Identification of Phosphatase That Dephosphorylates Xylose in the Glycosaminoglycan-Protein Linkage Region of Proteoglycans

Koike, Toshiyasu; Izumikawa, Tomomi; Sato, Ban; Kitagawa, Hiroshi

doi:10.1074/jbc.m113.520536

“…32 P]ATP (1.11 ϫ 10 5 dpm), 50 mM Tris buffer, pH 7.0, 10 mM MnCl 2 , 10 mM CaCl 2 , and 0.1% BSA as described (2,3). The products of each reaction were then separated by gel filtration chromatography on a Superdex peptide column that had been equilibrated with elution buffer (0.25 M NH 4 HCO 3 and 7% 1-propanol).…”

Section: Methodsmentioning

confidence: 99%

“…Enzyme Digestion-Enzyme digestion with chondroitinase ABC (EC 4.2.2.20) from Arthrobacter aurescens (10 mIU), chondroitinase AC-II (chondroitinase AC lyase; EC 4.2.2.5) from A. aurescens (10 mIU), or alkaline phosphatase (1 unit) (Roche Applied Science) was carried out in a total volume of 20 l of appropriate buffer at 37°C overnight (3).…”

Section: Methodsmentioning

confidence: 99%

“…Pulldown Assays-Pulldown assays were performed as described previously (3). The His-tagged and protein A-tagged expression vectors (3,4) were co-transfected into COS-1 cells grown on 100-mm plates.…”

Section: Methodsmentioning

confidence: 99%

“…The labeled oligosaccharides were analyzed by high performance liquid chromatography (HPLC) on an amine-bound PA-03 column as described previously (3).…”

Section: Methodsmentioning

confidence: 99%

“…Thus, it is suggested that ChGn-1 regulates the number of CS chains and the total amount of CS in these patients and in growth plate cartilage. More recently, we demonstrated that XYLP regulates the number of CS chains by dephosphorylating the Xyl residue in the GAG-protein linkage region of proteoglycans (PGs) (3). However, the relationship between ChGn-1 and XYLP in the biosynthesis of CS was not clear.…”

mentioning

confidence: 97%

See 3 more Smart Citations

GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate) Is the Preferred Substrate for Chondroitin N-Acetylgalactosaminyltransferase-1

Izumikawa

¹

,

Sato

²

,

Mikami

³

et al. 2015

Journal of Biological Chemistry

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Solid‐Phase‐Supported Chemoenzymatic Synthesis and Analysis of Chondroitin Sulfate Proteoglycan Glycopeptides

Lin,

Xu,

Bali

et al. 2024

Angewandte Chemie

1

0

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Proteoglycans (PGs), consisting of glycosaminoglycans (GAGs) linked with the core protein through a tetrasaccharide linkage region, play roles in many important biological events. The chemical synthesis of PG glycopeptides is extremely challenging. In this work, the enzymes required for synthesis of chondroitin sulfate (CS) PG (CSPG) have been expressed and the suitable sequence of enzymatic reactions has been established. To expedite CSPG synthesis, the peptide acceptor was immobilized on solid phase and the glycan units were directly installed enzymatically onto the peptide. Subsequent enzymatic chain elongation and sulfation led to the successful synthesis of CSPG glycopeptides. The CS dodecasaccharide glycopeptide was the longest homogeneous CS glycopeptide synthesized to date. The enzymatic synthesis was much more efficient than the chemical synthesis of the corresponding CS glycopeptides, which could reduce the total number of synthetic steps by 80%. The structures of the CS glycopeptides were confirmed by mass spectrometry analysis and NMR studies. In addition, the interactions between the CS glycopeptides and cathepsin G were studied. The sulfation of glycan chain was found to be important for binding with cathepsin G. This efficient chemoenzymatic strategy opens new avenues to investigate the structures and functions of PGs.

show abstract

Solid‐Phase‐Supported Chemoenzymatic Synthesis and Analysis of Chondroitin Sulfate Proteoglycan Glycopeptides

Lin,

Xu,

Bali

et al. 2024

Angew Chem Int Ed

0

View full text Add to dashboard Cite

Proteoglycans (PGs), consisting of glycosaminoglycans (GAGs) linked with the core protein through a tetrasaccharide linkage region, play roles in many important biological events. The chemical synthesis of PG glycopeptides is extremely challenging. In this work, the enzymes required for synthesis of chondroitin sulfate (CS) PG (CSPG) have been expressed and the suitable sequence of enzymatic reactions has been established. To expedite CSPG synthesis, the peptide acceptor was immobilized on solid phase and the glycan units were directly installed enzymatically onto the peptide. Subsequent enzymatic chain elongation and sulfation led to the successful synthesis of CSPG glycopeptides. The CS dodecasaccharide glycopeptide was the longest homogeneous CS glycopeptide synthesized to date. The enzymatic synthesis was much more efficient than the chemical synthesis of the corresponding CS glycopeptides, which could reduce the total number of synthetic steps by 80%. The structures of the CS glycopeptides were confirmed by mass spectrometry analysis and NMR studies. In addition, the interactions between the CS glycopeptides and cathepsin G were studied. The sulfation of glycan chain was found to be important for binding with cathepsin G. This efficient chemoenzymatic strategy opens new avenues to investigate the structures and functions of PGs.

show abstract

Identification of Phosphatase That Dephosphorylates Xylose in the Glycosaminoglycan-Protein Linkage Region of Proteoglycans

Cited by 62 publications

References 34 publications

GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate) Is the Preferred Substrate for Chondroitin N-Acetylgalactosaminyltransferase-1

GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate) Is the Preferred Substrate for Chondroitin N-Acetylgalactosaminyltransferase-1

Solid‐Phase‐Supported Chemoenzymatic Synthesis and Analysis of Chondroitin Sulfate Proteoglycan Glycopeptides

Solid‐Phase‐Supported Chemoenzymatic Synthesis and Analysis of Chondroitin Sulfate Proteoglycan Glycopeptides

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