Christian Prante

Human Xylosyltransferase II Is Involved in the Biosynthesis of the Uniform Tetrasaccharide Linkage Region in Chondroitin Sulfate and Heparan Sulfate Proteoglycans

Human xylosyltransferase I (XT-I) initiates the biosynthesis of the glycosaminoglycan (GAG) linkage tetrasaccharide in proteoglycans. Xylosyltransferase II (XT-II) is a protein homologous to XT-I but with hitherto unknown activity or physiological function. Here, we report the enzymatic activity of XT-II and provide evidence that XT-II initiates the biosynthesis of both heparan sulfate and chondroitin sulfate GAGs. Transfection of the xylosyltransferase-deficient Chinese hamster ovary mutant pgsA-745 with XT-I or XT-II coding cDNA completely restored GAG biosynthesis. GAG disaccharide analysis revealed that XT-I- and XT-II-transfected pgsA-745 cells produced similar amounts of chondroitin sulfate and heparan sulfate. Furthermore, a high xylosyltransferase activity was measured after transfection with cDNAs encoding either isozyme. Analysis of the enzyme activity revealed that XT-II catalyzes the transfer of xylose to similar peptide acceptors as XT-I but with different efficiency. The optimal XT-II acceptor was observed using a bikunin-related peptide (Km 5.2 μm). Analysis of XT-I and XT-II mRNA expression in murine tissues showed a differential expression pattern for both enzymes. In particular, XT-II is highly expressed in liver tissue, where XT-I transcripts were not detected. This is the first report on the enzyme activity of XT-II and its involvement in chondroitin sulfate and heparan sulfate biosynthesis.

Cloning and recombinant expression of active full-length xylosyltransferase I (XT-I) and characterization of subcellular localization of XT-I and XT-II.

Xylosyltransferase I (XT-I) catalyzes the transfer of xylose from UDP-xylose to serine residues in proteoglycan core proteins. This is the first and apparently rate-limiting step in the biosynthesis of the tetrasaccharide linkage region in glycosaminoglycan-containing proteoglycans. The XYLT-II gene codes for a highly homologous protein, but its physiological function is not yet known. Here we present for the first time the construction of a vector encoding the full-length GFP-tagged human XT-I and the recombinant expression of the active enzyme in mammalian cells. We expressed XT-I-GFP and various GFP-tagged XT-I and XT-II mutants with C-terminal truncations and deletions in HEK-293 and SaOS-2 cells in order to investigate the intracellular localization of XT-I and XT-II. Immunofluorescence analysis showed a distinct perinuclear pattern of XT-I-GFP and XT-II-GFP similar to that of α-mannosidase II, which is a known enzyme of the Golgi cisternae. Furthermore, a co-localization of native human XT-I and α-mannosidase II could also be demonstrated in untransfected cells. Using brefeldin A, we could also show that both xylosyltransferases are resident in the early cisternae of the Golgi apparatus. For its complete Golgi retention, XT-I requires the N-terminal 214 amino acids. Unlike XT-I, for XT-II, the first 45 amino acids are sufficient to target and retain the GFP reporter in the Golgi compartment. Here we show evidence that the stem regions were indispensable for Golgi localization of XT-I and XT-II.

Transforming Growth Factor β1-regulated Xylosyltransferase I Activity in Human Cardiac Fibroblasts and Its Impact for Myocardial Remodeling

In cardiac fibrosis remodeling of the failing myocardium is associated with a complex reorganization of the extracellular matrix (ECM). Xylosyltransferase I and Xylosyltransferase II (XT-I and XT-II) are the key enzymes in proteoglycan biosynthesis, which are an important fraction of the ECM. XT-I was shown to be a measure for the proteoglycan biosynthesis rate and a biochemical fibrosis marker. Here, we investigated the XT-I and XT-II expression in cardiac fibroblasts and in patients with dilated cardiomyopathy and compared our findings with nonfailing donor hearts. We analyzed XT-I and XT-II expression and the glycosaminoglycan (GAG) content in human cardiac fibroblasts incubated with transforming growth factor (TGF)-β1 or exposed to cyclic mechanical stretch. In vitro and in vivo no significant changes in the XT-II expression were detected. For XT-I we found an increased expression in parallel with an elevated chondroitin sulfate-GAG content after incubation with TGF-β1 and after mechanical stretch. XT-I expression and subsequently increased levels of GAGs could be reduced with neutralizing anti-TGF-β1 antibodies or by specific inhibition of the activin receptor-like kinase 5 or the p38 mitogen-activated protein kinase pathway. Usage of XT-I small interfering RNA could specifically block the increased XT-I expression under mechanical stress and resulted in a significantly reduced chondroitin sulfate-GAG content. In the left and right ventricular samples of dilated cardiomyopathy patients, our data show increased amounts of XT-I mRNA compared with nonfailing controls. Patients had raised levels of XT-I enzyme activity and an elevated proteoglycan content. Myocardial remodeling is characterized by increased XT-I expression and enhanced proteoglycan deposition. TGF-β1 and mechanical stress induce XT-I expression in cardiac fibroblasts and have impact for ECM remodeling in the dilated heart. Specific blocking of XT-I expression confirmed that XT-I catalyzes a rate-limiting step during fibrotic GAG biosynthesis.

