title: Reduced Expression of EXTL2, a Member of the Exostosin (EXT) Family of Glycosyltransferases, in Human Embryonic Kidney 293 Cells Results in Longer Heparan Sulfate Chains.
abstract: Heparan sulfate proteoglycans are ubiquitously located on cell surfaces and in the extracellular matrices. The negatively charged heparan sulfate chains interact with a multitude of different proteins, thereby influencing a variety of cellular and developmental processes, for example cell adhesion, migration, tissue morphogenesis, and differentiation. The human exostosin (EXT) family of genes contains five members: the heparan sulfate polymerizing enzymes, EXT1 and EXT2, and three EXT-like genes, EXTL1, EXTL2, and EXTL3. EXTL2 has been ascribed activities related to the initiation and termination of heparan sulfate chains. Here we further investigated the role of EXTL2 in heparan sulfate chain elongation by gene silencing and overexpression strategies. We found that siRNA-mediated knockdown of EXTL2 in human embryonic kidney 293 cells resulted in increased chain length, whereas overexpression of EXTL2 in the same cell line had little or no effect on heparan sulfate chain length. To study in more detail the role of EXTL2 in heparan sulfate chain elongation, we tested the ability of the overexpressed protein to catalyze the in vitro incorporation of N-acetylglucosamine and N-acetylgalactosamine to oligosaccharide acceptors resembling unmodified heparan sulfate and chondroitin sulfate precursor molecules. Analysis of the generated products revealed that recombinant EXTL2 showed weak ability to transfer N-acetylgalactosamine to heparan sulfate precursor molecules but also, that EXTL2 exhibited much stronger in vitro N-acetylglucosamine-transferase activity related to elongation of heparan sulfate chains.
authors.affiliation: From the Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway and.
authors.forename: Kirankumar
authors.initials: K
authors.lastname: Katta
authors.affiliation: From the Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway and.
authors.forename: Tabasum
authors.initials: T
authors.lastname: Imran
authors.affiliation: the Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.
authors.forename: Marta
authors.initials: M
authors.lastname: Busse-Wicher
authors.affiliation: From the Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway and.
authors.forename: Mona
authors.initials: M
authors.lastname: Grønning
authors.affiliation: From the Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway and.
authors.forename: Szymon
authors.initials: S
authors.lastname: Czajkowski
authors.affiliation: From the Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway and Marion.Kusche@biomed.uib.no.
authors.forename: Marion
authors.initials: M
authors.lastname: Kusche-Gullberg
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D005434:Flow Cytometry; D057809:HEK293 Cells; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D006497:Heparan Sulfate; D008565:Membrane Proteins; D017351:N-Acetylglucosaminyltransferases; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; C110774:EXTL3 protein, human; C121267:EXTL1 protein, human; C110834:EXTL2 protein, human
keywords: EXTL2; Exostosin-like 2; chondroitin sulfate; glycosaminoglycan; glycosyltransferase; heparan sulfate; proteoglycan
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