Department of Pathology and Molecular Medicine Faculty Publications

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    Development of a whole-cell biosensor for β-lactamase inhibitor discovery
    (The Royal Society of Chemistry, 2023-10-02) Jeffs, Mitchell A.; Gray, Rachel A. V.; Sheth, Prameet; Lohans, Christopher T.
    The production of β-lactamases by bacterial pathogens endangers antimicrobial therapy, and new inhibitors for β-lactamases are urgently needed. We report the development of a luminescentbased biosensor that quantifies β-lactamase inhibition in a cellular context, based on the activation of transcriptional factor AmpR following the exposure of bacterial cells to β-lactams. This rapid method can account for factors like membrane permeability and can be employed to identify new β-lactamase inhibitors.
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    The scavenger receptor SCARA5 is an endocytic receptor for von Willebrand factor expressed by littoral cells in the human spleen
    (Wiley, 2019-05-24) Swystun, Laura L.; Ogiwara, Kenichi; Lai, Jesse D.; Ojala, Juha R. M.; Rawley, Orla; Lassalle, Fanny; Notley, Colleen; Rengby, Olle; Michels, Alison; Nesbitt, Kate; Tryggvason, Karl; Lillicrap, David
    Background Scavenger receptors play a significant role in clearing aged proteins from the plasma, including the large glycoprotein coagulation factors von Willebrand factor (VWF) and factor VIII (FVIII). A large genome-wide association study meta-analysis has identified genetic variants in the gene SCARA5, which encodes the class A scavenger receptor SCARA5, as being associated with plasma levels of VWF and FVIII. Objectives The ability of SCARA5 to regulate the clearance of VWF-FVIII was characterized. Methods VWF-FVIII interactions with SCARA5 were evaluated by solid phase binding assays and in vitro cell based assays. The influence of SCARA5 deficiency on VWF:Ag and half-life was assessed in a murine model. The expression pattern of SCARA5 and its colocalization with VWF was evaluated in human tissues. Results VWF and the VWF-FVIII complex bound to human recombinant SCARA5 in a dose- and calcium-dependent manner. SCARA5 expressing HEK 293T cells bound and internalized VWF and the VWF-FVIII complex into early endosomes. In vivo, SCARA5 deficiency had a modest influence on the half-life of human VWF. mRNA analysis and immunohistochemistry determined that human SCARA5 is expressed in kidney podocytes and the red pulp, white pulp, and marginal zone of the spleen. VWF was found to colocalize with SCARA5 expressed by littoral cells lining the red pulp of the human spleen. Conclusions SCARA5 is an adhesive and endocytic receptor for VWF. In human tissues, SCARA5 is expressed by kidney podocytes and splenic littoral endothelial cells. SCARA5 may have a modest influence on VWF clearance in humans.
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    The endothelial lectin clearance receptor CLEC4M binds and internalizes factor VIII in a VWF-dependent and independent manner
    (2019-02-11) Swystun, Laura L.; Notley, Colleen; Georgescu, Ilinca; Lai, Jesse D.; Nesbitt, Kate; James, Paula D.; Lillicrap, David
    Essentials CLEC4M is an endocytic receptor for factor FVIII. CLEC4M interacts with FVIII in a VWF-dependent and independent manner. CLEC4M binds to mannose-containing glycans on FVIII. CLEC4M internalization of FVIII involves clathrin coated pits. Summary Background von Willebrand factor (VWF) and factor VIII (FVIII) circulate in the plasma as a non-covalent complex, and the majority of FVIII is likely to be cleared by VWF-dependent pathways. Clearance of VWF-free FVIII is rapid and underlies the pathological basis of some quantitative FVIII deficiencies. The receptor pathways that regulate the clearance of VWF-bound and VWF-free FVIII are incompletely uncharacterized. The human liver-expressed endothelial lectin CLEC4M has been previously characterized as a clearance receptor for VWF, although its influence on FVIII is unknown. Objective The interaction between FVIII and CLEC4M was characterized in the presence or absence of VWF. Methods FVIII interactions with CLEC4M were evaluated by in vitro cell-based and solid phase binding assays. Interactions between FVIII and CLEC4M or liver sinusoidal endothelial cells were evaluated in vivo by immunohistochemistry. Results CLEC4M-expressing HEK 293 cells bound and internalized recombinant and plasma-derived FVIII through VWF-dependent and independent mechanisms. CLEC4M binding to recombinant FVIII was dependent on mannose-exposed N-linked glycans. CLEC4M mediated FVIII internalization via a clathrin-coated pit-dependent mechanism, resulting in transport of FVIII from early and late endosomes for catabolism by lysosomes. In vivo hepatic expression of CLEC4M after hydrodynamic liver transfer was associated with a decrease in plasma levels of endogenous murine FVIII:C in normal mice, whereas infused recombinant human FVIII was associated with sinusoidal endothelial cells in the presence or absence of VWF. Conclusions These findings suggest that CLEC4M is a novel clearance receptor that interacts with mannose-exposed glycans on FVIII in the presence or absence of VWF.