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J. Biol. Chem., Vol. 278, Issue 12, 10523-10530, March 21, 2003
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From the Ceramidase is a key enzyme involved in regulating
cellular levels of ceramide, sphingosine, and possibly sphigosine
1-phosphate and thus could modulate sphingolipid signaling. Here
we report that O-glycosylation of the mucin-like domain of
neutral ceramidases was required for localization to the surface of
plasma membranes. The deduced amino acid sequences of the mammalian
enzymes contain a serine-threonine-rich domain (mucin box), which
follows the signal/anchor sequence, whereas those of bacterial and
invertebrate enzymes completely lack a mucin box, suggesting that the
specific domain has been acquired during evolution. In HEK293 cells
overexpressing ceramidase, the enzyme was not only secreted into the
medium after cleavage of the NH2-terminal signal/anchor
sequence but also localized at the plasma membrane as a type II
integral membrane protein. Lectin blot analysis using peanut agglutinin
revealed that the mucin box of the enzyme is highly glycosylated with
O-glycans. Interestingly, a mutant lacking the mucin box or
possible O-glycosylation sites in the mucin box was
secreted into the medium but not localized at the surface of the cells.
Furthermore, a mucin box-fused chimera green fluorescent protein (GFP),
but not GFP itself, with the signal/anchor sequence was distributed on
the surface of the cells. These results suggest that
O-glycosylation of the mucin box retains proteins on the
plasma membranes. We also found that the 112-kDa membrane-bound enzyme
from mouse kidney is O-glycosylated, whereas the 94-kDa
soluble enzyme from liver is not. These results clearly indicate that
post-translational modification of the enzyme with O-glycans is tissue-specific and helps the enzyme to
localize at the surface of plasma membranes as a type II membrane protein.
O-Glycosylation of Mucin-like Domain
Retains the Neutral Ceramidase on the Plasma Membranes as a Type II
Integral Membrane Protein*
,¶
Department of Bioscience and Biotechnology
and the § Department of Applied Genetics and Pest
Management, Graduate School of Bioresource and Bioenvironmental
Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka
812-8581, Japan
*
This work was supported by a Grant-in-Aid for Scientific
Research on Priority Areas (B) 12140204 and Special Coordination Funds
for Promoting Science and Technology from the Ministry of Education,
Culture, Sports, Science and Technology, Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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