Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays Reveal Functionally Relevant Homo- and Heteromeric Complexes among Hyaluronan Synthases HAS1, HAS2, and HAS3

• Published in: The Journal of biological chemistry Vol. 290; no. 18; pp. 11479 - 11490
• Main Authors: Bart, Geneviève, Vico, Nuria Ortega, Hassinen, Antti, Pujol, Francois M., Deen, Ashik Jawahar, Ruusala, Aino, Tammi, Raija H., Squire, Anthony, Heldin, Paraskevi, Kellokumpu, Sakari, Tammi, Markku I.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2015; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Carbohydrate Biosynthesis; Chlorocebus aethiops; COS Cells; Fluorescence Resonance Energy Transfer; Fluorescence Resonance Energy Transfer (FRET); Glucuronosyltransferase - chemistry; Glucuronosyltransferase - metabolism; Glycobiology and Extracellular Matrices; Golgi; Humans; Hyaluronan; Hyaluronan Synthase; Hyaluronan Synthases; Hyaluronic Acid - biosynthesis; Isoenzymes - chemistry; Mice; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Protein Complex; Protein Multimerization; Protein Structure, Quaternary; Protein Structure, Tertiary; Proximity Ligation Assay; Hyaluronan; Hyaluronan Synthase; Proximity Ligation Assay; Protein Complex; Fluorescence Resonance Energy Transfer (FRET); Golgi; Carbohydrate Biosynthesis
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M115.640581

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. Background: Co-immunoprecipitation experiments suggest the existence of HAS2-HAS2 and HAS2-HAS3 enzyme complexes. Results: FRET and proximity ligation assays show homo- and heteromeric complexes among all HASs. Conclusion: Like Golgi glycosyltransferases, HASs form homo- and heteromeric complexes. Significance: Different HAS complexes may have specific effects on hyaluronan synthesis.