O-Glycosylation of Mucin-like Domain
Retains the Neutral Ceramidase on the Plasma Membranes as a Type II
Integral Membrane Protein*
Motohiro
Tani
,
Hiroshi
Iida§, and
Makoto
Ito
¶
From the
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
Received for publication, August 5, 2002, and in revised form, December 20, 2002
 |
ABSTRACT |
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.
 |
INTRODUCTION |
Sphingolipids have emerged as a multifunctional lipid biomodulator
within or among cells. Ceramide (N-acylsphingosine,
Cer),1 a common lipid
backbone of sphingomyelin (SM) and glycosphingoslipids, has been shown
to mediate many cellular events, including cell growth arrest,
differentiation, and apoptosis (1, 2), possibly regulating various
cytoplasmic proteins such as protein kinases C-
(3), C-
, and
C-
(4), and protein phosphatases 1 and 2A (5). Sphingosine (Sph),
the N-deacylated product of Cer, exerts mitogenic and
apoptosis-inducing activities, depending on the cell type and cell
cycle (6, 7). Sph is known to be a potent inhibitor of protein kinase C
(8) and an activator of 3-phosphoinositide-dependent kinase
1, which is thought to be occasionally localized at the inner plasma
membrane (9). Notably, Sph can be phosphorylated to yield Sph
1-phosphate (S1P), which regulates cell proliferation (10), motility
(11), and morphology (12). In contrast to Cer and Sph, S1P appears to act extracellularly by interacting with cell surface G protein-coupled receptors, the endothelial differentiation gene (EDG) family
(13).
Ceramidase (CDase, EC 3.5.1.23), an enzyme that catalyzes hydrolysis of
the N-acyl linkage of Cer to produce Sph, has been classified into three types mainly based on catalytic pH optima, i.e. acidic, neutral, and alkaline. Neutral CDases,
which have an optimum pH of 6.5-8.5, have been cloned from bacteria
(14), Drosophila (15), mouse (16), rat (17), and human (18). Interestingly, phylogenetic analysis revealed that the three CDase isoforms having different pH optima may be derived from different ancestral genes (16). Mammalian neutral CDase seems to regulate the
balance of Cer/Sph/S1P in response to various stimuli, including cytokines (19, 20) and growth factor (21), and thus could modulate
sphingolipid-mediated signaling. Furthermore, the fact that Sph is not
produced by de novo synthesis (22) implies a significant
role for CDase in the generation of Sph and possibly S1P.
We found that neutral CDases of bacteria (14) and Drosophila
(15) were released from cells as a soluble form, whereas those of
mammalian origins were mainly recovered in membrane fractions (21).
Recently, it was found that the intracellular distribution of the
mammalian enzyme was cell/tissue-specific. In rat kidney, neutral CDase
was mainly localized at the apical membrane of proximal tubules, distal
tubules, and collecting dusts, whereas in liver the enzyme was
distributed among endosome-like organelles in hepatocytes (17).
Human neutral CDase was exclusively localized to mitochondria in HEK293
and MCF7 cells when overexpressed as a fusion construct with green
fluorescent protein (GFP) at the NH2 terminus of the enzyme
(18). Furthermore, both neutral and acid CDases were found to be
released by murine endothelial cells (23). However, the molecular
mechanism by which neutral CDases are localized to different organelles
is not well understood.
This report clearly shows that O-glycosylation of the
mucin-like domain (mucin box) is required to retain neutral CDases on the plasma membranes as a type II integral membrane protein. It was
found that the domain was occasionally lost by post-translational processing, resulting in a different localization of the enzyme. These
findings facilitate the understanding of the mechanism for cell/tissue-specific localization of neutral CDases and provide some
insight into sphingolipid metabolism at the cell surface or in the
extracellular milieu.
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EXPERIMENTAL PROCEDURES |
Materials--
CHOP cells and anti-Rab6 antibody were kindly
provided by Dr. K. Nara (Mitsubishi Kagaku Institute of Life Sciences,
Tokyo, Japan) and Dr. S. Tanaka (Shizuoka University, Shizuoka,
Japan). HRP-labeled anti-mouse IgG antibody was purchased from Nacalai Tesque (Tokyo, Japan). Cy3-labeled anti-mouse IgG antibody,
anti-FLAG M2 antibody, benzyl-GalNAc, brefeldin A, and
cytochalasin D were obtained from Sigma. ECL Plus, HRP-labeled, and
Cy3-labeled anti-rabbit IgG antibodies were from Amersham Biosciences.
