The Structural and Biochemical Effects of XFG-Associated Variants in LOXL1

PROJECT SUMMARY/ABSTRACT The goal of this project is to structurally and biochemically characterize wild-type LOXL1 and LOXL1 containing variants associated with pseudoexfoliation glaucoma (XFG). XFG is the most common form of secondary open- angle glaucoma and is characterized by abnormal accumulation of fibrillar deposits in the anterior segment of

the eye. The accumulation of these deposits obstructs normal outflow of aqueous humor, leading to elevated intraocular pressure and subsequent retinal damage and vision loss. The strongest genetic link to XFG is lysyl oxidase 1 (LOXL1), a protein responsible for the cross-linking of tropoelastin into elastin microfibers. LOXL1 is

comprised of a signal peptide, a disordered N-terminal domain, and a C-terminal copper oxidase domain. The copper oxidase domain contains the copper-containing active site and lysyl-tyrosylquinone (LTQ) cofactor necessary for cross-linking activity. Residues H449, H451, and H453 coordinate the copper (Cu(II)) ion while

residues K477 and Y512 form the LTQ cofactor. While the pro-region of LOXL1 interacts with elastin, fibulin-5 interacts with the 334-402 amino acid region of LOXL1 in order to properly orient LOXL1 with elastin fibers. While proteolytic processing by BMP1 at residues 151-152 is necessary for proper cross-linking activity of LOXL1,

additional cleavage at residues 216-217, 292-293, and 375-376 by ADAMTS14 may also occur. Genome-wide association analyses have identified two common coding variants (R141L and G153D) in LOXL1 associated with increased XFG risk and one rare coding variant (Y407F) associated with decreased XFG risk. In addition, a

number of variants (e.g., D292V, K477R) in LOXL1 have been identified in the Genome Aggregation Database (gnomAD). Little is known about how these variants impact the structure and function of currently structurally uncharacterized LOXL1. However, these variants lie in important regions of LOXL1: R141L and G153D lie near

the 151-152 BMP1 cleavage site while D292V lies within the 292-293 ADAMTS14 cleavage site. Meanwhile Y407F lies in the copper oxidase domain just downstream of the 334-402 fibulin-5 interaction region, and K477R changes the lysine residue responsible for LTQ cofactor formation. Therefore, we hypothesize that these variants

impact the structure, proteolytic processing, protein interactions, and catalytic activity of LOXL1. We will test this hypothesis with the following aims: In AIM 1, we will characterize the effects of Y407F and K477R variants on the copper oxidase domain, examining domain structure, Cu(II) metalation, LTQ formation, and catalytic activity.

In AIM 2, we will characterize the effects of R141L, G153D, and D292V variants on the ability of the N-terminal domain to fold and be cleaved by BMP1 and ADAMTS14. In AIM 3, we will examine the effects of the Y407F variant and proteolytic cleavage on fibulin-5 interactions. Successful completion of this project will identify the

effects of XFG-associated variants on LOXL1 structure and function, thereby clarifying our understanding of how LOXL1 contributes to XFG and laying groundwork for future therapeutic development.