Research on Modes of Antigen Recognition by Variable Lymphocyte Receptors (VLRs)

Abstract

In jawless vertebrates, variable lymphocyte receptors (VLRs) play a crucial role in recognition of antigens as part of the adaptive immune system. Leucine-rich repeat (LRR) modules and the highly variable insert (HVI) of VLRs contribute to the specificity and diversity of antigen recognition. VLR2913, whose antigen is not known, contains the same HVI amino acid sequence with that of VLR RBC36, which recognizes the H-trisaccharide from human blood type O erythrocytes. Since the HVI sequence is rarely identical among all known VLRs, I am attempting to identify the antigen for VLR2913 and the main contributing factors for antigen recognition based on comparison of VLR2913 and VLR RBC36. To initiate and facilitate the structural approach, the ectodomain of VLR2913 was fused with the N-terminal domain of Internalin B (InlB-VLR2913-ECD). I mutated three amino acid residues on the concave surface of LRR modules of InlB-VLR2913-ECD, considering important residues for hydrogen bonds in recognition of H-trisaccharide by VLR RBC36. The InlB-VLR2913-ECD was overexpressed in Escherichia coli, and crystallized at 295 K using sitting-drop vapour-diffusion methods. X-ray diffraction data were collected to 2.04 Å resolution. Crystal structure of InlB-VLR2913-ECD was determined by molecular replacement (MR). As a reference, VLR RBC36 ectodomain was fused with N-terminus of Internalin B (InlB-VLR RBC36-ECD). InlB-VLR RBC36-ECD was overexpressed in E. coli and purified up to 99% purity. In binding affinity experiment by ITC200, InlB-VLR2913-ECD with 7 residues mutation, which mimicked hydrogen bonds and van der Waals interaction based on the sequence alignment between VLR RBC36 and VLR2913, showed interaction with H-trisaccharide, but wild type and three residues mutant form of InlB-VLR2913-ECD were not. I am currently trying to determine how concave surface residues influence to antigen recognition through mutation of key factor and crystal structure of VLR2913 mutant complex with H-trisaccharide.