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1.
Biomedicines ; 12(3)2024 Mar 04.
Article En | MEDLINE | ID: mdl-38540185

Desbuquois dysplasia type 2 (DBQD2) and spondylo-ocular syndrome (SOS) are autosomal recessive disorders affecting the extracellular matrix (ECM) and categorized as glycosaminoglycan (GAG) linkeropathies. Linkeropathies result from mutations within glycosyltransferases involved in the synthesis of the tetrasaccharide linker, a linker between the core protein of proteoglycan (PG) and GAG. DBQD2 and SOS are caused by the isolated mutations of the xylosyltransferase (XT) isoforms. In this work, we successfully generated XYLT1- as well as XYLT2-deficient GAG linkeropathy model systems in human dermal fibroblasts using a ribonucleoprotein-based CRISPR/Cas9-system. Furthermore, it was possible to generate a complete XYLT-knockdown. Short- and long-term XT activity deficiency led to the mutual reduction in all linker transferase-encoding genes, suggesting a potential multienzyme complex with mutual regulation. Fibroblasts compensated for ECM misregulation initially by overexpressing ECM through the TGFß1 signaling pathway, akin to myofibroblast differentiation patterns. The long-term reduction in one XT isoform induced a stress response, reducing ECM components. The isolated XYLT1-knockout exhibited α-smooth muscle actin overexpression, possibly partially compensated by unaltered XT-II activity. XYLT2-knockout leads to the reduction in both XT isoforms and a strong stress response with indications of oxidative stress, induced senescence and apoptotic cells. In conclusion, introducing XYLT-deficiency revealed temporal and isoform-specific regulatory differences.

2.
Biochimie ; 218: 127-136, 2024 Mar.
Article En | MEDLINE | ID: mdl-37689257

Xylosyltransferase-I and -II (XT-I, -II) possess a central role during the glycosylation of proteoglycans (PGs). They catalyze the formation of an O-glycosidic bond between the xylosyl residue of uridinediphosphate-xylose and the core protein of a PG. Thereafter, three following glycosyltransferases lead to the generation of a tetrasaccharide linker, which connects the PG core protein to the respective glycosaminoglycan. The selective quantification of XT-I and XT-II activity is of biological and clinical interest due to their association with fibrotic processes and skeletal dysplasia. There is no assay available to date that simultaneously determines the activity of the two XT isoforms. Although an XT-I selective UPLC MS/MS-based assay was published by Fischer et al., in 2021, the determination of XT-II activity can only be performed simultaneously by the improved assay presented here. To establish the assay, two synthetic peptides, selectively xylosylated by the respective isoform, were identified and the associated measurement parameters for the mass spectrometer were optimized. In addition, the quantitative range of the xylosylated peptides were validated, and the incubation time of the enzyme reaction was optimized for cell culture samples and human sera. The specific enzyme kinetics (KM and Vmax) of the respective XT isoform for the two peptides were also determined. Subsequently, a mathematical model was developed, allowing the simultaneous determination of XT-I and XT-II activity from the chromatographic results. Summarized, a mass spectrometric method suitable for the simultaneous analysis of XT-I and XT-II activity in cell culture lysates, supernatants and human sera was successfully developed.


Pentosyltransferases , UDP Xylose-Protein Xylosyltransferase , Humans , Pentosyltransferases/chemistry , Tandem Mass Spectrometry , Chromatography, Liquid , Liquid Chromatography-Mass Spectrometry , Protein Isoforms , Peptides
3.
PLoS One ; 18(5): e0286334, 2023.
Article En | MEDLINE | ID: mdl-37235555

Arthrofibrosis following total knee arthroplasty is a fibroproliferative joint disorder marked by dysregulated biosynthesis of extracellular matrix proteins, such as collagens and proteoglycans. The underlying cellular events remain incompletely understood. Myofibroblasts are highly contractile matrix-producing cells characterized by increased alpha-smooth muscle actin expression and xylosyltransferase-I (XT-I) secretion. Human XT-I has been identified as a key mediator of arthrofibrotic remodeling. Primary fibroblasts from patients with arthrofibrosis provide a useful in vitro model to identify and characterize disease regulators and potential therapeutic targets. This study aims at characterizing primary synovial fibroblasts from arthrofibrotic tissues (AFib) regarding their molecular and cellular phenotype by utilizing myofibroblast cell culture models. Compared to synovial control fibroblasts (CF), AFib are marked by enhanced cell contractility and a higher XT secretion rate, demonstrating an increased fibroblast-to-myofibroblast transition rate during arthrofibrosis. Histochemical assays and quantitative gene expression analysis confirmed higher collagen and proteoglycan expression and accumulation in AFib compared to CF. Furthermore, fibrosis-based gene expression profiling identified novel modifier genes in the context of arthrofibrosis remodeling. In summary, this study revealed a unique profibrotic phenotype in AFib that resembles some traits of other fibroproliferative diseases and can be used for the future development of therapeutic interventions.