Polymorphisms in the xylosyltransferase genes cause higher serum XT-I activity in patients with pseudoxanthoma elasticum (PXE) and are involved in a severe disease course

Background: Pseudoxanthoma elasticum (PXE) is a heritable connective tissue disorder caused by mutations in the ABCC6 gene. Fragmentation of elastic fibres and deposition of proteoglycans result in a highly variable clinical picture. The altered proteoglycan metabolism suggests that enzymes from this pathway function as genetic co-factors in the severity of PXE. Therefore, we propose the XYLT genes encoding xylosyltransferase I (XT-I) as the chain-initiating enzyme in the biosynthesis of proteoglycans and the highly homologous XT-II as potential candidate genes. Methods: We screened all XYLT exons in 65 German PXE patients using denaturing high performance liquid chromatography and analysed the influence of the variations on clinical characteristics. Results: We identified 22 variations in the XYLT genes. The missense variation p.A115S (XT-I) is associated with higher serum XT activity (p = 0.005). The amino acid substitution p.T801R (XT-II; c.2402C>G) occurs with significantly higher frequency in patients under 30 years of age at diagnosis (43% v 26%; p = 0.04); all PXE patients with this variation suffer from skin lesions compared to only 75% of the wild type patients (p = 0.002). c.166G>A, c.1569C>T, and c.2402C>G in the XYLT-II gene were found to be more frequent in patients with higher organ involvement (p = 0.04, p = 0.01, and p = 0.02, respectively). Conclusions: Here we show for the first time that variations in the XYLT-II gene are genetic co-factors in the severity of PXE. Furthermore, the higher XT activity in patients with the exchange p.A115S (XT-I) indicates that this polymorphism is a potential marker for increased remodelling of the extracellular matrix.

Measurement of mycophenolic acid and its glucuronide using a novel rapid liquid chromatography–electrospray ionization tandem mass spectrometry assay

OBJECTIVES Mycophenolic acid (MPA), the active metabolite of the ester prodrug mycophenolate mofetil is an immunosuppressant which selectively inhibits inosine-monophosphate dehydrogenase. The requirement for therapeutic drug monitoring shown in previous studies raises the necessity of acquiring accurate and sensitive methods to measure MPA and also its metabolite mycophenolic acid glucuronide (MPAG). DESIGN AND METHODS We developed a robust, rapid, sensitive and highly specific HPLC-electrospray ionization mass spectrometry method to assay MPA and its metabolite MPAG in human plasma and serum. Ion suppression was investigated by a post column infusion experiment. RESULTS Determination of MPA and MPAG were performed during a 3.0-min run time. Multiple calibration curves for the analysis of MPA and MPAG exhibited consistent linearity and reproducibility in the range of 0.05 to 100 mg/L (r>0.999) and 6 to 400 mg/L r>0.998, respectively. Limits of detection were 0.009 mg/L for MPA and 4.5 mg/L for MPAG and lower limits of quantification were 0.011 mg/L for MPA and 4.9 mg/L for MPAG. Interassay imprecision was <6.0% for both substances. Mean recovery was 48.9% (range 43.3-60.0%) for MPA and 112.2% (range 95.0-127.0%) for MPAG. Agreement was relatively good for MPA (n=122) between the presented method and a validated ELISA method (Viva analyzer, Siemens Medicals Solutions Diagnostics, NY). The Passing-Bablok regression line was: EMIT=0.91 (LC-MS/MS)+0.17 [mg/L]; r=0.97. CONCLUSIONS This simple, robust and interference-free LC-MS/MS assay allows the rapid and accurate determination of MPA and MPAG in human plasma and other body fluids.

Analysis of the DXD Motifs in Human Xylosyltransferase I Required for Enzyme Activity

Human xylosyltransferase I (XT-I) is the initial enzyme involved in the biosynthesis of the glycosaminoglycan linker region in proteoglycans. Here, we tested the importance of the DXD motifs at positions 314–316 and 745–747 for enzyme activity and the nucleotide binding capacity of human XT-I. Mutations of the 314DED316 motif did not have any effect on enzyme activity, whereas alterations of the 745DWD747 motif resulted in reduced XT-I activity. Loss of function was observed after exchange of the highly conserved aspartic acid at position 745 with glycine. However, mutation of Asp745 to glutamic acid retained full enzyme activity, indicating the importance of an acidic amino acid at this position. Reduced substrate affinity was observed for mutants D747G (Km = 6.9 μm) and D747E (Km = 4.4 μm) in comparison with the wild-type enzyme (Km = 0.9 μm). Changing the central tryptophan to a neutral, basic, or acidic amino acid resulted in a 6-fold lower Vmax, with Km values comparable with those of the wild-type enzyme. Despite the major effect of the DWD motif on XT-I activity, nucleotide binding was not abolished in the D745G and D747G mutants, as revealed by UDP-bead binding assays. Ki values for inhibition by UDP were determined to be 1.9–24.6 μm for the XT-I mutants. The properties of binding of XT-I to heparin-beads, the Ki constants for noncompetitive inhibition by heparin, and the activation by protamine were not altered by the generated mutations.

