Functional Analyses of the Crohn’s Disease Risk Gene LACC1

Abstract

BackgroundGenetic variation in the Laccase (multicopper oxidoreductase) domain-containing 1 (LACC1) gene has been shown to affect the risk of Crohn’s disease, leprosy and, more recently, ulcerative colitis and juvenile idiopathic arthritis. LACC1 function appears to promote fatty-acid oxidation, with concomitant inflammasome activation, reactive oxygen species production, and anti-bacterial responses in macrophages. We sought to contribute to elucidating LACC1 biological function by extensive characterization of its expression in human tissues and cells, and through preliminary analyses of the regulatory mechanisms driving such expression.MethodsWe implemented Western blot, quantitative real-time PCR, immunofluorescence microscopy, and flow cytometry analyses to investigate fatty acid metabolism-immune nexus (FAMIN; the LACC1 encoded protein) expression in subcellular compartments, cell lines and relevant human tissues. Gene-set enrichment analyses were performed to initially investigate modulatory mechanisms of LACC1 expression. A small-interference RNA knockdown in vitro model system was used to study the effect of FAMIN depletion on peroxisome function.ResultsFAMIN expression was detected in macrophage-differentiated THP-1 cells and several human tissues, being highest in neutrophils, monocytes/macrophages, myeloid and plasmacytoid dendritic cells among peripheral blood cells. Subcellular co-localization was exclusively confined to peroxisomes, with some additional positivity for organelle endomembrane structures. LACC1 co-expression signatures were enriched for genes involved in peroxisome proliferator-activated receptors (PPAR) signaling pathways, and PPAR ligands downregulated FAMIN expression in in vitro model systems.ConclusionFAMIN is a peroxisome-associated protein with primary role(s) in macrophages and other immune cells, where its metabolic functions may be modulated by PPAR signaling events. However, the precise molecular mechanisms through which FAMIN exerts its biological effects in immune cells remain to be elucidated.

Laccase Properties, Physiological Functions, and Evolution

“…The laccase of Suberites domuncula showed high sequence similarity with the insect laccases, including Bombus impatiens, Apis mellifera, and Acromyrmex echinatior, and with nematode enzymes from Caenorhabditis remanei, Ancylostoma ceylanicum, and Haemonchus contortus [202]. Recently, genetic variation in the gene coding for human laccase has been associated with the risk of Crohn's disease, leprosy, ulcerative colitis, and juvenile idiopathic arthritis [10]. In many mammalian species, putative sequences coding for this enzyme are identified (Table S1).…”

“…Interestingly, the human LACC1 gene shows no similarity to the other mammalian proteins, since it contains a C-terminal domain homologous to bacterial multicopper polyphenol oxidases (PO) and laccase [10]. Alignment of laccase sequences across the tree of life shows that they are grouped into main clades: two-and three-domain oxidases (Figure 3).…”

“…However, it is hard to elucidate the origin of short sequences (c.a. 170 aa) from vertebrates, which were annotated in recent years and some of them are biochemically categorized as laccases [10]. There is a chance that they may form a separate branch of MCO evolution, as their structures are rather close to prokaryotic laccases (Figures 1 and 2).…”

LACC1 Required for NOD2-Induced, ER Stress-Mediated Innate Immune Outcomes in Human Macrophages and LACC1 Risk Variants Modulate These Outcomes

“…Understanding the consequences of genetic variants associated with immune-mediated diseases can often lead to unexpected elucidation of mechanisms and pathways required for maintaining immune homeostasis. LACC1 genetic variants are associated with inflammatory bowel disease (IBD) (Jostins et al, 2012), leprosy (Liu et al, 2015), and juvenile arthritis (Wakil et al, 2015), yet only a limited number of studies has examined the functional consequences of mammalian laccase domain containing-1 (LACC1) protein and its variants (Assadi et al, 2016;Cader et al, 2016;Lahiri et al, 2017;Skon-Hegg et al, 2019). We and others found that the common LACC1 Ile254Val variant (allele frequency 0.226-0.245 in European ancestry individuals per the dbSNP) results in a loss of function as manifested by decreased cytokine secretion, bacterial clearance, ROS production, and fatty-acid oxidation in macrophages (Cader et al, 2016;Lahiri et al, 2017).…”

“…Subcellular localization can provide important insight into mechanisms through which proteins mediate their effects, with localization to multiple subcellular compartments allowing for proteins to carry out distinct functions at these respective sites (Yogev and Pines, 2011). Previous studies showed LACC1 localized to the cytosol, mitochondria, and/or peroxisomes in human macrophages (Assadi et al, 2016;Cader et al, 2016;Lahiri et al, 2017). However, our in silico analysis showing LACC1 localization to the ER and a prior study showing that a small percent of LACC1 may also localize to the ER in THP-1 cells under homeostatic conditions (Assadi et al, 2016) set the foundation for additional LACC1 mechanisms.…”