Generation and biochemical validation of the sgshΔex5−6 mutant zebrafish. (A) Exon-intron diagram representations of wild type human SGSH, wild type zebrafish sgsh, and the zebrafish sgshΔex5−6 allele. Exons comprising sulfatase domain highlighted in red; exons comprising sulfatase-associated Domain of Unknown Function DUF4976 highlighted in blue. Downwards-facing arrows indicate gRNA target sites. Orange arrowheads reflect primer positions for RT-PCR; green arrowheads reflect primer positions for genotyping PCR. Scale bar is 1 kilobase. (B) Genotyping PCR of wild type (+/+), heterozygous (+/−) and homozygous sgshΔex5−6 (−/−) alleles. Wild type sgsh produces a band at 772 bp, while sgshΔex5−6 produces a band at 415 bp. (C) RT-PCR amplicons of sgsh in RNA from wild type (+/+) or homozygous sgshΔex5−6 (−/−) zebrafish embryos at 24 hpf using primers located in the first and last exons of sgsh. Wild type sgsh produces a prominent band at 1336 bp, while sgshΔex5−6 produces a band at 1050 bp corresponding to loss of exons 5 and 6. eef1a1l1 (amplicon size 181 bp) included for both genotypes as housekeeping control. (D) Overview of Sgsh enzyme activity assay. 4MU-αGlcNS is desulfated by Sgsh in total protein, then glucosamine is hydrolysed from UV-fluorescent 4MU by bacterial α-glucosidase. (E) Enzyme activity in wild type (+/+), sgshΔex5−6 heterozygous (+/−) and homozygous (−/−) whole embryos (n = 100 embryos per sample, 6 samples per group). (F) Enzyme activity in adult brain (n = 3 brains per sample, 3 samples per group) for each genotype. Negative arbitrary fluorescence values observed in sgshΔex5−6 homozygous samples result from absorbance of UV excitation wavelengths by protein input and blank correction. Data in (E,F) are presented as mean ± SEM and tested via ordinary one-way ANOVA with Tukey’s multiple comparisons test; **** p < 0.0001 and *** p = 0.0001. (G) HS disaccharide abundance in whole 24 hpf embryos from either wild type or sgshΔex5−6 genotypes as determined by LC-MS/MS (n = 50 embryos per sample, n = 3 samples per group). Data presented as mean ± SEM and tested by Welch’s t-test. ** p = 0.0021. (H) HS abundance in young (6-month-old) or aged (18-month-old) brains from either wild type or sgshΔex5−6 genotypes determined by LC-MS/MS (n = 3 brains per sample, n = 3 samples per group). Data presented as mean ± SEM and tested by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. * p = 0.0171, ** p = 0.0031.
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