Effects of single-nucleotide polymorphisms in the human holocarboxylase synthetase gene on enzyme catalysis

Abstract

Holocarboxylase synthetase (HLCS) is a biotin protein ligase, which has a pivotal role in biotin-dependent metabolic pathways and epigenetic phenomena in humans. Knockdown of HLCS produces phenotypes such as heat susceptibility and decreased life span in Drosophila melanogaster, whereas knockout of HLCS appears to be embryonic lethal. HLCS comprises 726 amino acids in four domains. More than 2500 single-nucleotide polymorphisms (SNPs) have been identified in human HLCS. Here, we tested the hypotheses that HLCS SNPs impair enzyme activity, and that biotin supplementation restores the activities of HLCS variants to wild-type levels. We used an in silico approach to identify five SNPs that alter the amino acid sequence in the N-terminal, central, and C-terminal domains in human HLCS. Recombinant HLCS was used for enzyme kinetics analyses of HLCS variants, wild-type HLCS, and the L216R mutant, which has a biotin ligase activity near zero. The biotin affinity of variant Q699R is lower than that of the wild-type control, but the maximal activity was restored to that of wild-type HLCS when assay mixtures were supplemented with biotin. In contrast, the biotin affinities of HLCS variants V96F and G510R are not significantly different from the wild-type control, but their maximal activities remained moderately lower than that of wild-type HLCS even when assay mixtures were supplemented with biotin. The V96 L SNP did not alter enzyme kinetics. Our findings suggest that individuals with HLCS SNPs may benefit from supplemental biotin, yet to different extents depending on the genotype.

Clinical Presentation and Positive Outcome of Two Siblings with Holocarboxylase Synthetase Deficiency Caused by a Homozygous L216R Mutation

“…The fibroblast K m in their study could not be differentiated from negative controls and the mutant HLCS enzyme was found to have a faster turnover rate than that of the wild-type enzyme (Bailey et al 2008). Other studies have also demonstrated that the L216R mutant has a biotin ligase activity near zero (Esaki et al 2012).…”

Holocarboxylase synthetase synergizes with methyl CpG binding protein 2 and DNA methyltransferase 1 in the transcriptional repression of long-terminal repeats

“…Full-length human HLCS (NM_000411) was subcloned from plasmid pET41a (+)-HLCS 44 into vector p3XFLAG-CMV-26 (Sigma-Aldrich, Cat# E7283) using NotI and XbaI, thereby creating plasmid FLAG/Myc-HLCS for studies of HLCS overexpression and its effects on H3K9me marks and LTR transcriptional activity. Plasmid pCMV-myc-HLCS was created as described previously 17 and was used for overexpression of Myc-tagged HLCS in co-immunoprecipitation studies.…”

Three promoters regulate the transcriptional activity of the human holocarboxylase synthetase gene

“…Unless diagnosed and treated early, HLCS deficiency is uniformly fatal [19]. Single nucleotide polymorphisms in the HLCS gene decrease catalytic activity, but the biological importance of these polymorphisms is unknown [20]. …”