Modulation and detection of transglutaminase 2 activity and gluten-derived peptides in vivo : implications for celiac disease

Celiac disease (CeD) is a complex autoimmune disorder that currently affects approximately 1 in 100 individuals. In CeD, dietary consumption of gluten (a class of proteins found in wheat, rye, and barley) drives an inflammatory response that classically presents as enteropathy of the small intestine. Unlike most dietary proteins, gluten resists digestion by gastrointestinal proteases and crosses the epithelial barrier of the gut in the form of relatively long peptides. In genetically susceptible individuals, these peptides bind to human leukocyte antigen (HLA)-DQ2 or DQ8 on the surface of antigen presenting cells and initiate a TH1-type immune response. The protein transglutaminase 2 (TG2) is central to CeD pathogenesis, as it is the autoantigen and is also responsible for deamidating gluten peptides, which markedly increases their affinity for HLA-DQ2/8. Avoidance of gluten is the only treatment for this lifelong aliment. Thus, there is a pressing need for development of non-dietary therapies. However, efforts to develop pharmacological treatments for CeD have been hindered by fundamental gaps in our understanding of its molecular pathogenesis, as well as by the lack of a pre-clinical animal model. The work in this thesis addresses these issues. In Chapter 1, we provide an overview of TG2 and discuss the role of chemical biology in furthering our understanding of this enigmatic protein. This review sets the context for Chapter 2, where we describe development of chemical biological tools for modulating extracellular TG2 activity in vivo. Previous evidence suggested that extracellular TG2 is held catalytically dormant by formation of an allosteric disulfide bond (Cys370-371). We hypothesized that a reduction reaction promoted by the protein thioredoxin-1 (TRX) would be sufficient to induce TG2 activity in vivo. This hypothesis was tested in several experiments. We first show that immune cells secrete endogenous TRX that activates their extracellular pools of TG2. The specificity of this protein-protein interaction is demonstrated by engineering of a TRX mutant to kinetically trap its substrate proteins. This mutant TRX labeled tissues from wild-type but not TG2-null mice, establishing TG2 as the preferred physiological substrate of TRX in the extracellular environment. Finally, intravenous administration of wild-type recombinant TRX to living mice promoted extracellular TG2 activity, as judged by incorporation of the activity probe 5-biotinamidopentylamine. This activity was blocked by ERW1041E, an active site directed TG2 inhibitor. At a minimum, this work provides a facile set of tools for activating, inhibiting, and detecting TG2 activity in vivo. More broadly, these data provide functional support for the emerging hypothesis that TRX is responsible for upregulating TG2 activity in CeD patients. After the identification of recombinant TRX as a selective reductant of TG2, we wished to identify an analogous tool to catalyze the reverse oxidation reaction. In Chapter 3, we hypothesized that cystamine, a symmetric disulfide compound, could serve as one such molecule. Through kinetic analysis and mass spectrometry-based disulfide mapping, it is shown that cystamine efficiently promotes formation of the Cys370-371 disulfide of TG2. Given that cystamine has widely been used as a TG2 inhibitor in animal models, we searched for clinically approved molecules that could inhibit TG2 by the same mechanism. We identify disulfiram, an oral thiuram disulfide compound, as a fairly potent TG2 inhibitor and a promising candidate for evaluating the effect of inhibiting intestinal TG2 in human CeD patients. In Chapters 4 and 5, we describe the development of the most advanced mouse model of CeD to date and its use in interrogating the role of TG2 activity in CeD pathogenesis. In humans, HLA-DQ2 or -8 alleles are necessary but not sufficient for development of CeD. Guided by observations that the majority of CeD patients overexpress the cytokine interleukin-15 (IL-15), we hypothesized that both an HLA-dependent adaptive immune response to gluten and intestinal stress induced by IL-15 are required for the onset of CeD. Accordingly, we engineered a mouse model that expresses HLA-DQ8 along with IL-15 in both the lamina propria and epithelium of the small intestine. These mice develop the key molecular and phenotypic features of human CeD, including gluten-dependent enteropathy of the small intestine. Comparative transcriptional analysis of our model with human patient biopsy samples showed a strikingly high degree of overlap in differentially expressed genes upon gluten feeding, providing evidence that these mice are a reliable pre-clinical disease model. Using the protocols validated in Chapter 2, we then studied the effects of pharmacological inhibition of TG2. As shown in Chapter 5, TG2 inhibition significantly reduced the extent of gluten-induced intestinal damage, providing strong evidence for the essential role of TG2 in CeD pathogenesis and highlighting TG2 as a promising target for treatment in humans. Lastly, in Chapters 6 and 7, we develop a rapid and robust method for analysis of the human urinary peptidome by liquid chromatography-mass spectrometry (LC/MS) and apply it to analyze gluten peptides excreted in human urine. We show that cleanup of urine can be achieved using hydrophobic interaction/strong cation exchange solid phase extraction. This simple technique relieves throughput limitations imposed by previously reported methods for removal of urinary matrix interferents. Analysis of urine from healthy donors revealed the presence of approximately four times more human peptides than reported in the literature to date. Relevant to our interest in CeD, antibody-based methods have indicated that peptides from dietary gluten survive gastrointestinal degradation and accumulate in urine. We therefore hypothesized that our peptidomic method would be capable of revealing the specific sequences of these peptides. To test this hypothesis, volunteers were challenged with a gluten-containing meal, and urine was subsequently collected. LC/MS analysis revealed a rich repertoire of gluten peptide sequences in gluten-challenged individuals but not in controls who maintained a gluten-free diet. Some of the identified peptides are known to stimulate the adaptive or innate immune response in CeD, whereas others are novel sequences that warrant further study. In a small cohort of CeD patients, levels of gluten-derived peptides were altered, suggesting that CeD patients may digest, absorb, and excrete gluten differently from healthy individuals. Further studies with larger sample sizes are needed to more clearly elucidate these differences. Collectively, the projects presented in this thesis advance our molecular understanding of the roles of TG2 activity and gluten digestion in CeD pathogenesis. Future avenues for study include determining if TG2 inhibition is a viable therapeutic option for CeD in humans and if specific gluten-derived peptides can serve as non-invasive biomarkers of CeD status.

Keywords: Chemistry.
Advisor: Khosla Chaitan
Kool Eric T. 
Zare Richard N.