The Usher Syndrome Type IIIB Histidyl-tRNA Synthetase Mutation Confers Temperature Sensitivity

Abstract

Histidyl-tRNA synthetase (HARS) is a highly conserved translation factor that plays an essential role in protein synthesis. HARS has been implicated in the human syndromes Charcot-Marie-Tooth (CMT) Type 2W and Type IIIB Usher (USH3B). The USH3B mutation, which encodes a Y454S substitution in HARS, is inherited in an autosomal recessive fashion and associated with childhood deafness, blindness, and episodic hallucinations during acute illness. The biochemical basis of the pathophysiologies linked to USH3B is currently unknown. Here, we present a detailed functional comparison of wild-type (WT) and Y454S HARS enzymes. Kinetic parameters for enzymes and canonical substrates were determined using both steady state and rapid kinetics. Enzyme stability was examined using differential scanning fluorimetry. Finally, enzyme functionality in a primary cell culture was assessed. Our results demonstrate that the Y454S substitution leaves HARS amino acid activation, aminoacylation, and tRNA binding functions largely intact compared with those of WT HARS, and the mutant enzyme dimerizes like the wild type does. Interestingly, during our investigation, it was revealed that the kinetics of amino acid activation differs from that of the previously characterized bacterial HisRS. Despite the similar kinetics, differential scanning fluorimetry revealed that Y454S is less thermally stable than WT HARS, and cells from Y454S patients grown at elevated temperatures demonstrate diminished levels of protein synthesis compared to those of WT cells. The thermal sensitivity associated with the Y454S mutation represents a biochemical basis for understanding USH3B.

Substrate interaction defects in histidyl-tRNA synthetase linked to dominant axonal peripheral neuropathy

“…HEK293 cells were transiently transfected with plasmids expressing N‐terminal FLAG‐conjugated HARS gene for either WT or neuropathy‐associated HARS mutants. Enzymes were purified by affinity and ion exchange chromatography as previously described (Abbott et al., ). Purified proteins were visualized by on a 10% SDS‐PAGE gel.…”

“…Multiple turnover aminoacylation assays were performed using a modified version of the Uhlenbeck‐Wolfson assay as previously described (Abbott et al., ; Wolfson, Pleiss, & Uhlenbeck, ). Multiple turnover experiments were conducted in a buffer composed of (50 mM HEPES pH 7.5, 150 KCl, 10 mM MgCl 2 , 5 mM ß‐ME, 2 U/ml PPiase and 32 P‐labeled tRNA His ) at a fixed concentration of enzyme 5 nM for WT and (20 nM) for CMT variants with saturating concentrations of two of the three substrates.…”

“…HARS is unusual among the ARS in that different mutations in the gene encoding the cytoplasmic enzyme lead to two distinct inherited neurological syndromes. Initially, a single HARS mutation (P.Tyr454Ser) in the homozygous state was linked to recessive Usher Syndrome Type 3B in a small number of Amish children presenting with early onset loss of auditory and visual function (Puffenberger et al., ) (Abbott et al., ). Initial characterization of the mutant enzyme did not reveal a significant loss of aminoacylation, change in expression level, or change in intracellular localization.…”

Peripheral neuropathy and cognitive impairment associated with a novel monoallelic HARS variant

“…Among ARSs CMT genes, the histidyl‐tRNA synthetase ( HARS ) gene (OMIM*142810) is unique in that deleterious variants in the cytoplasmic isoform of the enzyme can lead to autosomal dominant CMT disease (OMIM #616625), but also to Usher syndrome type B3 (OMIM *614504). In this inherited neurological syndrome, a single HARS variant in homozygous state is reported to be the cause of early onset of blindness and deafness . In this report, we describe an unusual association between a novel variant in the HARS gene and a clinical phenotype initially suggestive of adult polyglucosan body disease (APBD, OMIM #263570), a condition affecting both the central and the peripheral nervous system, in an adult individual.…”

“…(A–C) Localization of reported pathogenic HARS variants on its protein domains. In orange, variants associated with Charcot‐Marie‐Tooth disease and in green with Usher syndrome . In red, localization of our newly discovered variant (A).…”