Abstract
Purpose :
Glaucoma is the leading cause of irreversible blindness worldwide. Pseudoexfoliation glaucoma (XFG) is the most common form of secondary open-angle glaucoma. The strongest genetic link to XFG is lysyl oxidase like 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. Aside from its role in elastin crosslinking, little is known about LOXL1. Currently, the structure of LOXL1 has not been solved, biophysical characterization of human LOXL1 is lacking, and the role of XFG-associated variants in LOXL1 is not well understood.
Methods :
This project sought to express and purify LOXL1 for future structural, biophysical, and biochemical characterization using two expression systems: a mammalian ExpiCHO expression system for full-length LOXL1 and a BL21 E. coli expression system for N- and C-terminal constructs of LOXL1 for a divide-and-rebuild approach. Structural characterization was conducted using circular dichroism (CD) and metal analysis was conducted using total reflection X-ray fluorescence (TXRF).
Results :
Stable full-length LOXL1 protein (~67kDa) was obtained from the soluble intracellular fraction of ExpiCHO cells, while a naturally proteolytically cleaved product (~35kDa) containing the copper oxidase domain was obtained from ExpiCHO spent media. Meanwhile, N- and C-terminal domains of LOXL1 were obtained from a BL21 E. coli expression system. Structural characterization by CD suggests that the C-terminal end of LOXL1 is predominantly comprised of beta-sheets, while full-length LOXL1 exhibits a strong alpha helical signature. Metal analysis of LOXL1 has shown that supplementing growth media with copper is necessary for LOXL1 incorporation of enzymatically critical Cu(II).
Conclusions :
Taken together, this work has laid the foundation for future biochemical characterization of LOXL1 in both its wild-type and XFG-associated variant forms. Biochemical characterization of variant LOXL1 will allow us to address the critical knowledge gap of how variant LOXL1 contributes to this severe form of glaucoma.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.