טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentAlmog May
SubjectPlatelet-Activating Receptor CLEC-2: Characterization and
Modulation of Protein-Ligand Interactions
Promoting Cancer Metastases
DepartmentDepartment of Biology
Supervisor Professor Meytal Landau
Full Thesis textFull thesis text - English Version


Abstract

Platelet C-type lectin like receptor 2 (CLEC-2) is a lectin-like transmembrane platelet receptor that is activated and functions as a homodimer. Under physiological conditions, CLEC-2 plays a role in hemostasis and thrombosis, in the development of lymphatic vessels and their separation from blood vessels during embryogenesis and in promoting megakaryocyte growth. Apart from its physiological roles, CLEC-2 also mediates snake envenomation via interactions with the snake toxin Rhodocytin from the Malayan pit viper, resulting in massive platelet aggregation and tissue damage leading to dysfunctional and amputated limbs.  In pathophysiology, CLEC-2 is involved in passive transfer of HIV infected cells after budding, and in promoting cancer metastases by interacting with its endogenous ligand, Podoplanin, a heavily glycosylated mucin, which is over-expressed on the surface of cancer cells in more than a hundred types of cancers. The extracellular part of Podoplanin includes four PLAG (EDxxVTPG) domains, with a sialo-sugar moiety, termed disialyl-core 1, on Threonine 52, which is essential for the binding and for subsequent oligomerization of CLEC-2, leading to platelet-aggregation. The specific binding site of Podoplanin and the dimerization interface are yet to be clarified. 

Computational analysis performed in our lab by Einav Tayeb-Fligelman predicted a binding pocket for Podoplanin's disialyl-core 1 within the structure of CLEC-2, including a triad of conserved residues within the pocket. Indeed, docking simulations with disialyl-core 1 as a ligand indicated preference to bind to this predicted binding site.  Einav Tayeb-Fligelman was already able to crystallize and solve the structure of the C-type lectin-like ectodomain of CLEC-2, expressed and purified in a bacterial system from inclusion bodies. The CLEC-2 lectin domain showed a monomeric form, similar to a previous structure determined at lower resolution. Crystallization of the CLEC-2 lectin domain in the presence of a commercial sialo-sugar ligand, which resembles the Podoplanin disialyl-core 1, showed formation of dimers in crystals, with our predicted disialyl-core 1 binding pocket located at the dimerization interface between the two monomers. Unfortunately, probably due to either low occupancy or high flexibility of the ligand within the binding pocket, the ligand was not evident in the structure.

CLEC-2 has two N-glycosylated sites in the C-type lectin-like domain, and a third glycosylation site at the stalk region connecting the lectin domain to the transmembrane region. Glycosylations can change and determine the physicochemical properties of the protein and play a key role in protein stability, affinity and activity. Dimerization of CLEC-2 was also shown to be stabilized by post-translational glycosylation. For this reason, to gain biologically relevant insights, this study focused on the production of glycosylated CLEC-2 in a mammalian expression system and characterizing dimerization. I managed to obtain purified CLEC-2 as a soluble ectodomain construct produced in human cells. I was then able to use the produced CLEC-2 for analysing its oligomerization state upon introduction of a commercial sialo-sugar in solution, and to initiate crystallization attempts. Further optimizations are needed for obtaining a crystal structure of the glycosylated CLEC-2, for uncovering its yet elusive dimerization interface, and for validating the binding interfaces with its proteinaceous ligands.