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Processing of mutant N-acetyl-alpha-glucosaminidase in mucopolysaccharidosis type IIIB fibroblasts cultured at low temperature. / Meijer, O. L. M.; te Brinke, H.; Ofman, R. et al.

In: Molecular genetics and metabolism, Vol. 122, No. 1-2, 2017, p. 100-106.

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@article{8a071ea50856433abc90590dbb0e385e,
title = "Processing of mutant N-acetyl-alpha-glucosaminidase in mucopolysaccharidosis type IIIB fibroblasts cultured at low temperature",
abstract = "Background: The autosomal recessive, neurodegenerative disorder mucopolysaccharidosis type IIIB (MPSIIIB) is caused by a deficiency of the lysosomal enzyme N-acetyl-alpha-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 sldn fibroblasts of SP patients when cultured at 30 degrees C which may be relevant for development of novel therapeutic strategies. Here we report on the processes involved in this phenomenon. Methods: Fibroblasts 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 degrees C to 27 degrees 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. Results: In control fibroblasts NAGLU was present is a 85 kDa precursor and a 82 kDa mature form. In SP patients' fibroblasts cultured at 37 degrees 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. Conclusion: We 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 degrees C. Culturing at lower temperatures resulted in the formation of the mature, enzymatically active form, due to higher mRNA levels and improved processing. (C) 2017 Elsevier Inc. All rights reserved",
author = "Meijer, {O. L. M.} and {te Brinke}, H. and R. Ofman and L. IJlst and Wijburg, {F. A.} and {van Vlies}, N.",
year = "2017",
doi = "10.1016/j.ymgme.2017.07.005",
language = "English",
volume = "122",
pages = "100--106",
journal = "Molecular genetics and metabolism",
issn = "1096-7192",
publisher = "Academic Press Inc.",
number = "1-2",

}

RIS

TY - JOUR

T1 - Processing of mutant N-acetyl-alpha-glucosaminidase in mucopolysaccharidosis type IIIB fibroblasts cultured at low temperature

AU - Meijer, O. L. M.

AU - te Brinke, H.

AU - Ofman, R.

AU - IJlst, L.

AU - Wijburg, F. A.

AU - van Vlies, N.

PY - 2017

Y1 - 2017

N2 - Background: The autosomal recessive, neurodegenerative disorder mucopolysaccharidosis type IIIB (MPSIIIB) is caused by a deficiency of the lysosomal enzyme N-acetyl-alpha-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 sldn fibroblasts of SP patients when cultured at 30 degrees C which may be relevant for development of novel therapeutic strategies. Here we report on the processes involved in this phenomenon. Methods: Fibroblasts 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 degrees C to 27 degrees 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. Results: In control fibroblasts NAGLU was present is a 85 kDa precursor and a 82 kDa mature form. In SP patients' fibroblasts cultured at 37 degrees 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. Conclusion: We 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 degrees C. Culturing at lower temperatures resulted in the formation of the mature, enzymatically active form, due to higher mRNA levels and improved processing. (C) 2017 Elsevier Inc. All rights reserved

AB - Background: The autosomal recessive, neurodegenerative disorder mucopolysaccharidosis type IIIB (MPSIIIB) is caused by a deficiency of the lysosomal enzyme N-acetyl-alpha-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 sldn fibroblasts of SP patients when cultured at 30 degrees C which may be relevant for development of novel therapeutic strategies. Here we report on the processes involved in this phenomenon. Methods: Fibroblasts 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 degrees C to 27 degrees 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. Results: In control fibroblasts NAGLU was present is a 85 kDa precursor and a 82 kDa mature form. In SP patients' fibroblasts cultured at 37 degrees 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. Conclusion: We 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 degrees C. Culturing at lower temperatures resulted in the formation of the mature, enzymatically active form, due to higher mRNA levels and improved processing. (C) 2017 Elsevier Inc. All rights reserved

U2 - 10.1016/j.ymgme.2017.07.005

DO - 10.1016/j.ymgme.2017.07.005

M3 - Article

C2 - 28751108

VL - 122

SP - 100

EP - 106

JO - Molecular genetics and metabolism

JF - Molecular genetics and metabolism

SN - 1096-7192

IS - 1-2

ER -

ID: 3990240