposed to IL-22:Fc alone (n = 269). Cells were fixed and stained with fluorescein isothiocyanate-labeled wheat germ agglutinin to quantify EG. Total RNA was collected, and select messenger RNAs were quantified by real time - quantitative polymerase chain reaction (RT-qPCR) using SYBR green fluorescence.ResultsExposure of HUVECs to LPS resulted in degradation of the EG compared with control (5.86 vs. 6.09 arbitrary unit [AU], p = 0.01). Interleukin-22:Fc alone also resulted in degradation of EG (5.08 vs. 6.09 AU, p = 0.01). Treatment with IL-22:Fc after LPS injury resulted in less degradation of EG compared with LPS injury alone (5.86 vs. 5.08 AU, p = 0.002). Expression of the IL-22Ra1 receptor was not different for IL-22:Fc treated compared with LPS injury only (0.69 vs. 0.86 relative expression, p = 0.10). Treatment with IL-22:Fc after LPS injury resulted in less matrix metalloproteinase 2 (0.79 vs. 1.70 relative expression, p = 0.005) and matrix metalloproteinase 14 (0.94 vs. 2.04 relative expression, p = 0.02).ConclusionsInterleukin-22:Fc alone induces EG degradation. However, IL-22:Fc treatment after LPS injury appears to mitigate EG degradation. This protective effect appears to be mediated via reduced expression of metalloproteinases.

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Cell surface photoengineering enables modeling of glycocalyx shedding dynamics.

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