1 Fluorescence Resonance Energy Transfer ( FRET ) and Proximity Ligation Assays Reveal Functionally Relevant Homo-and Heteromeric Complexes among Hyaluronan Synthases HAS 1 , HAS 2 and HAS 3

In vertebrates, hyaluronan is produced in plasma membrane from cytosolic UDPsugar substrates by HAS1-3 isoenzymes that transfer N-Acetyl glucosamine (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 et al. 2010 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 Cterminal parts. Of all 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. http://www.jbc.org/cgi/doi/10.1074/jbc.M115.640581 The latest version is at JBC Papers in Press. Published on March 20, 2015 as Manuscript M115.640581