Anti-myc and anti-GFP antibodies were purchased from
Invitrogen Co. Anti-neutral CDase antibody was raised in a rabbit using
the recombinant rat CDase as the antigen (17). C12-NBD-Cer was prepared
as described in a previous study (24). HEK293 cells (JCRB9068,
established by F. L. Graham) were obtained from the Human Science
Research Resource Bank. All other reagents were of the highest purity available.
Plasmid Construction--
The vector pcDNA3.1/Myc-His(+)
containing a full-length rat neutral CDase gene (pcDNAkCD) was
constructed as described previously (17). To generate a construct
expressing the GFP-fused neutral CDase, the vector pcDNAkCD was
treated with KpnI and XhoI, and subcloned into
pBluescript II SK (Stratagene). The product of the full-length neutral
CDase gene digested with KpnI and SmaI was cloned
into pEGFP-N2 (Clontech). A mutant, which lacks the mucin-like domain (
mucin), was constructed by fusing the
NH2-terminal fragment of
Met1-Lys42 with the COOH-terminal fragment of
Asn79-Thr761. The NH2-terminal
fragment was amplified by PCR using a 5' primer with a KpnI
restriction site
(5'-AGGGTACCGAAATGGCAAAGCGAACCTTCTCC-3') and a 3'
primer (5'-ACACCAATGTAGTAGCCACTGAAGTTTTTGTGGTTTTCGATGGTCCC-3'). The COOH- terminal fragment was amplified with a 5' primer
(5'-GGGACCATCGAAAACCACAAAAACTTCAGTGGCTACTACATTGGTGT-3') and a 3' primer
with an XhoI restriction site
(5'-GCCGCTCGAGAGTAGTGACAATTTCAAAAGGGGAAGA-3'). These
fragments were extended with Pyrobest DNA polymerase (Takara Shuzo Co.)
and subcloned into the vector pcDNA3.1/Myc-His(+) (Invitrogen). To
obtain wild-type and
mucin CDases tagged with the FLAG epitope (MDYKDDDDK) at the NH2 terminus, DNA fragments of the
FLAG-tagged CDases were amplified by PCR using a 5' primer with a
KpnI restriction site
(5'-AGGTGGTACCATGGACTACAAAGACGATGACGACAAGGCAAAGCGAACCTTCTCCTCC-3') and a 3' primer with an XhoI restriction site
(5'-GCCGCTCGAGAGTAGTGACAATTTCAAAAGGGGAAGA-3') and
subcloned into the vector pcDNA3.1/Myc-His(+). The mucin box-fused chimera GFP with signal/anchor sequence (S-M-GFP) and GFP with signal/anchor sequence (S-GFP) were designed using the two sequences of
the NH2-terminal fragments of the enzyme,
Met1-Gln78 and
Met1-Lys42, respectively. Each sequence was
amplified by PCR using a 5' primer with a KpnI restriction
site (5'-AGGGTACCGAAATGGCAAAGCGAACCTTCTCC-3') and a
3' primer with a BamHI restriction site
(5'-CCACGGATCCCCTGAGAGGGAGGGAGGTCTGG-3') or
(5'-TTCCGGATCCCTTTGTGGTTTTCGATGGTCCC-3'). The
amplified fragments were subcloned into pEGFP-N2. Construction of a
mutant (Muc-Ala), in which all Ser and Thr residues of mucin box were
replaced with Ala residues, is described below. An
NH2-terminal fragment (Met1-Pro62)
was amplified with a 5' primer with a KpnI restriction site (5'-AGGGTACCGAAATGGCAAAGCGAACCTTCTCC-3'), and a 3'
primer
(5'-TGGAGCTGCCTGGGCGGCTGCGGGTCCCTGCGCGGCTGCAACCCAATGATTCCCTGCATCTT-3'). A COOH-terminal fragment (Asp43-Asn330) was
amplified with a 5' primer
(5'-AAGATGCAGGGAATCATTGGGTTGCAGCCGCGCAGGGACCCGCAGCCGCCCAGGCAGCTCCAGCCGCACAAGCTCCAGCCGCACAAGCTCCAGACCTCCCTCCCGCTCAG-3') and a 3' primer (5'-GCAGGCTTTGCTTCATCAA-3'). These fragments were extended with Pyrobest DNA polymerase, digested with KpnI
and BamHI, and subcloned into pcDNAkCD. The sequences of
these constructs were verified with a DNA sequencer (model 377, Applied
Biosystems, Japan).
