Lysyl Oxidase Like 2 Processing by Factor Xa Modulates its Activity and Localization
Authors: Wang Huilei , Chen James , Poe Alan , Hong Yurie , Jandu Sandeep , Nandakumar Kavitha , Steppan Jochen , Santhanam Lakshmi 
Published: June 2021

IntroductionLysyl oxidase like 2 (LOXL2) is a member of the lysyl oxidase (LOX) family of amine oxidases that catalyzes the oxidative deamination of lysine residues to crosslink extracellular matrix (ECM) proteins, thereby forming a stable and highly organized matrix. LOXL2 is shown to be involved in a diverse set of diseases including hepatic fibrosis and cancer metastasis, due to its critical role in tubulogenesis, matrix assembly, and angiogenesis. Despite the significant advances made in linking LOXL2 to fibrosis initiation and disease progression, the mechanisms of LOXL2 regulation are still largely unknown.Prior studies have shown that LOXL2 is processed extracellularly and LOXL2 processing putatively regulates its catalytic function. However, the exact identity of the protease that processes LOXL2 remains unknown. Specifically, LOXL2 is composed of a C‐terminal prototypic LOX domain that has the enzymatic function to catalyze ECM protein crosslinking, and four N‐terminal scavenger receptor cysteine rich (SRCR) domains, which are typically involved in protein‐protein recognition and signaling. Extracellular processing releases an N‐terminus fragment of 317 amino acids containing SRCR domains 1–2 and a C‐terminus fragment of SRCR domains 3–4 along with the LOX domain.Methods and ResultsIn silico digest of LOXL2 using the ExPASy peptide cutter showed Factor Xa as the potential protease that cleaves LOXL2 at position 317. Therefore, we first tested processing of exogenously expressed LOXL2 in cell culture media in vitro. Factor Xa dose dependently increased LOXL2 processing, and this was inhibited using aprotinin (non‐specific inhibitor) and rivaroxaban (specific inhibitor). We next determined the effect of LOXL2 processing on its sub‐cellular localization. Comparing the full‐length and processed LOXL2 content in the conditioned cell‐culture media and ECM showed that full length LOXL2 is predominantly found in the ECM and the processed form is found predominantly in the media. Next, we investigated whether LOXL2 processing regulates its catalytic activity. To this end, we measured the in‐situ assembly of FITC‐labeled collagen I or elastin, the main substrates of LOXL2, by processed and full length LOXL2. The incorporation of collagen I/elastin and the deposition of LOXL2 were visualized by confocal epifluorescence microscopy. Our findings indicate that LOXL2 processing markedly reduces collagen I/elastin incorporation when compared to that by full‐length LOXL2.ConclusionsFactor Xa is the protease responsible for LOXL2 processing in vitro. LOXL2 processing inhibits its ability to utilize collagen I and elastin as substrates. Future studies will continue to 1) investigate whether LOXL2 processing regulates its substrate specificity by shifting LOXL2 catalysis to Collagen IV, 2) determine the functions of the cleaved SRCR1–2 propeptide, 3) identify the specific mechanism by which processed and full length LOXL2 are trafficked to the cell‐derived ECM, and 4) evaluate the in vivo significance of this processing in the vascular compartment.Support or Funding InformationFunded by JHMI ACCM StAAR Grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.