Investigating ps surface sensitivity and calcium dependency of hTIM-1 and hTIM-4.

Abstract

Transmembrane immunoglobulin and mucin domain (TIM) protein plays an important role in the immune system. TIM protein utilizes the phosphatidylserine (PS) binding pocket of the IgV domain to recognize PS lipids on the surface of target cells, and the binding is calcium-mediated. Although all members of the TIM protein family possess PS-binding pockets, they are involved in different immune response and are expressed on the surface of different immune cells. To understand the variation of intercellular recognition mechanisms, it is important to investigate their surface sensitivities to PS and their dependency on calcium for binding. Previous work in our group showed that murine TIM1 (mTIM1) and TIM4 (mTIM4) have different PS surface sensitivities. Moreover, mTIM1 and mTIM4 can bind, to different extent, to the membrane without calcium ions, but calcium ions enhance binding. Since literature shows that genes of mTIM1 and mTIM4 are orthologs of human TIM1 (hTIM1) and TIM4 (hTIM4), respectively, we aim to understand the PS surface sensitivities and calcium dependency of hTIM1 and hTIM4, and elucidate if the difference of their PS surface sensitivities and calcium dependency preserve between murine and human TIM. hTIM1 and hTIM4 have three tryptophan amino acids, but only one of them buries into the membrane when they bind to the membrane. Using a tryptophan fluorescence assay, we quantify PS surface sensitivities and calcium dependency of hTIM1 and hTIM4. We have observed that hTIM1 binds to 7:3 POPC:POPS vesicles without calcium and the fluorescence spectrum blue shifts. By investigating factors of membrane-binding mechanism for hTIM1 and hTIM4, we help elucidate difference in TIM binding between human and murine, and may provide further insights on intercellular recognition mechanisms.