Department of Bioscience and Laboratory Medicine, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, 036-8564, Japan; Research Center for Biomedical Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, 036-8564, Japan.
Division of Glyco-signal Research, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aobaku, Sendai, Miyagi, 981-8558, Japan.
Biochem Biophys Res Commun. 2020 Dec 17;533(4):732-738. doi: 10.1016/j.bbrc.2020.08.093. Epub 2020 Sep 29.
Hyaluronan synthase 2 (HAS2) is an integral membrane protein composed of multi-membrane-spanning regions and a large intracellular loop (HAS2-loop). We previously examined the effect of phorbol 12-myristate 13-acetate (PMA) and/or 4-methylumbelliferone (4-MU) on the synthesis of hyaluronan (HA) in human skin fibroblasts and found that TPA and 4-MU have opposing effects on HA synthesis by phosphorylation and O-linked β-N-acetylglucosaminylation of HAS2, respectively. In this study, we constructed an expression vector for the HAS2-loop and analyzed its post-translational modification by PMA and 4-MU using mass spectrometry. We identified a phosphorylation site at the position corresponding to the Thr position of full-length HAS2, which was detected in the cells regardless of the presence of PMA or 4-MU. We next prepared T328A site-directed mutagenesis construct-transfected cells and investigated HA synthesis. The amount of HA was increased in cells expressing full-length HAS2 compared to in mock cells, whereas the amount of HA synthesized by cells transfected with the T328A site-directed mutagenesis construct was the same as that in mock cells. This phosphorylation site corresponded with the casein kinase 1 substrate motif. These results suggest that Thr phosphorylation is an essential factor for HA synthesis by HAS2 and the role of HAS2-loop may be useful in analyzing the regulation of HAS2 synthesis in physiological and pathological conditions.
透明质酸合酶 2（HAS2）是一种完整的跨膜蛋白，由多个跨膜区和一个大的细胞内环（HAS2 环）组成。我们之前研究了佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）和/或 4-甲基伞形酮（4-MU）对人皮肤成纤维细胞中透明质酸（HA）合成的影响，发现 TPA 和 4-MU 通过 HAS2 的磷酸化和 O-连接β-N-乙酰氨基葡萄糖基化分别对 HA 合成产生相反的影响。在这项研究中，我们构建了 HAS2 环的表达载体，并通过质谱分析了 PMA 和 4-MU 对其翻译后修饰的影响。我们鉴定了一个与全长 HAS2 的 Thr 位置相对应的磷酸化位点，该位点在细胞中无论是否存在 PMA 或 4-MU 都可检测到。我们接下来制备了 T328A 定点突变构建体转染的细胞，并研究了 HA 的合成。与mock 细胞相比，表达全长 HAS2 的细胞中 HA 的含量增加，而转染 T328A 定点突变构建体的细胞中合成的 HA 与 mock 细胞相同。该磷酸化位点与酪蛋白激酶 1 底物基序相对应。这些结果表明 Thr 磷酸化是 HAS2 合成 HA 的必要因素，HAS2 环的作用可能有助于分析生理和病理条件下 HAS2 合成的调节。
