(546f) A Novel Mechanism for Gluten Transport across the Cell Membrane: Implications for Celiac Disease,

Celiac disease (CeD) is an autoimmune disease of the small intestine known to affect approximately 1% of the population in most parts of the world. Ingestion of gluten proteins from wheat, barley or rye by a patient with CeD causes mucosal inflammation and villous atrophy. The primary environmental and genetic factors that cause CeD, dietary gluten and the expression of HLA-DQ2.5 or HLA-DQ8 respectively, have been identified and characterized. However, short of complete and lifelong gluten exclusion there is currently no pharmacological treatment for CeD.

The site specific deamidation of gluten peptides by transglutaminase 2 (TG2) is necessary for the HLA-dependent antigen presentation of these peptides to CeD specific inflammatory T-cells. TG2 catalyzes the deamidation of gluten peptides leading to high affinity immunogenic HLA-DQ2.5 ligands. TG2 is ubiquitously expressed, and its activity is allosterically and redox regulated by other proteins and small molecules both intra- and extracellularly. TG2 is only capable of deamidating gluten peptides in its active “open” conformation.

A long unanswered question in our understanding of CeD pathogenesis is the localization of TG2 activity as well as where TG2 meets its substrates to deamidate them and how these substrates then encounter HLA-DQ2.5 in order to be presented on the cell surface. In order to address these questions, we sought to develop a probe that would allow us to directly localize active TG2 in cell culture, rather than using indirect probes. Such probes include the widely used 5-biotinamidopentylamine (5-BP), a TG2 substrate with relatively low specificity which labels other TG2 substrates rather than the enzyme itself. To this end, we developed RZ-5, a DON compound with a substituted leucine residue tagged with a fluorescent label. This probe covalently binds the cysteine residue (C277) of the TG2 active site fluorescently labeling only the active conformation of the enzyme.

Using this probe, we uncovered a distinctive localization of active TG2 in cell culture; both in mouse embryonic fibroblasts (MEFs) and HT1080 fibrosarcoma cells RZ-5 appears in punctate dots primarily located intracellularly, near the nucleus. Contrarily, 5-BP staining is strictly extracellular and diffuse, present across most of the extracellular matrix. In both cases, the TG2 activity is only observable when cells are treated with TRX to activate TG2 and is fully eliminated in the presence of TG2 specific inhibitors such as CK805. The seemingly intracellular localization of RZ-5 and the distinctive morphology of the staining point toward a yet undescribed internalization pathway for TG2 and a bound substrate indicating a potential uptake mechanism of gluten peptides in the context of CeD.

We seek to explore the mechanisms of this pathway and uncover the implications that is has for our understanding of the ways gluten is shuttled across the cell membrane in patients with CeD.