R01AI170844

Determining the Origins of Nonclassical Class I Molecules through Molecular and Functional Approaches - Summary

Sharks are members of the oldest class of living vertebrates with adaptive immunity based on immunoglobulin, T cell receptors, and the major histocompatibility complex. Recently, the Flajnik Lab uncovered eight class I lineages, including the classical class I (UAA) and seven nonclassical lineages (UBA to UHA). Surprisingly, two of these MHC-linked shark nonclassical class I (class IB) molecules show striking similarities to the human nonclassical class I molecules CD1 and HLA-E.

Preliminary data on genetics, expression, and sequence similarity strongly suggest that UFA is the orthologue of CD1. Structural modeling and in silico biophysical analyses in the Adams Lab support UFA's clear similarity to CD1, revealing a strongly hydrophobic binding site. The second shark class IB molecule, UDA, is predicted to bind peptides like classical class I, yet it is monomorphic, single-copy, and has properties similar to mammalian HLA-E/QA-1.

The aim of this study is to biochemically define UFA and UDA ligands and elucidate their structures. Recombinant versions of UDA and UFA will be expressed and purified for biochemical analysis, including ligand elution and preparation of tetramers to identify the shark cells recognizing these nonclassical class I molecules. Highly purified forms of recombinant proteins will be used for structural analysis through X-ray crystallography.

Using in-house technologies, nanobody binding reagents will be generated to these proteins for structural analysis. Additionally, the antisera and monoclonal antibodies already generated for tissue-expression analysis will be complemented. In situ hybridization and antibody/nanobody staining will be used to define UDA/UFA cellular expression and examine their upregulation in cells under stress.

UDA and UFA tetramers will reveal which lymphocytes interact with UDA/UFA and, with single-cell analysis, the adaptive and/or innate receptors that interact with them. Biochemical analysis of shark primary cells will be performed to characterize UDA/UFA biosynthetic pathways.

This proposal combines the expertise and experience of two investigators, Martin Flajnik, who has studied the genetics, biochemistry, and function of the shark immune system for 35 years and generally the evolution of adaptive immunity, and Erin Adams, a structural biologist with deep knowledge of the human class I system and evolutionary biology.

In summary, the hypothesis is that CD1 and HLA-E analogues arose at the big bang of adaptive immunity 500 million years ago. This suggests that peptide-binding by the classical class I, lipid-binding by "CD1," and peptide-binding by a jack-of-all-trades "HLA-E-like" nonclassical class I were significant features in the earliest adaptive immune system.

University Of Maryland, Baltimore

NOT APPLICABLE

93.855 - Allergy and Infectious Diseases Research

National Institute of Allergy and Infectious Diseases (NIAID) [HHS - NIH]

Baltimore, Maryland 21201 United States

Single Zip Code

Processing and Presentation of Non-Conventional MHC Ligands (R01 Clinical Trial Not Allowed) (PA-19-067)

Amendment Since initial award the total obligations have increased 392% from $710,408 to $3,497,540.