UDP-D-Xylose : Proteoglycan Core Protein β-D-Xylosyltransferase: A New Marker of Cartilage Destruction in Chronic Joint Diseases

Kleesiek, Knut; Reinards, R.; Okusi, J.; Wolf, Bernhard; Greiling, H.

doi:10.1515/cclm.1987.25.8.473

Cited by 18 publications

(16 citation statements)

References 21 publications

(19 reference statements)

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“…We found highly increased XT activities in synovial fluids of patients with chronic joint diseases (10). The elevation of enzyme activity in synovial fluids indicates the increased synthesis of proteoglycans in inflammatory processes.…”

mentioning

confidence: 54%

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First Isolation of Human UDP-d-Xylose: Proteoglycan Core Protein β-d-Xylosyltransferase Secreted from Cultured JAR Choriocarcinoma Cells

Kühn

Götting

Schnölzer

et al. 2001

Journal of Biological Chemistry

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Human UDP-D-xylose:proteoglycan core protein ␤-Dxylosyltransferase (EC 2.4.2.26, XT) initiates the biosynthesis of glycosaminoglycan lateral chains in proteoglycans by transfer of xylose from UDP-xylose to specific serine residues of the core protein. In this study, we report the first isolation of the XT and present the first partial amino acid sequence of this enzyme. We purified XT 4,700-fold with 1% yield from serum-free JAR choriocarcinoma cell culture supernatant. The isolation procedure included a combination of ammonium sulfate precipitation, heparin affinity chromatography, ion exchange chromatography, and protamine affinity chromatography. Among other proteins an unknown protein was detected by matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis in the purified sample. The molecular mass of this protein was determined as 120 kDa by SDS-polyacrylamide gel electrophoresis. The isolated protein was enzymatically cleaved by trypsin and endoproteinase Lys-C. Eleven peptide fragments were sequenced by Edman degradation. Searches with the amino acid sequences in protein and EST data bases showed no homology to known sequences. XT was enriched by immunoaffinity chromatography with an immobilized antibody against a synthetic peptide deduced from the sequenced peptide fragments and was specifically eluted with the antigen. In addition, XT was purified alternatively with an aprotinin affinity chromatography and was detected by Western blot analysis in the enzyme-containing fraction.

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confidence: 54%

“…Previous studies have shown that heparin reduced XT activity indicating a strong interaction of heparin with the enzyme (10). Therefore, we used a heparin matrix as an affinity ligand for the XT.…”

Section: Table IImentioning

confidence: 99%

First Isolation of Human UDP-d-Xylose: Proteoglycan Core Protein β-d-Xylosyltransferase Secreted from Cultured JAR Choriocarcinoma Cells

Kühn

Götting

Schnölzer

et al. 2001

Journal of Biological Chemistry

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“…Enzyme-linked immunosorbent assay (ELISA) techniques have been used to measure carboxy-terminal crosslinked collagen I-telopeptide in serum or amino-terminal crosslinked collagen Itelopeptide in urine (18), but these markers are largely bone specific. Other proteins and estimated enzyme activities (e.g., collagenase, stromelysin, and Dxylosyltransferase) are not specific for the detection or description of degradation processes of cartilage (19,20). HPLC, unlike ELISA, is capable of adequately discriminating between the crosslinks.…”

