TY - JOUR
T1 - CD109 is a component of exosome secreted from cultured cells
AU - Sakakura, Hiroki
AU - Mii, Shinji
AU - Hagiwara, Sumitaka
AU - Kato, Takuya
AU - Yamamoto, Noriyuki
AU - Hibi, Hideharu
AU - Takahashi, Masahide
AU - Murakumo, Yoshiki
N1 - Funding Information:
We thank Mr. K. Imaizumi, Mr. K. Uchiyama and Mrs. K. Ushida for their technical assistance. This work was supported by Grants-in-Aid for Global Center of Excellence (GCOE) research commissioned by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to MT) and for Scientific Research (C) commissioned by MEXT of Japan (21590435 to YM).
Publisher Copyright:
© 2015 Elsevier Inc. All rights reserved.
PY - 2016/1/22
Y1 - 2016/1/22
N2 - Exosomes are 50-100-nm-diameter membrane vesicles released from various types of cells. Exosomes retain proteins, mRNAs and miRNAs, which can be transported to surrounding cells. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, and is released from the cell surface to the culture medium in vitro. Recently, it was reported that secreted CD109 from the cell surface downregulates transforming growth factor-β signaling in human keratinocytes. In this study, we revealed that CD109 is a component of the exosome in conditioned medium. FLAG-tagged human CD109 (FLAG-CD109) in conditioned medium secreted from HEK293 cells expressing FLAG-CD109 (293/FLAG-CD109) was immunoprecipitated with anti-FLAG affinity gel, and the co-precipitated proteins were analyzed by mass spectrometry and western blotting. Exosomal proteins were associated with CD109. We revealed the presence of CD109 in exosome fractions from conditioned medium of 293/FLAG-CD109. Moreover, the localization of CD109 in the exosome was demonstrated using immuno-electron microscopy. When we used HEK293 cells expressing FLAG-tagged truncated CD109, which does not contain the C-terminal region, the association of truncated CD109 with exosomes was not detected in conditioned medium. These findings indicate that CD109 is an exosomal protein and that the C-terminal region of CD109 is required for its presence in the exosome.
AB - Exosomes are 50-100-nm-diameter membrane vesicles released from various types of cells. Exosomes retain proteins, mRNAs and miRNAs, which can be transported to surrounding cells. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, and is released from the cell surface to the culture medium in vitro. Recently, it was reported that secreted CD109 from the cell surface downregulates transforming growth factor-β signaling in human keratinocytes. In this study, we revealed that CD109 is a component of the exosome in conditioned medium. FLAG-tagged human CD109 (FLAG-CD109) in conditioned medium secreted from HEK293 cells expressing FLAG-CD109 (293/FLAG-CD109) was immunoprecipitated with anti-FLAG affinity gel, and the co-precipitated proteins were analyzed by mass spectrometry and western blotting. Exosomal proteins were associated with CD109. We revealed the presence of CD109 in exosome fractions from conditioned medium of 293/FLAG-CD109. Moreover, the localization of CD109 in the exosome was demonstrated using immuno-electron microscopy. When we used HEK293 cells expressing FLAG-tagged truncated CD109, which does not contain the C-terminal region, the association of truncated CD109 with exosomes was not detected in conditioned medium. These findings indicate that CD109 is an exosomal protein and that the C-terminal region of CD109 is required for its presence in the exosome.
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U2 - 10.1016/j.bbrc.2015.12.063
DO - 10.1016/j.bbrc.2015.12.063
M3 - Article
C2 - 26707640
AN - SCOPUS:84957434767
VL - 469
SP - 816
EP - 822
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 4
ER -