Utilizing SULT4A1 Mutant Mouse Models to Characterize SULT4A1

Abstract

Identified by our lab in 2000, sulfotransferase 4A1 (SULT4A1) belongs to the cytosolic sulfotransferase (SULT) gene superfamily of phase II conjugation enzymes based on key sequence and structural similarities. Broadly, SULTs catalyze the transfer of a sulfonate moiety (SO3−) to a diverse set of endogenous and exogenous substrates in tandem with the obligate cofactor 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS). Expression studies have shown SULT4A1 to be localized to neuronal tissues, and SULT4A1 orthologs have been found in all vertebrate species examined. Remarkably, human SULT4A1 shares a high degree of sequence homology with other vertebrate SULT4A1 orthologs, more than any other human SULT. Despite the conserved nature of SULT4A1 among vertebrates, all attempts to demonstrate sulfotransferase activity have failed and the neuronal function of this orphan enzyme remains unclear. To elucidate an understanding of SULT4A1, CRISPR‐Cas9 gene editing technology was performed to disrupt SULT4A1 function in a C57BL/6J mouse background and two mutations of interest were pursued. A 28 bp deletion (Δ28) in exon 1 resulted in a frameshift mutation and a premature stop codon at amino acid (AA) 62. A second mutation of interest was a 12 bp (Δ12) in‐frame deletion in exon 3 of four AA immediately adjacent to the active site histidine. Dynamic modeling studies of the Δ12 mutation suggested a structural disruption to the substrate binding pocket. Interestingly, homozygous mutant pups of both lines displayed a severe and progressive neurological pathology beginning 8–10 days post‐natal, until death at day 21–25. Neurological symptoms included persistent tremor, ataxia, absence seizures, and decreased weight gain. Homozygous mutant mice of both strains were confirmed to lack detectable SULT4A1, while heterozygous mice of both strains showed a modest decrease compared to wild‐type (WT) mice. Since little is known about SULT4A1 function, subcellular fractionation was performed to determine if SULT4A1 localizes to other cellular compartments. Indeed, SULT4A1 was detected in mitochondrial, cytosolic, and microsomal fractions, a previously unreported finding. Due to the early onset of neurological symptoms, SULT4A1 expression was assessed in primary cortical neurons (PCNs) harvested from gestational day 15 mice. PCNs were cultured in vitro to day 12 and robust expression of SULT4A1 was observed in these cells by immunostaining. In summary, SULT4A1 is a critical protein for neurofunction and normal development, as mice lacking functional SULT4A1 displayed a progressively severe and lethal neurological pathology. These mutant mouse models will be instrumental into characterizing the function of SULT4A1.Support or Funding InformationThis work was supported by GM113980 to CNF and NS08695301 to SAA.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.