Mutation of threonin 34 in mouse podoplanin-Fc reduces CLEC-2 binding and toxicity in vivo while retaining anti-lymphangiogenic activity [Molecular Bases of Disease]

June 6th, 2014 by Bianchi, R., Fischer, E., Yuen, D., Ernst, E., Chen, L., Otto, V. I., Detmar, M.

The lymphatic system plays an important role in cancer metastasis and inhibition of lymphangiogenesis could be valuable in fighting cancer dissemination. Podoplanin (Pdpn)4 is a small, transmembrane glycoprotein expressed on the surface of lymphatic endothelial cells (LEC). During mouse development, binding of Pdpn to the C-type lectin-like receptor 2 (CLEC-2) on platelets is critical for the separation of the lymphatic and blood vascular systems. Competitive inhibition of Pdpn functions with a soluble form of the protein, Pdpn-Fc, leads to reduced lymphangiogenesis in vitro and in vivo. However, the transgenic overexpression of human Pdpn-Fc in mouse skin causes disseminated intravascular coagulation (DIC) due to platelet activation via CLEC-2. In the present study, we produced and characterized a mutant form of mouse Pdpn-Fc, in which threonin 34, which is considered essential for CLEC-2 binding, was mutated to alanine (PdpnT34A-Fc). Indeed, PdpnT34A-Fc displayed a 30-fold reduced binding affinity for CLEC-2 compared to Pdpn-Fc. This also translated into fewer side effects due to platelet activation in vivo. Mice showed less prolonged bleeding time and fewer embolized vessels in the liver, when PdpnT34A-Fc was injected intravenously. However, PdpnT34A-Fc was still as active as wild-type Pdpn-Fc in inhibiting lymphangiogenesis in vitro and also inhibited lymphangiogenesis in vivo. These data suggest that the function of Pdpn in lymphangiogenesis does not depend on threonine 34 in the CLEC-2 binding domain and that PdpnT34A-Fc might be an improved inhibitor of lymphangiogenesis with fewer toxic side effects.
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