title: Mutation of threonine 34 in mouse podoplanin-Fc reduces CLEC-2 binding and toxicity in vivo while retaining antilymphangiogenic activity.
abstract: The lymphatic system plays an important role in cancer metastasis and inhibition of lymphangiogenesis could be valuable in fighting cancer dissemination. Podoplanin (Pdpn) 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 due to platelet activation via CLEC-2. In the present study, we produced and characterized a mutant form of mouse Pdpn-Fc, in which threonine 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 with 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.
authors.forename: Roberta
authors.initials: R
authors.lastname: Bianchi
authors.forename: Eliane
authors.initials: E
authors.lastname: Fischer
authors.forename: Don
authors.initials: D
authors.lastname: Yuen
authors.forename: Ellen
authors.initials: E
authors.lastname: Ernst
authors.forename: Alexandra M
authors.initials: AM
authors.lastname: Ochsenbein
authors.forename: Lu
authors.initials: L
authors.lastname: Chen
authors.forename: Vivianne I
authors.initials: VI
authors.lastname: Otto
authors.forename: Michael
authors.initials: M
authors.lastname: Detmar
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000235:genetics / Q000494:pharmacology; D037181:Lectins, C-Type / Q000235:genetics / Q000378:metabolism*; D042583:Lymphangiogenesis / Q000187:drug effects* / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D020125:Mutation, Missense; D015539:Platelet Activation / Q000187:drug effects / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D000098853:Podoplanin
chemical_list: D007127:Immunoglobulin Constant Regions; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C529518:CLEC-2 protein, mouse; C490441:Pdpn protein, mouse; D000098853:Podoplanin