The Formation of Extracellular Matrix During Chondrogenic Differentiation of Mesenchymal Stem Cells Correlates with Increased Levels of Xylosyltransferase I

In vitro differentiation of mesenchymal stem cells (MSCs) into chondrogenic cells and their transplantation is promising as a technique for the treatment of cartilaginous defects. But the regulation of extracellular matrix (ECM) formation remains elusive. Therefore, the objective of this study was to analyze the regulation of proteoglycan (PG) biosynthesis during the chondrogenic differentiation of MSCs. In different stages of chondrogenic differentiation, we analyzed mRNA and protein expression of key enzymes and PG core proteins involved in ECM development. For xylosyltransferase I (XT‐I), we found maximum mRNA levels 48 hours after chondrogenic induction with a 5.04 ± 0.58 (mean ± SD)‐fold increase. This result correlates with significantly elevated levels of enzymatic XT‐I activity (0.49 ± 0.03 μU/1 × 106 cells) at this time point. Immunohistochemical staining of XT‐I revealed a predominant upregulation in early chondrogenic stages. The highly homologous protein XT‐II showed 4.7‐fold (SD 0.6) increased mRNA levels on day 7. To determine the differential expression of heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS) chains, we analyzed the mRNA expression of EXTL2 (α‐4‐N‐acetylhexosaminyltransferase), GalNAcT (β‐1,4‐N‐acetylgalactosaminyltransferase), and GlcAC5E (glucuronyl C5 epimerase). All key enzymes showed a similar regulation with temporarily downregulated mRNA levels (up to −87‐fold) after chondrogenic induction. In accordance to previous studies, we observed a similar increase in the expression of PG core proteins. In conclusion, we could show that key enzymes for CS, DS, and HS synthesis, especially XT‐I, are useful markers for the developmental stages of chondrogenic differentiation.

Analytical evaluation of the automated galectin-3 assay on the Abbott ARCHITECT immunoassay instruments.

Abstract Background: Galectin-3 is secreted from macrophages and binds and activates fibroblasts forming collagen. Tissue fibrosis is central to the progression of chronic heart failure (CHF). We performed a European multicentered evaluation of the analytical performance of the two-step routine and Short Turn-Around-Time (STAT) galectin-3 immunoassay on the ARCHITECT i1000SR, i2000SR, and i4000SR (Abbott Laboratories). Methods: We evaluated the assay precision and dilution linearity for both routine and STAT assays and compared serum and plasma, and fresh vs. frozen samples. The reference interval and biological variability were also assessed. Measurable samples were compared between ARCHITECT instruments and between the routine and STAT assays and also to a galectin-3 ELISA (BG Medicine). Results: The total assay coefficient of variation (CV%) was 2.3%–6.2% and 1.7%–7.4% for the routine and STAT assays, respectively. Both assays demonstrated linearity up to 120 ng/mL. Galectin-3 concentrations were higher in plasma samples than in serum samples and correlated well between fresh and frozen samples (R=0.997), between the routine and STAT assays, between the ARCHITECT i1000 and i2000 instruments and with the galectin-3 ELISA. The reference interval on 627 apparently healthy individuals (53% male) yielded upper 95th and 97.5th percentiles of 25.2 and 28.4 ng/mL, respectively. Values were significantly lower in subjects younger than 50 years. Conclusions: The galectin-3 routine and STAT assays on the Abbott ARCHITECT instruments demonstrated good analytical performance. Further clinical studies are required to demonstrate the diagnostic and prognostic potential of this novel marker in patients with CHF.

Cystic fibrosis transmembrane conductance regulator can export hyaluronan.

Objectives: Hyaluronan, a major water binding component of the extracellular matrix, is synthesised within the cytosol and exported across the plasma membrane by the ABC-transporter MRP5 in fibroblasts. Although its synthesis is vital for embryogenesis, MRP5-deficient mice are without phenotype, suggesting that another transporter had substituted for the MRP5 protein. Thus, we searched for a compensatory exporter in fibroblasts from MRP5 deficient mice and found that cystic fibrosis transmembrane conductance regulator (CFTR) mRNA was upregulated. Methods: Hyaluronan export was measured in cell culture. The CFTR transporter was knocked out using si-RNA. Blockers of the ABC-transporter family were used to ascertain the hyaluronan transport capabilities functionally. Results: CFTR specific siRNA inhibited hyaluronan export. The tetrasaccharide was exported in undegraded form only from normal human epithelial cells and not from human epithelial cells carrying ΔF508 CFTR. The CFTR inhibitors GlyH-101 and CFTR172 reduced hyaluronan export from CFTR-expressing mouse fibroblasts and from human breast cancer cell lines. Bronchial secretions from patients with cystic fibrosis that consist mainly of necrotic epithelia contained at least 40-fold higher concentration of hyaluronan than secretions from patients with acute bronchitis. Conclusions: CFTR transports hyaluronan across the plasma membrane of epithelial cells and this transport mechanism is defective in cystic fibrosis patients.

Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme.

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.