Defining the functional sites in LRG1 that are responsible for its role in ocular angiogenesis

Abstract

Purpose : Elevated levels of extracellular matrix protein leucine-rich repeat alpha 2 glycoprotein 1 (LRG1) have been implicated in the progression of a number of vasoproliferative conditions including neovascular age-related macular degeneration and proliferative diabetic retinopathy. LRG1 was reported to direct the TGFβ pathway towards pro-angiogenic smad 1/5 signalling in endothelial cells. Despite this knowledge, little is known about the structure of LRG1 and its specific interactions with the TGFβ pathway and other extracellular matrix (ECM) proteins. We aimed to collect structural information on LRG1 and elucidate LRG1 active sites that are involved in the pro-angiogenic TGFβ signalling pathway and angiogenesis.

Methods : We have used small angle X-ray scattering (SAXS), multi-angle light scattering (MALS) and analytical ultracentrifugation (AUC) to collect structural data from purified human LRG1. Using a homology model and mutagenesis, we designed and expressed a number of LRG1 variants, each with a single amino acid point mutation at a predicted functional site. The variants were screened for binding to TGFβ1 and ECM proteins in surface plasmon resonance (SPR) and coprecipitation assays. Variants of interest that exhibited altered binding capabilities to known LRG1 binding partners were taken forward in to in vitro and ex vivo angiogenesis assays including the endothelial tube formation assay and the aortic ring assay.

Results : Using SAXS, MALS and AUC, we determined that LRG1 is monomeric, rigid and mostly spherical. A number of the LRG1 variants have a reduced binding affinity to TGFβ1 or ECM proteins as shown by SPR and coprecipitation assays. Of particular interest, one variant is no longer able to bind TGFβ1, however, its association with other LRG1 binding partners is well preserved. In functional studies, this variant loses its promoting effect on endothelial cell tube formation and vessel sprouting from mouse aortic rings.

Conclusions : We show that LRG1 contains discrete functional sites for different protein-protein interactions. The capability of LRG1 to bind TGFβ1 is critical for its angiogenic function as demonstrated in in vitro and ex vivo assays. This study provides novel insights into the mechanism of action of LRG1 and this may be exploited when designing anti-LRG1 therapeutics.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

LRG1 homology model with N-linked glycans fitted in to a LRG1 bead model map generated from SAXS data