Identification and characterisation of novel ligands presented by major histocompatibility complex class I-related protein 1(MR1)

Mathias, Edward J. (2026). Identification and characterisation of novel ligands presented by major histocompatibility complex class I-related protein 1(MR1). University of Birmingham. Ph.D.

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Abstract

Major histocompatibility complex class I-related protein 1 (MR1) presents bacterially derived and endogenous metabolite antigens at the mammalian cell surface to MR1-restrictedT(MR1T) cells. A limited number of endogenously derived metabolites have been identified which can be stabilised within the binding groove of MR1. Evidence has implicated MR1 in autoimmune diseases and cancer, however, no associated metabolite antigens presented in these conditions have been identified. Furthermore, cellular studies have discovered MR1T cells activated by endogenously derived metabolites presented by MR1, but these compounds have not been elucidated highlighting that the ligandome of MR1 is not fully characterised. Only a limited number of studies have employed ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) analysis techniques to identify novel, endogenously derived ligands of MR1. In this thesis, methods to produce, isolate, and identify ligands bound toMR1 expressed in mammalian cell lines and human cells have been developed and applied.
Intact and native MS analyses of MR1 expressed in a mammalian cell line were first carried out to identify the m/z of ligands in complex with the protein. To remove protein-induced complexities during analysis, a solvent extraction method was optimised to isolate both weakly and tightly associated metabolites from the protein for UHPLC-MS analysis. UHPLC-MS assays were then developed for the detection of ligands belonging to suspected and predicted metabolite classes as well as for the unbiased analysis of MR1-bound metabolites. The optimised assays were applied to samples of ligands released from MR1 expressed in mammalian cell lines and healthy donor monocyte and B cells.
An extraction method following a dilution of MR1 with a basic buffer followed by protein precipitation and metabolite extraction with methanol was optimal for the isolation of MR1-bound ligands. A Phenyl and a Z-HILIC column provided complementary detection and separation of the evaluated chemical standards and the intracellular metabolite extracts used to develop the untargeted analysis method. These UHPLC-MS assays, as well as the intact and native MS analysis, have uncovered m/z values, putative metabolite annotations, and metabolite identifications of endogenously derived MR1-ligands. The experimental assays and analysis methods developed in this thesis can be employed in future MR1 studies focused on identifying novel ligands. The profiling of intracellular metabolomes of the cell lines used to generate MR1 samples has been carried out to assist metabolite annotation and identification but due to word count restrictions has not been included in this thesis.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):