Characterisation of LRG1 Structure and Function

Abstract

Leucine-rich alpha-2 glycoprotein 1 (LRG1) has been implicated in a variety of inflammatory and vasoproliferative diseases. This leucine-rich repeat family protein interacts with active TGF-beta1 and modulates TGFbeta signalling to promote pathological neovascularisation through activation of the pro-angiogenic SMAD 1/5/8 pathway. Emerging data continues to reveal TGFbeta-associated functions of LRG1 in disease, such as regulation of fibrosis. Despite this knowledge, little is known about the structure or molecular mechanism of LRG1. In this study, biophysical techniques were employed to characterise the LRG1 protein. In addition, a series of LRG1 variants were designed and expressed to test specific amino acid activity in TGFbeta pathway protein interaction studies, and in models of in vitro and ex vivo angiogenesis. Findings from small angle X-ray scattering, multi-angle light scattering and analytical ultracentrifugation experiments demonstrated that LRG1 exists as a mostly spherical monomer in solution. These data combined with glycosylation state findings showed that LRG1 expressed in HEK cells is glycosylated at 4 N-linked glycan sites. This project also revealed a previously unknown interaction between LRG1 and the BMP-2 ligand of the TGFbeta superfamily. Variant LRG1 studies located a functional amino acid (S297) in the LRG1 concave face, that when mutated altered the LRG1-TGF-beta1 interaction, decreased LRG1-activation of SMAD 1/5/8 signalling, and significantly inhibited LRG1s pro-angiogenic function in the metatarsal sprouting assay (p

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Clair Baldock (Supervisor)