Atrial Fibrillation , Joint Diseases , Humans , Atrial Fibrillation/metabolism , Fibroblasts/metabolism , Myofibroblasts/metabolism , Extracellular Matrix/metabolism , Collagen/metabolism , Actins/genetics , Actins/metabolism
4.
Front Biosci (Landmark Ed) ; 28(3): 55, 2023 03 20.
Article En | MEDLINE | ID: mdl-37005749

BACKGROUND: Pseudoxanthoma elasticum (PXE) is a rare autosomal recessive disorder caused by mutations in the ATP-binding cassette sub-family C member 6 (ABCC6) gene. Patients with PXE show molecular and clinical characteristics of known premature aging syndromes, such as Hutchinson-Gilford progeria syndrome (HGPS). Nevertheless, PXE has only barely been discussed against the background of premature aging, although a detailed characterization of aging processes in PXE could contribute to a better understanding of its pathogenesis. Thus, this study was performed to evaluate whether relevant factors which are known to play a role in accelerated aging processes in HGPS pathogenesis are also dysregulated in PXE. METHODS: Primary human dermal fibroblasts from healthy donors (n = 3) and PXE patients (n = 3) and were cultivated under different culture conditions as our previous studies point towards effects of nutrient depletion on PXE phenotype. Gene expression of lamin A, lamin C, nucleolin, farnesyltransferase and zinc metallopeptidase STE24 were determined by quantitative real-time polymerase chain reaction. Additionally, protein levels of lamin A, C and nucleolin were evaluated by immunofluorescence and the telomere length was analyzed. RESULTS: We could show a significant decrease of lamin A and C gene expression in PXE fibroblasts under nutrient depletion compared to controls. The gene expression of progerin and farnesyltransferase showed a significant increase in PXE fibroblasts when cultivated in 10% fetal calf serum (FCS) compared to controls. Immunofluorescence microscopy of lamin A/C and nucleolin and mRNA expression of zinc metallopeptidase STE24 and nucleolin showed no significant changes in any case. The determination of the relative telomere length showed significantly longer telomeres for PXE fibroblasts compared to controls when cultivated in 10% FCS. CONCLUSIONS: These data indicate that PXE fibroblasts possibly undergo a kind of senescence which is independent of telomere damage and not triggered by defects of the nuclear envelope or nucleoli deformation.


Aging, Premature , Progeria , Pseudoxanthoma Elasticum , Humans , Progeria/genetics , Progeria/metabolism , Progeria/pathology , Aging, Premature/genetics , Aging, Premature/metabolism , Aging, Premature/pathology , Lamin Type A/genetics , Lamin Type A/metabolism , Pseudoxanthoma Elasticum/genetics , Pseudoxanthoma Elasticum/metabolism , Pseudoxanthoma Elasticum/pathology , Farnesyltranstransferase/metabolism , Metalloproteases/metabolism , Zinc/metabolism , Fibroblasts/metabolism
5.
Biomedicines ; 11(2)2023 Feb 04.
Article En | MEDLINE | ID: mdl-36830996

The human xylosyltransferase isoform XT-I catalyzes the initial step in proteoglycan biosynthesis and represents a biomarker of myofibroblast differentiation. Furthermore, XT-I overexpression is associated with fibrosis, whereby a fibrotic process initially develops from a dysregulated wound healing. In a physiologically wound healing process, extracellular matrix-producing myofibroblasts enter acute senescence to protect against fibrosis. The aim of this study was to determine the role of XT-I in acute senescent proto-myofibroblasts. Normal human dermal fibroblasts were seeded in a low cell density to promote myofibroblast differentiation and treated with H2O2 to induce acute senescence. Initiation of the acute senescence program in human proto-myofibroblasts resulted in a suppression of XYLT mRNA expression compared to the control, whereby the isoform XYLT1 was more affected than XYLT2. Moreover, the XT-I protein expression and enzyme activity were also reduced in H2O2-treated cells compared to the control. The examination of extracellular matrix remodeling revealed reduced expression of collagen I, fibronectin and decorin. In summary, acute senescent proto-myofibroblasts formed an anti-fibrotic phenotype, and suppression of XT-I during the induction process of acute senescence significantly contributed to subsequent ECM remodeling. XT-I therefore plays an important role in the switch between physiological and pathological wound healing.

