論文の公開元へSecretory expression of mammalian NOTCH tandem epidermal growth factor-like repeats based on increased O-glycosylation
概要
主論文の要旨
Secretory expression of mammalian NOTCH
tandem epidermal growth factor-like repeats based
on increased O-glycosylation
O-グリコシル化の増加に基づく哺乳類NOTCHタンデム
上皮成長因子様リピートの分泌発現
名古屋大学大学院医学系研究科
生体管理医学講座
麻酔･蘇生医学分野
（指導：西脇 公俊
张
総合医学専攻
爱玲
教授）
【Introduction】
Dysregulation of Notch signaling is associated with various cancer. Although Notch
signaling has both tumor-promoting and tumor-suppressive roles, it is considered a potential
therapeutic target for tumors because tumor angiogenesis depends on Notch signaling;
disruption of Notch signaling inhibits tumor growth by impairing angiogenesis. Thus,
establishing effective strategies to inhibit Notch signaling is of therapeutic importance. A
previous study developed a soluble form of the NOTCH1 receptor (NOTCH1 decoy) that
inhibits ligand-induced Notch signaling and tumor angiogenesis in vivo. Although the
NOTCH1 decoy molecule consists exclusively of a tandem array of epidermal growth factorlike (EGF) repeats, the expressed proteins are poorly secreted into the culture media and are
predominantly retained within the cells. NOTCH EGF repeats are modified with specific Oglycans, including O-fucose, O-glucose, and O-GlcNAc, which are catalyzed by POFUT1,
POGLUT1, and EOGT, respectively. Previous studies have revealed that POFUT1 and
POGLUT1 affect the cell surface expression of endogenous NOTCH1 in various cell types,
including HEK293T cells. Thus, optimal expression of EGF domain-specific glycosyltransferases
may improve the production of full-length NOTCH EGF repeats.
In this study, we evaluated the effects of overexpressed EGF domain-specific Oglycosyltransferases, including POFUT1, POGLUT1, and EOGT, on the secretion of NOTCH
EGF repeats in HEK293T cells. Our results reveal a novel function of EOGT in promoting the
secretion of recombinantly expressed NOTCH1 EGF repeats.
【Method】
We exogenously expressed EOGT in HEK293T cells to prepare a culture medium containing
NOTCH1 EGF repeats. The plasmid construct encoding the entire NOTCH1 EGF repeat with
an N-terminal FLAG tag (FLAG:N1-EGF) was generated and transfected into HEK293T cells
with or without EOGT. Culture media containing FLAG:N1-EGF were incubated with DLL4
ligand-expressing cells and analyzed by FACS. Next, we examined the combined effects of
glycosyltransferases acting on NOTCH EGF domains in the endoplasmic reticulum (ER),
including POFUT1, POGLUT1, and EOGT. Because a previous study on Drosophila POFUT1
homologue indicated that glycosyltransferases could promote NOTCH secretion in an enzyme
activity-independent manner, we analyzed the impact of enzymatically inactive mutants of
EOGT and POGLUT1. To evaluate changes in the O-glycosylation states of EGF repeats
mediated by the co-expression of EOGT and POGLUT1, mass spectrometric analysis was
performed. We selected FLAG-tagged NOTCH3 EGF repeats (FLAG:N3-EGF) for analysis
because of the abundance of the purified proteins.
【Result】
First, we confirmed that with EOGT overexpression the amount of FLAG:N1-EGF bound to
-1-
ligand-expressing cells was significantly increased. At the same time, the amount of FLAG:N1EGF in the culture media was elevated. These results suggest that EOGT possesses a novel but
seemingly physiologically irrelevant ability to promote the secretion of EGF repeats of
NOTCH1, which at least partially contributes to increased binding to DLL4. Next, we prepared
culture media and cell lysates to analyze the amount of FLAG:N1-EGF, FLAG:N3-EGF
secretion. Immunoblotting results indicated that FLAG:N1-EGF secreted in the culture media
was increased by co-expression of EOGT. Despite the roles of POFUT1 and POGLUT1 in the
secretory pathway, the effect of POFUT1 or POGLUT1 overexpression on FLAG:N1-EGF
NOTCH1 secretion was not statistically significant in HEK293T cells. In contrast, coexpression of EOGT/POGLUT1, but not EOGT/POFUT1, further increased secretion
compared with EOGT alone. Also, FLAG:N3-EGF secreted in culture media was increased by
co-expression of EOGT/POGLUT1. Quantification of secreted FLAG:N1-EGF from EOGT
mutant HEK293T cells co-expressing inactive EOGT harboring the R377Q mutation showed
that EOGT R377Q failed to facilitate secretion into the culture media. Co-expression of
POGLUT1 R279W slightly increased the secretion of FLAG:N1-EGF, but to a lesser extent than
that of wild-type POGLUT1. These data suggested that enzyme activity is essential for the
effect of EOGT on the promotion of FLAG:N1-EGF secretion. The optimal combination of
ER-resident glycosyltransferases serves as a strategy for efficiently producing EGF repeats of
various NOTCH family members based on the engineered expression of glycosyltransferases.
Finally, we adopted the label-free quantitation workflow in Proteome Discoverer with Byonic
node, a search engine suitable for glycopeptide identification from MS/MS spectra. Consistent
with the O-GlcNAc transferase activity of EOGT, the abundance ratio of HexNAc-modified
glycopeptides corresponding to O-GlcNAc, O-GlcNAc-Gal, or O-GlcNAc-Gal-NeuAc glycoforms
was markedly increased at EGF9, 10, 25, 31, and 34 by EOGT/POGLUT1 overexpression. In
contrast, the effect on EGF4, 14, 18, and 30 was limited because O-GlcNAcylation occurs at
high stoichiometries regardless of the exogenously expressed glycosyltransferases. These data
support our proposal that elevated O-GlcNAc modifications promote the secretion of NOTCH
EGF repeats. Lack of these constitutive modifications may contribute to the reduced secretion
efficiency of NOTCH EGF repeats in EOGT mutant cells. Unlike in the case of O-GlcNAc
modification, the effect of EOGT/POGLUT1 on O-Glc modification was obscure because all
detectable
O-Glc
sites
on
FLAG:N3-EGF
were
mostly
O-glycosylated
without
EOGT/POGLUT1 overexpression. However, that NOTCH EGF repeats with insufficient Oglycosylation are not secreted and, therefore, not detected by mass spectrometry of secreted
proteins in the culture medium. Expression of POGLUT1 may reduce the proportion of
insufficiently O-glucosylated NOTCH EGF, which is otherwise retained in the cells.
【Conclusion】
This study suggests that combining ER-resident EGF domain-specific glycosyltransferases
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improved the amount of secreted NOTCH EGF repeats in the culture media. Notably, we
showed for the first time that EOGT has the potential to promote the secretory expression of
NOTCH EGF repeats. We speculated that overexpressing these glycosyltransferases is
effective in the sensitized condition where NOTCH EGF repeats are prone to be under-Oglycosylated. The altered O-glycosylation states of NOTCH EGF repeats were evaluated by
mass spectrometry using the simplified label-free quantitation workflow of Proteome
Discoverer. These strategies are applicable to the production and quality control of full-length
NOTCH EGF repeats for use as pharmaceuticals effective against diseases, including various
cancers.
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