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
From the Institute of Biomedicine/Anatomy, University of Eastern Finland, FI-70211 Kuopio, Finland.
the Faculty of Biochemistry and Molecular Medicine, University of Oulu, FI-90014 Oulu, Finland.
J Biol Chem. 2015 May 1;290(18):11479-90. doi: 10.1074/jbc.M115.640581. Epub 2015 Mar 20.
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.
在脊椎动物中，透明质酸由细胞质中的尿苷二磷酸糖底物通过透明质酸合酶1 - 3（HAS1 - 3）同工酶在质膜上产生，这些同工酶在将不断增长的多糖链同时挤出到细胞外空间的过程中，将N - 乙酰葡糖胺（GlcNAc）和葡糖醛酸（GlcUA）交替连接到多糖链的不同位置。研究表明，HAS2免疫沉淀物中含有功能性的HAS2同型二聚体以及与HAS3形成的异型二聚体（Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) 透明质酸合酶2的活性受二聚化和泛素化调节。《生物化学杂志》285, 23647 - 23654）。在此，我们利用荧光共振能量转移（FRET）和流式细胞术定量分析，在活细胞中系统地筛选了HAS同工酶之间潜在的相互作用。我们发现所有的HAS同工酶都能相互形成同型二聚体和异型二聚体复合物。通过FRET显微镜和受体光漂白法在高尔基体和质膜中均检测到了相同的复合物。用HAS抗体进行的邻近连接分析证实了内源性表达的HAS之间存在HAS1 - HAS2、HAS2 - HAS2和HAS2 - HAS3复合物。C末端缺失分析表明，这些酶主要通过未明确的N末端86个氨基酸结构域相互作用，但在其C末端部分可能还存在其他结合位点。在所有同型二聚体复合物中，HAS1的合成活性最低，HAS3的合成活性最高。有趣的是，转染HAS1会降低由HAS2和HAS3产生的透明质酸的合成，这表明这些同工酶之间存在功能协同作用。这些数据表明，HAS同工酶普遍倾向于形成同型二聚体和异型二聚体复合物，这可能对透明质酸的合成产生重要的功能影响。