6.
Biomedicines ; 10(11)2022 Nov 09.
Article En | MEDLINE | ID: mdl-36359389

Chronic inflammation and excessive synthesis of extracellular matrix components, such as proteoglycans (PG), by fibroblast- or macrophage-derived myofibroblasts are the hallmarks of fibrotic diseases, including systemic sclerosis (SSc). Human xylosyltransferase-I (XT-I), which is encoded by the gene XYLT1, is the key enzyme that is involved in PG biosynthesis. Increased cellular XYLT1 expression and serum XT-I activity were measured in SSc. Nothing is known so far about the regulation of XT-I in immune cells, and their contribution to the increase in measurable serum XT-I activity. We utilized an in vitro model, with primary human CD14+CD16+ monocyte-derived macrophages (MΦ), in order to investigate the role of macrophage polarization on XT-I regulation. The MΦ generated were polarized towards two macrophage phenotypes that were associated with SSc, which were classified as classical pro-inflammatory (M1-like), and alternative pro-fibrotic (M2-like) MΦ. The fully characterized M1- and M2-like MΦ cultures showed differential XT-I gene and protein expressions. The fibrotic M2-like MΦ cultures exhibited higher XT-I secretion, as well as increased expression of myofibroblast marker α-smooth muscle actin, indicating the onset of macrophage-to-myofibroblast transition (MMT). Thus, we identified XT-I as a novel macrophage polarization marker for in vitro generated M1- and M2-like MΦ subtypes, and broadened the view of XT-I as a myofibroblast marker in the process of MMT.

7.
Biomedicines ; 10(6)2022 Jun 19.
Article En | MEDLINE | ID: mdl-35740472

Inflammation plays a vital role in regulating fibrotic processes. Beside their classical role in extracellular matrix synthesis and remodeling, fibroblasts act as immune sentinel cells participating in regulating immune responses. The human xylosyltransferase-I (XT-I) catalyzes the initial step in proteoglycan biosynthesis and was shown to be upregulated in normal human dermal fibroblasts (NHDF) under fibrotic conditions. Regarding inflammation, the regulation of XT-I remains elusive. This study aims to investigate the effect of lipopolysaccharide (LPS), a prototypical pathogen-associated molecular pattern, and the damage-associated molecular pattern adenosine triphosphate (ATP) on the expression of XYLT1 and XT-I activity of NHDF. We used an in vitro cell culture model and mimicked the inflammatory tissue environment by exogenous LPS and ATP supplementation. Combining gene expression analyses, enzyme activity assays, and targeted gene silencing, we found a hitherto unknown mechanism involving the inflammasome pathway components cathepsin B (CTSB) and caspase-1 in XT-I regulation. The suppressive role of CTSB on the expression of XYLT1 was further validated by the quantification of CTSB expression in fibroblasts from patients with the inflammation-associated disease Pseudoxanthoma elasticum. Altogether, this study further improves the mechanistic understanding of inflammatory XT-I regulation and provides evidence for fibroblast-targeted therapies in inflammatory diseases.

8.
Int J Mol Sci ; 23(9)2022 May 02.
Article En | MEDLINE | ID: mdl-35563435

BACKGROUND: Xylosyltransferases-I and II (XT-I and XT-II) catalyze the initial and rate limiting step of the proteoglycan (PG) biosynthesis and therefore have an import impact on the homeostasis of the extracellular matrix (ECM). The reason for the occurrence of two XT-isoforms in all higher organisms remains unknown and targeted genome-editing strategies could shed light on this issue. METHODS: XT-I deficient neonatal normal human dermal fibroblasts were generated by using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated proteins (Cas) 9 system. We analyzed if a reduced XT-I activity leads to abnormalities regarding ECM-composition, myofibroblast differentiation, cellular senescence and skeletal and cartilage tissue homeostasis. RESULTS: We successfully introduced compound heterozygous deletions within exon 9 of the XYLT1 gene. Beside XYLT1, we detected altered gene-expression levels of further, inter alia ECM-related, genes. Our data further reveal a dramatically reduced XT-I protein activity. Abnormal myofibroblast-differentiation was demonstrated by elevated alpha-smooth muscle actin expression on both, mRNA- and protein level. In addition, wound-healing capability was slightly delayed. Furthermore, we observed an increased cellular-senescence of knockout cells and an altered expression of target genes knowing to be involved in skeletonization. CONCLUSION: Our data show the tremendous relevance of the XT-I isoform concerning myofibroblast-differentiation and ECM-homeostasis as well as the pathophysiology of skeletal disorders.


CRISPR-Cas Systems , Pentosyltransferases , Skin , CRISPR-Cas Systems/genetics , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Editing , Humans , Infant, Newborn , Pentosyltransferases/genetics , Pentosyltransferases/metabolism , Skin/metabolism , UDP Xylose-Protein Xylosyltransferase
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