CDase Assay--
CDase activity was measured using C12-NBD-Cer
as a substrate as described in a previous study (25).
Cell Culture and cDNA Transfection--
HEK293 cells, human
embryonic kidney cells, were grown in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and 60 µg/ml kanamycin in a humidified incubator containing 5%
CO2. CHOP cells, Chinese hamster ovary cells that express
polyoma LT antigen, to support the replication of eukaryotic expression
vectors (26), were grown in
-minimal essential medium (
-MEM)
supplemented with 10% FBS, 100 µg/ml streptomycin, and 100 units/ml
penicillin in a humidified incubator containing 5% CO2.
cDNA transfection was carried out using LipofectAMINETM
Plus (Invitrogen) according to the instructions of the manufacturer.
Preparation of Culture Supernatants and Cell Lysates--
At
18 h after transfection with the CDase cDNA, the medium was
replaced with serum-free Opti-MEM (Invitrogen, 500 µl/well on 24-well
plates) and cultured for an additional 24 h. The cell culture
medium was collected and subjected to centrifugation at 13,000 × g for 5 min. The supernatant, supplemented with a 1/10 volume of 200 mM Tris-HCl, pH 7.5, containing 1% Triton
X-100 and 3.3 µg/ml proteinase inhibitors (leupeptin, pepstatin, and chymostatin), was used as cell supernatant. Cells attached to the
culture plate were rinsed with PBS and then lysed by adding 200 µl of
10 mM Tris-HCl, pH 7.5, containing 0.5% Triton X-100 and
3.3 µg/ml of the proteinase inhibitors described above. Lysates were
collected by pipette and used as cell lysates.
Protein Assay, SDS-PAGE, and Western Blotting--
Protein
content was determined by the bicinchoninic acid protein assay (Pierce)
with bovine serum albumin as standard. SDS-PAGE was carried out
according to the method of Laemmli (27). Protein transfer onto a
polyvinylidene difluoride membrane was performed using
Trans-Blot SD (Bio-Rad) according to the method described in a previous
study (28). After treatment with 3% skim milk in Tris-buffered saline
(TBS) containing 0.1% Tween 20 (T-TBS) for 1 h, the membrane was
incubated with primary antibody for 1 day at 4 °C. After a wash with
T-TBS, the membrane was incubated with HRP-conjugated secondary
antibody for 2 h. After another wash with T-TBS, the ECL reaction
was performed for 2-3 min as recommended by the manufacturer, and
chemiluminescent signals were visualized on ECLTM
Mini-camera (Amersham Biosciences).
Amino Acid Sequencing--
The NH2-terminal sequence
of the 94-kDa soluble CDase was determined by the method described
previously (25), i.e. the 94-kDa band was visualized by
SDS-PAGE with GelCode Blue Stain reagent (Pierce), extracted from the
gel, and determined by automated Edman degradation using an amino acid
sequencer model 477A (PE Biosystems).
Immunoprecipitation of CDase--
Anti-neutral CDase antibody at
a dilution of 1:100 was conjugated with 10 µl of protein A-agarose
(Santa Cruz Biotechnology) in 100 µl of reaction buffer (10 mM Tris-HCl, pH 7.5, containing 150 mM NaCl,
1% Triton X-100, and 0.1% bovine serum albumin) at 4 °C for 2 h. After five washes with reaction buffer, 10 µl of protein A-agarose
conjugated with antibody was resuspended in 900 µl of reaction
buffer, to which 100 µl of denatured CDase was added, and then
incubation conducted at 4 °C for 18 h. Before immunoprecipitation, CDase was denatured by boiling for 5 min in 100 µl of SDS sample buffer (20 mM Tris-HCl, pH 7.5 containing 1% SDS and 1% 2-mercaptoethanol). The precipitate was spun
down by centrifugation, washed with reaction buffer five times, and then suspended in 20 µl of SDS sample buffer. After boiling at 100 °C for 5 min, the sample was subjected to SDS-PAGE, followed by
Western blotting analysis as described above.
Immunocytochemistry and Fluorescence Microscopy--
Transfected
cells were cultured on cover glass and then fixed with 3%
paraformaldehyde in PBS for 15 min. After being rinsed with PBS and 50 mM NH4Cl in PBS, cells were permeabilized, if necessary, by 0.1% Triton X-100 in PBS. After treatment with blocking buffer (5% skim milk in PBS) for 15 min, the samples were incubated with primary antibody (diluted 1:1000 with blocking buffer) at 4 °C
for 1 day followed by Cy3-labeled secondary antibody at room temperature for 2 h. Immunostained samples were examined with a
confocal laser-scanning microscope (Digital Eclipse C1, Nikon, Japan).
