Isolation and identification of histone H3 protein enriched in microvesicles secreted from cultured sebocytes
Isolation and identification of histone H3 protein enriched in microvesicles secreted from cultured sebocytes
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DOI：
10.1210/en.2004-1478
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发表时间：
2005-06-01
期刊：
影响因子：
4.8
通讯作者：
Sumida, M
中科院分区：
文献类型：
--
作者：
Nagai, A;Sato, T;Sumida, M
在培养的皮脂细胞中首次发现分泌含有脂质颗粒的微囊泡，即皮脂体。鼠克隆皮脂细胞达到合流状态后，释放出直径0.5 ~ 5.0 μ m的泡状微泡，其结构复杂，含有多个油红o染色颗粒。仓鼠克隆和大鼠原代培养的脂质细胞微泡的脂质成分均为大量角鲨烯。微泡含有浓缩的17 kda阳离子蛋白，可溶于pH为1.5的硫酸盐缓冲液（包括Nonidet P-40）中。由于该蛋白与肝素- sepharose结合紧密，用1.5 M NaCl洗脱，进一步用SDS-PAGE凝胶纯化。肽段测序鉴定该蛋白为组蛋白H3。针对纯化蛋白的多克隆抗体在仓鼠克隆和大鼠原代培养的皮脂细胞的微泡中检测到抗原。抗体显示该蛋白分布于细胞核、细胞质和前体微泡内。将编码组蛋白h3增强绿色荧光蛋白的基因构建体转染到脂细胞中，在细胞核和细胞质前体微泡中都检测到融合蛋白的荧光。硫酸肝素在微泡中的分布明显，提示组蛋白H3可能被募集后被分子凝聚到分泌的微泡中。此外，在微泡中检测到的14-3-3蛋白也可能有助于将组蛋白H3蛋白整合到微泡中，因为它可以结合组蛋白和脂质颗粒。
Secretion of microvesicles, defined as sebosomes, containing lipid particles were discovered for the first time in cultured sebocytes. After reaching confluency, hamster-cloned sebocytes released bubble-like microvesicles with a diameter range of 0.5-5.0 mu m. They had a complex structure containing multiple Oil Red O-stainable particles. The lipid components of the microvesicles were large amounts of squalene both of hamster-cloned and rat primary cultured sebocytes. The microvesicles contained a concentrated 17-kDa cationic protein, which was soluble in sulfate buffer including Nonidet P-40 at pH 1.5. As the protein bound tightly to heparin-Sepharose and eluted with 1.5 M NaCl, it was further purified from a SDS-PAGE gel. Peptide sequencing identified the protein to be histone H3. Polyclonal antibodies against the purified protein detected the antigen in the microvesicles both in the hamster-cloned and rat primary cultured sebocytes. The antibodies demonstrated a distribution of the protein within the nucleus, cytoplasm, and precursor microvesicles. When a gene construct encoding histone H3-enhanced green fluorescent protein was transfected to the sebocytes, fluorescence of the fusion proteins was detected within both the nucleus and the precursor microvesicles of the cytoplasm. The distribution of heparan sulfate was evident in the microvesicles, and it suggested the possibility that the histone H3 protein was recruited and then condensed to the secreted microvesicles by the molecules. In addition, the 14-3-3 protein, which was detected in the microvesicles, also may help incorporate the histone H3 protein in the microvesicles because it can bind to both histone and lipid particles.