Perforin is a Ca²⁺-dependent highly cytotoxic pore-forming protein that is stored in secretory granules of cytotoxic lymphocytes (CL). Perforin is essential for protection against viral infections and cellular transformation. In humans, the loss of perforin expression leads to fatal immune dysregulation, infection and/or cancers.

When naive CLs activate in response to an infection or cancerous cells, they rapidly synthesise large quantities of perforin. How they survive this stress and protect themselves against perforin’s inherent toxicity remains unknown.

Two decades ago, it was hypothesised that the N-glycosylated C-terminal peptide of perforin interferes with its Ca²⁺-dependent membrane-binding C2 domain necessitating its cleavage to activate the molecule. When we determined the X-ray crystal structure of perforin (Law, Nature 2010), it became apparent that such an interaction was highly unlikely. Furthermore, we discovered that the C-terminus of perforin is critical for its export from the endoplasmic reticulum, where at the neutral pH and high concentrations of Ca²⁺ perforin can unleash its lethal force (Brennan, Immunity 2011). Still, the C-terminus of perforin is invariably cleaved in secretory granules, but why and how this occurred remained a mystery.

We have discovered that the C-terminal glycan does not influence perforin membrane binding, but instead interferes with the formation of a pore. This is a unique example of deglycosylation being used in this manner to regulate protein function, effectively by modulating the homotypic protein-protein interaction necessary for pore formation. Remarkably, we found that despite an invariable conservation of the C-terminus, its cleavage is not determined by a specific protease consensus site but rather by the susceptibility of the unstructured C-terminal ‘tail’ to a range of peptidases.

Our work defines a unique mechanism of CL protection from their own essential yet lethal molecule, perforin. This mechanism is critical to CL function and, more broadly, immune homoeostasis. This study has resolved a critical remaining question in CL biology and has reconciled numerous studies, spanning two decades of research.