Flow Cytometric Analysis--
Transfected cells were harvested
and incubated with primary antibody (1:1000) in 100 µl of PBS
containing 50% FBS on ice for 1.5 h. After being washed twice
with PBS, cells were treated with Cy3-labeled secondary antibody in 100 µl of PBS containing 50% FBS on ice for 1 h and then analyzed
by flow cytometry (EPICS XL System-IC, Beckman Coulter).
 |
RESULTS |
Subcellular Localization and Topology of Neutral CDase--
The
neutral CDase was reported to be localized at apical membranes of rat
kidney (17), whereas the enzyme was actively released from murine
endothelial cells (23). We found that the CDase fused with GFP at the
COOH terminus of the enzyme (Fig.
1E, all constructs used in
this study are illustrated in Fig. 1) was distributed in ER/Golgi
compartments as well as the plasma membranes of HEK293 cells using a
confocal laser microscope (Fig.
2A). The fluorescent signal
for GFP-fused CDase was partly co-localized with that for Rab6, a
marker protein for Golgi apparatus (Fig. 2B). To clarify the
topology of the CDase, a double-tagged CDase, with a FLAG tag at the
NH2 terminus and myc tag at the COOH terminus,
was constructed (Fig. 1C) and expressed in HEK293 cells. The
FLAG signal was observed in ER/Golgi compartments as well as on plasma membranes when cells were permeabilized with Triton X-100 (Fig. 2C, panel a). However, the signal was not
detected in intact cells (Fig. 2C, panel b). On
the other hand, the COOH-terminal myc signal was observed on
plasma membranes with and without treatment of Triton X-100 (Fig.
2C, panels c and d). These results
indicate that the COOH terminus of the CDase resides on the
extracellular side of the plasma membrane, whereas the NH2
terminus is on the cytoplasmic side. In conclusion, the neutral CDase
was expressed on plasma membranes as a type II integral membrane
protein in HEK293 cells, being anchored to the membranes with an
internal uncleaved NH2-terminal signal/anchor sequence.

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Fig. 1.
Schematic diagram of cDNA
constructs. The diagram shows the structures of cDNA
constructs used in this study. A, wild-type CDase (wild-type
CDase tagged with myc at the COOH terminus); B,
mucin CDase (mucin box-deleted mutant CDase tagged with
myc at the COOH terminus); C, FLAG-tagged CDase
(neutral CDase tagged with FLAG at the NH2 terminus and
myc at the COOH terminus); D, FLAG-tagged
mucin CDase (mucin box-deleted mutant CDase tagged with FLAG at the
NH2 terminus and myc at the COOH terminus);
E, GFP-fused CDase (neutral CDase fused with GFP at the COOH
terminus); F, S-M-GFP (mucin box with signal/anchor sequence
fused with GFP); G, S-GFP (signal/anchor sequence fused with
GFP).
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Fig. 2.
Immunocytochemical analysis of the neutral
CDase overexpressed in HEK293 cells. A, HEK293
cells overexpressing GFP-fused neutral CDase. Cells were fixed
and examined for GFP fluorescence under a confocal laser microscope.
Arrows and an arrowhead indicate the expression
of CDase at plasma membrane and ER/Golgi compartments, respectively.
B, HEK293 cells expressing GFP-fused neutral CDase
(left) were fixed, and immunostained with anti-Rab6 antibody
followed by anti-rabbit IgG-Cy3 (center). Images were merged
(right). An arrow indicates the co-localization
of neutral CDase and Rab6, a marker protein for the Golgi apparatus.
C, analysis of membrane topology of neutral CDase.
a, stained with anti-FLAG antibody after permeabilization
with Triton X-100. b, same as a but before
treatment with Triton X-100. c, stained with
anti-myc antibody after permeabilization with Triton X-100.
d, same as c but before treatment with Triton
X-100. HEK293 cells were transformed with plasmid vector containing
FLAG-tagged CDase cDNA, fixed, and then stained with corresponding
antibody before and after treatment with Triton X-100 as described
under "Experimental Procedures."
