The HACL2 Knouckout SK-HEP-1 Polyclonal Cells product provides a heterogeneous population of SK-HEP-1 human liver sinusoidal endothelial cells (LSECs) engineered via CRISPR/Cas9-mediated disruption of the HACL2 gene. This polyclonal knockout pool enables loss-of-function studies of 2-hydroxyacyl-CoA lyase 2, a peroxisomal enzyme critical for the alpha-oxidation of 3-methyl-branched fatty acids, including phytanic acid. As a mixed population, the cells reflect diverse editing outcomes, offering a robust model for examining the functional consequences of HACL2 deficiency without the confounding effects of clonal selection.
The SK-HEP-1 host cell line was derived from the ascites of a patient with liver adenocarcinoma and exhibits hallmark hepatic endothelial features, including fenestrated monolayer formation, endocytic activity, and immune surveillance functions. These cells endogenously express peroxisomal fatty acid oxidation components, providing a physiologically relevant human LSEC model for studying alpha-oxidation pathways.
The HACL2 protein catalyzes the cleavage of 2-hydroxyacyl-CoA thioesters into formate and a fatty aldehyde, a reaction central to the peroxisomal alpha-oxidation pathway. This enzyme operates downstream of phytanoyl-CoA hydroxylase (PHYH) and works in concert with the paralog HACL1 to process 3-methyl-branched substrates. HACL2 is imported into peroxisomes via the PEX5 receptor and is transcriptionally regulated by PPAR-alpha and retinoid X receptors, which sense lipid ligands such as phytanic acid and respond to fibrate drugs like fenofibrate. The formate produced directly feeds into one-carbon metabolism, linking peroxisomal lipid breakdown to nucleotide synthesis and methylation reactions. Additionally, fatty aldehydes generated are further oxidized by aldehyde dehydrogenases such as ALDH3A2 to yield acetyl-CoA, bridging alpha-oxidation and beta-oxidation pathways.
Disruption of HACL2 in the SK-HEP-1 endothelial background creates a pertinent model for exploring how impaired alpha-oxidation affects liver sinusoidal endothelial cell function. Loss of 2-hydroxyacyl-CoA lyase activity results in accumulation of 2-hydroxyphytanoyl-CoA and reduced formate production, potentially perturbing one-carbon homeostasis and fatty aldehyde-dependent signaling. Given the role of LSECs in regulating the exchange of metabolites between blood and hepatocytes, such alterations may compromise endothelial integrity, fenestration dynamics, and endocytic capacity. This model is particularly valuable for studying peroxisomal disorders such as Refsum disease, where phytanic acid accumulation leads to neurological deficits, as well as broader aspects of hepatic lipid toxicity and endothelial dysfunction.
Researchers can employ this polyclonal HACL2 knockout cell population for diverse functional assays. Western blotting and RT-qPCR enable confirmation of reduced HACL2 expression, while enzyme activity assays using 2-hydroxyphytanoyl-CoA substrate measure catalytic output. LC-MS-based metabolomics can quantify formate and fatty aldehyde levels, and Oil Red O staining may reveal lipid accumulation under phytanic acid challenge. Immunofluorescence for peroxisomal markers such as PEX5 or catalase assesses organelle integrity, and migration/invasion assays evaluate endothelial behavior. This model supports investigations into peroxisomal alpha-oxidation in hepatic endothelium, phytanic acid toxicity, and the role of formate in one-carbon metabolism. For technical inquiries or ordering details, please contact Ascent Research.
