title: Fluorescence resonance energy transfer (FRET) and proximity ligation assays reveal functionally relevant homo- and heteromeric complexes among hyaluronan synthases HAS1, HAS2, and HAS3.
abstract: In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1-3 (HAS1-3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647-23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis.
authors: From the Institute of Biomedicine/Anatomy, University of Eastern Finland, FI-70211 Kuopio, Finland.
authors: Geneviève
authors: G
authors: Bart
authors: the Faculty of Biochemistry and Molecular Medicine, University of Oulu, FI-90014 Oulu, Finland.
authors: Nuria Ortega
authors: NO
authors: Vico
authors: the Faculty of Biochemistry and Molecular Medicine, University of Oulu, FI-90014 Oulu, Finland.
authors: Antti
authors: A
authors: Hassinen
authors: the Faculty of Biochemistry and Molecular Medicine, University of Oulu, FI-90014 Oulu, Finland.
authors: Francois M
authors: FM
authors: Pujol
authors: From the Institute of Biomedicine/Anatomy, University of Eastern Finland, FI-70211 Kuopio, Finland.
authors: Ashik Jawahar
authors: AJ
authors: Deen
authors: the Ludwig Institute for Cancer Research, Uppsala University, SE-75124, Uppsala, Sweden, and.
authors: Aino
authors: A
authors: Ruusala
authors: From the Institute of Biomedicine/Anatomy, University of Eastern Finland, FI-70211 Kuopio, Finland.
authors: Raija H
authors: RH
authors: Tammi
authors: the Institute for Experimental Immunology and Imaging, University Clinic Essen, 45147 Essen, Germany.
authors: Anthony
authors: A
authors: Squire
authors: the Ludwig Institute for Cancer Research, Uppsala University, SE-75124, Uppsala, Sweden, and.
authors: Paraskevi
authors: P
authors: Heldin
authors: the Faculty of Biochemistry and Molecular Medicine, University of Oulu, FI-90014 Oulu, Finland.
authors: Sakari
authors: S
authors: Kellokumpu
authors: From the Institute of Biomedicine/Anatomy, University of Eastern Finland, FI-70211 Kuopio, Finland, tammi@uef.fi.
authors: Markku I
authors: MI
authors: Tammi
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000096:biosynthesis; D007527:Isoenzymes / Q000737:chemistry; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
chemical_list: D007527:Isoenzymes; D010446:Peptide Fragments; D006820:Hyaluronic Acid; D014453:Glucuronosyltransferase; C523794:HAS1 protein, human; C101754:HAS2 protein, human; C105458:HAS3 protein, human; D000076002:Hyaluronan Synthases
keywords: Carbohydrate Biosynthesis; Fluorescence Resonance Energy Transfer (FRET); Golgi; Hyaluronan; Hyaluronan Synthase; Protein Complex; Proximity Ligation Assay