Analysis of Perforin Assembly by Quartz Crystal Microbalance Reveals a Role for Cholesterol and Calcium-independent Membrane Binding

Sarah E Stewart,Catherina H Bird,Rico F Tabor,Michael E D'Angelo,Stefania Piantavigna,James C Whisstock,Joseph A Trapani,Lisandra L Martin,Phillip I Bird

doi:10.1074/jbc.m115.683078

Abstract

Perforin is an essential component in the cytotoxic lymphocyte-mediated cell death pathway. The traditional view holds that perforin monomers assemble into pores in the target cell membrane via a calcium-dependent process and facilitate translocation of cytotoxic proteases into the cytoplasm to induce apoptosis. Although many studies have examined the structure and role of perforin, the mechanics of pore assembly and granzyme delivery remain unclear. Here we have employed quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate binding and assembly of perforin on lipid membranes, and show that perforin monomers bind to the membrane in a cooperative manner. We also found that cholesterol influences perforin binding and activity on intact cells and model membranes. Finally, contrary to current thinking, perforin efficiently binds membranes in the absence of calcium. When calcium is added to perforin already on the membrane, the QCM-D response changes significantly, indicating that perforin becomes membranolytic only after calcium binding.

Highlights

Cytotoxic lymphocytes have the capacity to eliminate virally infected or otherwise compromised cells by releasing stored cytotoxins from granules and/or by engaging death receptors
Perforin synergizes with granzymes to induce apoptosis; the mechanism by which perforin facilitates granzyme entry into the target cell remains a topic of debate
Perforin Binding and Assembly Measured by quartz crystal microbalance with dissipation monitoring (QCM-D)—To assess the binding and assembly of perforin, gold-coated sensors were modified with mercaptopropionic acid (MPA) and treated with DMPC: Chol liposomes to form a lipid bilayer on each of the four sensors (31)

Summary

Introduction

Cytotoxic lymphocytes have the capacity to eliminate virally infected or otherwise compromised cells by releasing stored cytotoxins from granules and/or by engaging death receptors. One stored cytotoxin is the pore-forming protein, perforin, which facilitates entry of other cytotoxins such as serine proteases (granzymes) into the target cell (1). Perforin is a 60 kDa member of the membrane attack complex/perforin (MACPF) family of proteins It comprises a MACPF domain, an epidermal growth factor-like domain, and a C2 calcium-binding domain (6, 7). Perforin monomers oligomerize to form 16 –20 nm diameter pores in the plasma membrane of target cell (8, 9). Conformational changes take place within each subunit This involves the unfurling of two key ␣-helices to form ␤-strands, together with vertical collapse of the pre-pore toward the membrane surface. In the case of the well studied CDC, Streptolysin O (SLO), a large ϳ30 nm pore forms in the membrane, allowing molecules of up to 150 kDa to enter cells (13–16). In contrast to CDCs, perforin pores show no indication of vertical collapse, suggesting that there are significant differ-

Results

Discussion

Conclusion