Recombinant, truncated B. circulans keratanase-II: Description and characterisation of a novel enzyme for use in measuring urinary keratan sulphate levels via LC–MS/MS in Morquio A syndrome

Steward, M. W.; Berezovskaya, Yana; Zhou, Huiyu; Shediac, Renée; Sun, Cynthia Q; Miller, Nicole L.; Rendle, Phillip M.

doi:10.1016/j.clinbiochem.2015.03.024

Cited by 8 publications

(4 citation statements)

References 25 publications

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“…and Chondroitinase ABC ( Proteus vulgaris ) were from Amsbio (Cambridge, MA). A recombinant truncated form of Keratanase II ( Bacillus circulans ) was produced in Escherichia coli using an expression plasmid kindly provided by Dr. Andrew Muscroft-Taylor, GlycoSyn, New Zealand and expressed and purified essentially as described ( Steward et al 2015 ). Recombinant Vibrio cholerae sialidase was kindly provided by Dr Garry Taylor, University of St. Andrews, and purified produced in E. coli from an expression plasmid as described ( Moustafa et al 2004 ; Mountney et al 2010 ).…”

Section: Methodsmentioning

confidence: 99%

Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways

et al. 2018

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Human siglecs are a family of 14 sialic acid-binding proteins, most of which are expressed on subsets of immune cells where they regulate immune responses. Siglec-8 is expressed selectively on human allergic inflammatory cells-primarily eosinophils and mast cells-where engagement causes eosinophil apoptosis and inhibits mast cell mediator release. Evidence supports a model in which human eosinophils and mast cells bind to Siglec-8 sialoglycan ligands on inflammatory target tissues to resolve allergic inflammation and limit tissue damage. To identify Siglec-8-binding sialoglycans from human airways, proteins extracted from postmortem human trachea were resolved by size-exclusion chromatography and composite agarose-acrylamide gel electrophoresis, blotted and probed by Siglec-8-Fc blot overlay. Three size classes of Siglec-8 ligands were identified: 250 kDa, 600 kDa and 1 MDa, each of which was purified by affinity chromatography using a recombinant pentameric form of Siglec-8. Proteomic mass spectrometry identified all size classes as the proteoglycan aggrecan, a finding validated by immunoblotting. Glycan array studies demonstrated Siglec-8 binding to synthetic glycans with a terminal Neu5Acα2-3(6-sulfo)-Gal determinant, a quantitatively minor terminus on keratan sulfate (KS) chains of aggrecan. Treating human tracheal extracts with sialidase or keratanase eliminated Siglec-8 binding, indicating sialylated KS chains as Siglec-8-binding determinants. Treating human tracheal histological sections with keratanase also completely eliminated the binding of Siglec-8-Fc. Finally, Siglec-8 ligand purified from human trachea extracts induced increased apoptosis of freshly isolated human eosinophils in vitro. We conclude that sialylated KS proteoglycans are endogenous human airway ligands that bind Siglec-8 and may regulate allergic inflammation.

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Section: Methodsmentioning

confidence: 99%

Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways

et al. 2018

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“…Before electrophoresis, as indicated, some nasal lavage samples were treated with hydrolytic enzymes to identify functional binding determinants. To equal aliquots we added buffer alone (control), 7 mU/mL of keratanase I (Pseudomonas spp [Amsbio, Abingdon, United Kingdom]), 7 mU/mL of keratanase II (Bacillus circulans expressed in Escherichia coli) prepared as described, 13 0.25 U/mL of chondroitinase ABC, 0.25 U/mL of heparinase (H3917, MilliporeSigma), or 76 mU/mL of sialidase (V cholerae expressed in E coli as described). 12 Samples were incubated at 378C for 20 hours (except for sialidase, which was incubated for 1.5 hours) before electrophoresis.…”

Section: Molecular Analyses Of Human Fluids and Tissuesmentioning

confidence: 99%

Isolation, identification, and characterization of the human airway ligand for the eosinophil and mast cell immunoinhibitory receptor Siglec-8

Gonzalez-Gil

Porell

et al. 2021

Journal of Allergy and Clinical Immunology

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“…9 Similarly, to degrade keratan sulfate glycosaminoglycans, sections were incubated with 50 µg/ml (dissolved in 30 mM sodium acetate, pH 5.2) of recombinant keratanase II (from Bacillus circulans) prepared as described. 10 To cleave internal β1,4galactosidic linkages in non-sulfated or GlcNAc-6-Osulfated oligo-or poly-N-acetyllactosamine, sections were incubated with 50 µg/ml (dissolved in 30 mM sodium acetate, pH 5.2) of endo-β-galactosidase (from Citrobacter freundii) purified as described. 11,12 As positive controls for enzymatic digestion, we performed immunohistochemistry with 5D4 antibody using porcine corneal tissues rich in N-glycosidically linked keratan sulfate glycosaminoglycans (Supplemental Figs.…”

Section: Pretreatment Of Tissue Sectionsmentioning

confidence: 99%

Apical Membrane Expression of Distinct Sulfated Glycans Is a Characteristic Feature of Ductules and Their Reactive and Neoplastic Counterparts

Hoshino

Akama

Uchimura

et al. 2021

J Histochem Cytochem.

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Intrahepatic bile ducts transport bile between bile canaliculi and the extrahepatic bile duct. The luminal surface of this tract is lined by a layer of biliary epithelial cells, or cholangiocytes, which secrete mucins consisting of scaffold proteins and O-glycosidically linked carbohydrate side chains. Although mucin core proteins have been extensively investigated, the structure and function of carbohydrate side chains have not. Here, we demonstrate that distinct sulfated glycans positive for MECA-79, R-10G, and 297-11A, but not 5D4, monoclonal antibodies are expressed in the cytoplasm of cells of large-sized ducts and in the apical membrane of cells in ductules, and that R-10G immunolabeling is partially eliminated by endo-β-galactosidase digestion, supporting the presence of N-acetylglucosamine-6- O-sulfated N-acetyllactosamine structures. We observed comparable apical membrane-predominant staining in ductular reactions seen during regeneration that occurs in various liver diseases and in cholangiolocarcinoma, a subtype of small duct-type intrahepatic cholangiocarcinoma (iCCA). Apical membrane expression of distinct sulfated glycans in large duct-type iCCA was negligible. Intriguingly, under pathological conditions, endo-β-galactosidase digestion almost completely eliminated R-10G immunoreactivity. These findings suggest that apical membrane expression of distinct sulfated glycans is a characteristic feature of ductules and their reactive and neoplastic counterparts

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Recombinant, truncated B. circulans keratanase-II: Description and characterisation of a novel enzyme for use in measuring urinary keratan sulphate levels via LC–MS/MS in Morquio A syndrome

Abstract: This novel, recombinant keratanase-II meets all performance requirements and can be produced in a rapid and reproducible manner. We speculate that other related bacterial enzymes of biomedical or industrial interest may be amenable to similar engineered enhancements.

Cited by 8 publications

References 25 publications

Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways

Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways

Isolation, identification, and characterization of the human airway ligand for the eosinophil and mast cell immunoinhibitory receptor Siglec-8

Apical Membrane Expression of Distinct Sulfated Glycans Is a Characteristic Feature of Ductules and Their Reactive and Neoplastic Counterparts

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