Autor:innen:
A. Kleine (Bad Oeynhausen, DE)
P. Lange (Bad Oeynhausen, DE)
V. Schmidt (Bad Oeynhausen, DE)
T. Ly (Bad Oeynhausen, DE)
I. Faust-Hinse (Bad Oeynhausen, DE)
C. Knabbe (Bad Oeynhausen, DE)
B. Fischer (Bad Oeynhausen, DE)
Background: The xylosyltransferase isoforms XT-I and XT-II catalyse the initial step of the proteoglycan (PG) tetrasaccharide linker region to bind glycosaminoglycans (GAG) to the PG-core protein. Through this essential step, the XT isoforms crucially contribute to the homeostasis of ECM remodelling.
Spondylo-ocular syndrome is a rare autosomal recessive inherited disease with features such as skeletal fragility, short stature, developmental delay as well as cardiac and ocular manifestations. Various XYLT2 mutations have been identified to be causative concerning disease manifestation. Due to the low availability of patient cells, the aim of this work was to generate an isolated CRISPR/Cas9-mediated XYLT2 knockout in neonatal human dermal fibroblasts to analyse the effects of XT-II deficiency on the cells. In addition, this approach could help to better understand the hitherto unexplained relevance of two different XT-isoforms in all higher organisms.
Methods: CRISPR/Cas9 based XYLT2 knockout: normal human dermal fibroblasts (NHDF) were revers transfected with 10 nM of a ribonucleoprotein (RNP)-complex targeting the XYLT2 gene. After 24 h the cells were sorted using FACS technology, genomic DNA was isolated and afterwards analysed using sanger sequencing.
Analysis of relative mRNA expression and XT activity: relative mRNA expression levels were determined using quantitative real-time PCR. Intra- (cell culture lysates) and extracellular (cell culture supernatants) XT-activity was measured using an in-house UPLC-MS-assay by specifically detecting the xylosylated peptide.
Migration assay: An artificial scratch within a confluent monolayer of control and XYLT2-deficient fibroblasts was created. The migration of cells into the area was observed for 96 h.
Senescence assay: NHDF and XYLT2-deficient cells were cultivated for 72 h and subsequently harvested. The senescence-associated-β-galactosidase-activity was observed photometrically.
Results: CRISPR/Cas9-based genome editing was successful due to a single thymine base deletion, resulting in a shortened amino acid sequence (p.Val95Glyfs*3). Compared to controls, XT-II deficient cells showed reduced mRNA expression levels of different glycosyltransferases, as well as a significantly reduced XT activity. In addition, the XYLT2-deficient cells closed the artificial gap more slowly and showed a significantly increased senescence-associated-β-galactosidase activity in comparison to the control fibroblasts.
Conclusion: Using the CRISPR/Cas9 system, a XYLT2-deficient fibroblast culture was successfully obtained. The lack of XT activity also lowers the mRNA expression of the remaining transferases involved in the tetrasaccharide linker synthesis. The migration capacity of the cells is impaired and there is a marked increase in cellular senescence. Our data reveal an impact of XYLT2 expression on the cellular metabolism. Further effects need to be analysed in the future.