Processing of mutant N-acetyl-α-glucosaminidase in mucopolysaccharidosis type IIIB fibroblasts cultured at low temperature

•Mucopolysaccharidosis type IIIB (MPSIIIB) is caused by a deficiency of the lysosomal enzyme N-acetyl-α-glucosaminidase (NAGLU).•The disease spectrum comprises a severe, rapidly progressing (RP) phenotype and a more attenuated, slowly progressing (SP) phenotype.•NAGLU protein consists of a precursor and a mature form•In SP patients' fibroblasts only the precursor protein is present at 37 °C culture conditions which is enzymatically inactive•Mature, enzymatically active NAGLU is formed at low culturing temperatures, due to higher mRNA levels and improved protein processing•These processes may be relevant for the development of novel therapeutic strategies for MPS IIIB

BackgroundThe autosomal recessive, neurodegenerative disorder mucopolysaccharidosis type IIIB (MPSIIIB) is caused by a deficiency of the lysosomal enzyme N-acetyl-α-glucosaminidase (NAGLU), resulting in accumulation of heparan sulfate. The disease spectrum comprises a severe, rapidly progressing (RP) phenotype and a more attenuated, slowly progressing (SP) phenotype. Previous studies showed significantly higher NAGLU activity in skin fibroblasts of SP patients when cultured at 30 °C which may be relevant for development of novel therapeutic strategies. Here we report on the processes involved in this phenomenon.MethodsFibroblasts from controls, one RP patient (homozygous for the p.R297* mutation) and three SP MPSIIIB patients (homozygous for the mutation p.S612G or p.R643C, or compound heterozygous for the mutations p.A72_G79dup8 and p.R565Q) were cultured at temperatures ranging from 37 °C to 27 °C and harvested at different time points to assess NAGLU activity, mRNA and protein levels, and NAGLU glycosylation. Intracellular localization of wild-type and mutant mCherry-tagged NAGLU was analyzed by immunofluorescence.ResultsIn control fibroblasts NAGLU was present as a 85 kDa precursor and a 82 kDa mature form. In SP patients' fibroblasts cultured at 37 °C, only the 85 kDa form was detected. Culturing at lower temperatures resulted in higher NAGLU mRNA levels, increased levels of both precursor and mature NAGLU protein and improved processing. The formation of mature NAGLU corresponded with higher NAGLU activity levels.ConclusionWe show that the NAGLU protein consists of a precursor and a mature form and that in SP MPSIIIB patients' fibroblasts only the precursor protein is present at 37 °C. Culturing at lower temperatures resulted in the formation of the mature, enzymatically active form, due to higher mRNA levels and improved processing.