PB0413 - Analyzing Moesin and Human TREM-like Transcript-1 (hTLT-1) Binding and Interaction

Background: Platelets are the primary characters in coagulation and hemostasis, they also play a vital role in illnesses like cardiovascular diseases making them a prime target for developing novel treatments. TREM-like transcript-1 (TLT-1), a membrane bound platelet protein found in platelet alpha granules, plays a role in the clotting process; however, not much is known about this protein. Moesin is a protein found in platelets that form a link between the platelet’s cytoskeleton and membrane bound proteins. This binding could hint at one of TLT-1’s potential roles in platelet activation and clotting.

Aims: Determine the amino acid residues on hTLT-1 that serve as the target binding site for moesin.

Methods: Anti TLT-1 antibodies were used to immunoprecipitate TLT-1 from stably transfected cell lines containing one of three truncated clones of TLT-1 of varying lengths (280 aa, 290 aa, and 300 aa long), as shown in Figure 1. Co-immunoprecipitations of HEK-293 lysates were performed for hTLT-1 and samples probed for moesin to determine binding between all clones

Results: We have characterized each of our clones and confirmed that they have the proper size by western and that they express TLT-1 by flow cytometry. Preliminary results suggest that binding is abrogated between construct 290 and 300. These studies are on going and the current state of this project will be reported here.

Conclusion(s): Human TLT-1 constructs were successfully generated. Initial results suggest binding to moesin is dependent on the region 290-300 aa located in the cytoplasmic tail of TLT-1. To further understand the contribution of each amino acid to the binding strength, we can perform an alanine scanning to replace each aa with a non-reactive alanine and test binding.