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Mucin-like Domain of Mammalian Neutral CDases--
Alignment of
the deduced amino acid sequences of rat and mouse neutral CDases with
those of Pseudomonas aeruginosa, Mycobacterium tuberculosis, Dictyostelium discoideum, and
Drosophila melanogaster neutral CDases, revealed that the
rat and mouse enzymes have a Ser/Thr-rich domain (amino acids 43-78)
downstream of the NH2-terminal hydrophobic region. The
hydrophobic region, which is a putative signal sequence, was observed
in all CDases except that from M. tuberculosis. However, the
Ser/Thr-rich domains were exclusively observed in CDases from mammals
(Fig. 3A), suggesting this
domain has been given to the enzyme during evolution. This domain shows a characteristic mucin-type repeating structure that contains not only
Ser and Thr but also Pro and Gln and thus was tentatively designated
the mucin box in this study. Sequences homologous to the rat and mouse
mucin box were found in rat sucrase isomaltase, human MUC2, and insect
intestinal mucin peptide (Fig. 3B).

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Fig. 3.
Alignments of deduced amino acid sequences of
neutral CDases based on NH2-terminal regions
(A) and mucin-like domains (B).
The identification of the mucin box was performed by a BLAST search
program (43).
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Expression and Processing of Wild-type and Mucin Box-deleted
CDases--
To investigate the effect of the mucin box on the
expression and processing of the enzyme, two constructs were generated; a wild-type CDase, which has the entire sequence of rat neutral CDase,
and a deletion mutant (
mucin), which lacks the mucin box from the
wild-type CDase. Both constructs have a signal/anchor sequence at the
NH2 terminus and are tagged with myc at the COOH terminus (Fig. 1, A and B). When the wild-type
construct was transfected into HEK293 cells, two protein bands having
molecular masses of 113 and 133 kDa on SDS-PAGE were detected in cell
lysate after visualization with anti-myc antibody (Fig.
4A). The 113- and 133-kDa proteins seem to be glycosylated with high mannose type
N-glycans and high mannose/complex/hybrid type
N-glycans, respectively, as reported previously (17),
suggesting that they are a developing form in the ER and a mature form
in the Golgi apparatus, respectively. A single band of ~105 kDa was
detected in cell lysate when the
mucin CDase was expressed in HEK293
cells and stained with anti-myc antibody (Fig.
4A). Notably, both wild-type and
mucin CDases were
secreted into the medium (Fig. 4A). To investigate whether a
NH2-terminal signal/anchor sequence was removed by
proteolysis, NH2-terminal FLAG-tagged wild-type (Fig.
1C) and
mucin (Fig. 1D) CDases were
constructed and expressed in HEK293 cells. Both constructs were also
tagged with myc at the COOH terminus. Western blotting
analysis using anti-FLAG antibody revealed that both overexpressed
wild-type and
mucin CDases were FLAG-positive in cell lysates, but
those in culture media were FLAG-negative (Fig. 4B). On the
other hand, myc-positive CDases were detected in both the
lysates and culture media of cells overexpressing wild-type and
mucin enzymes (Fig. 4B). These results indicate that the NH2-terminal signal/anchor sequence remains intact before
secretion, and the enzyme is secreted possibly after cleavage of the
signal/anchor sequence, regardless of the presence or absence of mucin
box.

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Fig. 4.
Expression, processing, and secretion of
wild-type and mucin box-deleted CDases. A, Western
blotting of the CDase expressed in HEK293 cells. Cells were transfected
with plasmid vector containing wild-type or mucin CDase cDNA.
The cell lysates and the culture supernatants were separately subjected
to SDS-PAGE (7.5% gels), followed by Western blotting using
anti-myc antibody. B, the same experiment was
carried out as shown in A, but cells were transfected with
plasmid vector containing FLAG-tagged wild-type or mucin CDase
cDNA. The cell lysates and the culture supernatants were separately
subjected to Western blotting using anti-myc antibody
(upper) or anti-FLAG antibody (lower).
C and M indicate cell lysate and medium (culture
supernatant), respectively.
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O-Glycosylation and Secretion of the CDase--
To verify whether
or not the mucin box is actually glycosylated with
O-glycans, wild-type and
mucin CDases were subjected to
lectin blotting using peanut agglutinin (PNA), which specifically binds
to the Gal
1,3GalNAc sequence of O-glycans (29). For
wild-type enzyme, the 133-kDa mature form, but not the 113-kDa ER form, was stained with the lectin, whereas no band was stained for
mucin CDase (Fig. 5A). Furthermore,
treatment of CDase-overexpressing cells with benzyl-GalNAc, an
inhibitor of O-glycosylation, resulted in a reduction in the
molecular mass of the 133-kDa, but not the 113-kDa, wild-type CDase.