Section: Discussionmentioning

confidence: 99%

Collagen crosslinks in fibromyalgia

Sprott¹,

Müller²,

Heine³

1997

Arthritis & Rheumatism

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Objective. To determine if abnormal collagen metabolism is a characteristic of fibromyalgia.Methods. The diagnosis of fibromyalgia was made according to the American College of Rheumatology criteria. Skin biopsy samples were obtained from the trapezius region of 8 patients with fibromyalgia. Urine was collected under standardized conditions from 55 control subjects and 39 patients with fibromyalgia, and serum was obtained from 17 controls and 22 patients with fibromyalgia. Pyridinoline (Pyd), an indicator of connective tissue disease, and deoxypyridinoline (Dpyd), an indicator of bone degradation, both of which represent products of lysyl oxidase-mediated crosslinking in collagen, were analyzed by ion-paired and gradient high-performance liquid chromatography (HPLC) methods with fluorescence detection. Levels of hydroxyproline (Hyp), a collagen turnover marker, were also measured. The findings were related to creatinine levels, and the PydDpyd ratio was determined.Results. Highly ordered cuffs of collagen were observed around the terminal nerve fibers by electron microscopic examination of biopsy tissue from all 8 patients with fibromyalgia, but were not observed in any of the control skin samples. The Pyd:Dpyd ratios in the urine and serum and the Hyp levels in the urine were significantly lower in patients with fibromyalgia than in healthy controls.Conclusion. Decreased levels of collagen crosslinking in fibromyalgia may contribute to remodeling of the extracellular matrix and collagen deposition around the nerve fibers, and may contribute to the lower pain Dr. Miiller

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“…All other peptides were purchased from Bachem Biochemica (Heidelberg, Germany). Silk fibroin and hydrofluoric acidand phenylmethylsulfonyl fluoride-degraded proteoglycan from bovine nasal septum cartilage were prepared as described previously (8). All other chemicals in pro analysi quality were purchased from Merck (Darmstadt, Germany).…”

Section: Udp-[mentioning

confidence: 99%

Recognition of Acceptor Proteins by UDP-D-xylose Proteoglycan Core Protein β-D-Xylosyltransferase

Brinkmann

Weilke

Kleesiek

1997

Journal of Biological Chemistry

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The formation of chondroitin sulfate is initiated by xylosyltransferase (XT) transferring xylose from UDPxylose to consensus serine residues of proteoglycan core proteins. Our alignment of 51 amino acid sequences of chondroitin sulfate attachment sites in 19 different proteins resulted in a consensus sequence for the recognition signal of XT. The complete recognition sequence is composed of the amino acids a-a-a-a-G-S-G-a-b-a, with a ‫؍‬ E or D and b ‫؍‬ G, E, or D. This sequence was confirmed by determination of the Michaelis-Menten constants for in vitro xylosylation of different synthetic proteins and peptides using an enriched XT preparation from conditioned cell culture supernatant of human chondrocytes. The highest acceptor activity was determined by the sequence Q-E-E-E-E-G-S-G-G-G-Q, which was found in the single chondroitin sulfate attachment site of bikunin, the inhibitory active component of the human inter-␣-trypsin inhibitor.We determined the Michaelis-Menten constant (K m ) of xylosylation of the synthetic bikunin analogous peptide Q-E-E-E-G-S-G-G-G-Q-K to be 22 M, which was 9-fold decreased in comparison to deglycosylated core protein from bovine cartilage (188 M), which was previously used as acceptor for the XT activity assay. The best XT acceptors were nonglycosylated recombinant wildtype bikunin (K m ‫؍‬ 0. These results imply that the primary structure of the acceptor is not the only determinant for recognition by xylosyltransferase. Thus, protein conformation is also a main factor in determining xylosylation.

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UDP-D-Xylose : Proteoglycan Core Protein β-D-Xylosyltransferase: A New Marker of Cartilage Destruction in Chronic Joint Diseases

Cited by 18 publications

References 21 publications

First Isolation of Human UDP-d-Xylose: Proteoglycan Core Protein β-d-Xylosyltransferase Secreted from Cultured JAR Choriocarcinoma Cells

First Isolation of Human UDP-d-Xylose: Proteoglycan Core Protein β-d-Xylosyltransferase Secreted from Cultured JAR Choriocarcinoma Cells

Collagen crosslinks in fibromyalgia

Recognition of Acceptor Proteins by UDP-D-xylose Proteoglycan Core Protein β-D-Xylosyltransferase

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