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Lingwood Lab

Research interests

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My laboratory is concerned with the molecular basis and physiology of glycosphingolipid (GSL) receptor function. Globotriaosyl ceramide (Gb3) binding is the mechanism by which verotoxin(VT) targets renal endothelial cells to initiate hemolytic uremic syndrome. Our studies on this interaction have led to our definition of a protective role for Gb3 in HIV infection also. The chemokine co receptor and GSL binding sites within the V3 loop of gp120 overlap. We are using molecular modeling to design small molecule inhibitors which prevent HIV -host cell binding.  Our interests are focused on the role of the GSL receptor lipid heterogeneity in determining the sensitivity of cells to VT-induced pathology/trafficking and HIV binding and membrane fusion. We have made soluble (adamantyl) GSL analogues which in large part mimic the properties of GSLs and prove effective as novel strategies against microbial pathogenesis.

Gb3 is upgregulated in certain human tumour cells and VT is a potential novel antineoplastic. The role of the drug resistance efflux pump MDR1 in GSL biosynthesis and the soluble GSL mimics we have generated allow us to manipulate cellular GSL metabolism to provide new approaches to several genetic diseases in which GSL metabolism is aberrant.

Indeed, we now show ABC transporters are key players in GSL biosynthesis. The first (precursor) GSL, glucosyl ceramide is made on the cytosolic Golgi membrane leaflet but all subsequent GSLs are made in the Golgi lumen. The mechanism by which GlcCer is flipped into the Golgi has been a mystery for more than 25 years. Using a photolabile GlcCer crosslinker we made, we identify ABC transporters as important in complex GSL biosynthesis, suggesting different GlcCer pools in the Golgi.

GSL and cholesterol form a complex in lipid rafts. In our generation of model lipid raft vesicle we found that GSLs became undetectable ("invisible GSLs") in these vesicles. Molecular modeling showed that within the GSL-cholesterol complex, the GSL sugar becomes reoriented from a membrane perpendicular to parallel conformation, less available for trans-ligand binding. This is important for many reasons, one being that the increased cholesterol of tumours results in the masking of tumour associated GSLs, to reduce efficacy of immune surveillance and response to anti GSL anineoplastic Mabs used clinically. Cyclodextrin extraction of tumour cholesterol results in a massive increase in the binding of such Mabs. There is a cholesterol gradient across the secretion/GSL biosynthetic pathway from ER to Golgi to PM which may affect precursor GSL availability for GSL anabolism. In the GSL/cholesterol complex the GSL sugar forms an umbrella over the steroid hydroxyl masking the cholesterol from some binding ligands e.g filipin. This mutual masking within the complex provides the basis of a new 'transistor-like' mechanism for the amplification of GSL and cholesterol mediated membrane signaling.

The retrograde trafficking of GSL binding bacterial subunit toxins (cholera toxin, Vero (Shiga)toxin) provide a prokaryotic basis for a new therapeutic approach to treat genetic protein misfolding diseases. The toxins traffic to the ER where the subunits separate and the A subunits hijack the membrane translocon (dislocon) used in ER associated degradation (ERAD) of misfolded proteins for cytosolic access and cellular pathology. ERAD elimination initiates or exacerbates deficiency disease symptoms in many genetic diseases in which the mutation does not completely delete activity but rather causes minor misfolding in the 3D protein structure. By providing an exogenous dislocon substrate, such toxins containing an inactivated A subunit, temporarily occupy the dislocon and thereby protect the mutant protein from ERAD, enabling an increased fraction of the mutant protein to escape destruction, mature and rescue the mutant disease phenotype. We have primarily focused on F508del CFTR cystic fibrosis and N370S GCC Gaucher Disease and found that this approach works in cell and animal modes of these diseases.

Future research

We will continue to develop these toxoids as clinical therapeutics for misfolding diseases and, as drug resistance becomes of increasing concern, the use of small molecular inhibitors of HIV. The interplay between GSLs and cholesterol provides a new perspective to study disease.