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Isolation and Identification of Histone H3 Protein Enriched in Microvesicles Secreted from Cultured Sebocytes

Department of Molecular and Cellular Biology (A.N., M.S.), Division of Biochemistry and Molecular Genetics, Ehime University School of Medicine, Shitsukawa, Toon-city, Ehime 791-0295, Japan; and Department of Biochemistry and Molecular Biology (T.S., N.A., A.I.), School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan

Address all correspondence and requests for reprints to: Michihiro Sumida Ph.D.,¹Department of Molecular and Cellular Biology, Division of Biochemistry and, Molecular Genetics, Ehime University School of Medicine, Shitsukawa, Toon-city, Ehime 791-0295, Japan, E-mail: sumida{at}m.ehime-u.ac.jp

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 µm. 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.