The inhibitor did not affect the molecular mass of the
mucin CDase
(Fig. 5B). These results suggest that O-glycosylation crucially occurred in the mucin box of
the CDase possibly at the Golgi apparatus. The secreted wild-type CDase was also stained with PNA (data not shown), suggesting that the mucin box is not removed by proteolytic processing of the signal/anchor sequence in HEK293 cells. The wild-type as well as
mucin CDase was
continuously released from cells into the culture medium (Fig. 6A). It should be noted,
however, that the
mucin CDase was found to be secreted much faster
than the wild-type enzyme. The secretion of both enzymes was strongly
inhibited by brefeldin A and treatment at 5 °C but not by
cytochalasin D (Fig. 6B), suggesting that the two CDases
were processed and secreted by a pathway via ER/Golgi compartments.

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Fig. 5.
O-Gylcosylation of neutral
CDase. A, lectin blotting of wild-type and mucin
box-deleted CDases. The lysates of HEK293 cells transfected with
plasmid vector containing wild-type or mucin CDase cDNA were
subjected to immunoprecipitation using anti-neutral CDase antibody as
described under "Experimental Procedures." Thereafter, the
immunoprecipitants were subjected to SDS-PAGE, followed by Western
blotting using anti-myc antibody or lectin blotting using
HRP-labeled PNA. B, inhibition of O-glycosylation
by benzyl-GalNAc. At 4 h after transfection, the cells were
treated with or without 5 mM benzyl-GalNAc in DMEM
supplemented with 10% FBS and then cultured for an additional 20 h. Cells were lysed and subjected to SDS-PAGE, followed by Western
blotting using anti-myc antibody.
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Fig. 6.
Secretion of wild-type and mucin box-deleted
CDases. A, time course for the secretion of wild-type
and mucin CDases. At 18 h after transfection, the medium was
replaced with serum-free Opti-MEM and the cells were incubated at
37 °C for the period indicated. The culture supernatant was employed
for the determination of neutral CDase activity using C12-NBD-Cer as a
substrate and Western blotting with anti-myc antibody.
B, effects of brefeldin A (BFA), cytochalasin D
(CytoD), and temperature on the secretion of CDase. At
18 h after transfection, cells were incubated with 10 µg/ml BFA
or 5 µM CytoD in DMEM supplemented with 10% FBS at
37 °C for 1 h. The medium was replaced with Opti-MEM containing
the inhibitors at the same concentrations and then cultured at 37 °C
for an additional 3 h. To examine the effect of temperature,
transfected cells were incubated at 5 °C for 3 h in fresh
Opti-MEM without inhibitors.
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Mucin Box-mediated Cell Surface Expression of Neutral
CDase--
To investigate the role of the mucin box in the subcellular
localization of the neutral CDase, immunocytochemical analysis of
wild-type and
mucin CDases (Fig. 1, A and B),
both of which were tagged with myc at the COOH terminus, was
performed. Interestingly, the myc signal of the wild-type
CDase was much stronger than that of the
mucin CDase when the
transformed cells were not permeabilized (Fig.
7A, panels a
versus c), although the expression levels of the
enzymes were almost the same when cells were permeabilized with Triton
X-100 (Fig. 7A, panels b versus
d). Flow cytometric analysis also showed a clear difference
in the cell surface expression of myc signal of the enzyme
due to the presence of a mucin box, i.e. wild-type CDase
localized at the surface of plasma membranes of HEK293 as well as CHOP
cells but
mucin enzyme did not (Fig. 7, B and
C).

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Fig. 7.
Cell surface expression of neutral
CDase. A, expression of neutral CDase on the surface
(a and c) or inside (b and
d). Wild-type- (a and b) and
mucin-expressing HEK293 cells (c and d) were
fixed, permeabilized by Triton X-100 (b and d) or
not (a and c), and stained with
anti-myc antibody. B and C, cell
surface expression of neutral CDase in HEK293 (B) and CHOP
(C) cells. Cells (3 × 105) were
transfected with plasmid vector containing wild-type or mucin CDase
cDNA, or empty vector (Mock) and then cultured at
37 °C for 24 h. Cells were harvested and incubated with
anti-myc antibody followed by anti-mouse IgG-Cy3 at 4 °C
and analyzed by flow cytometry as described under "Experimental
Procedures."
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Significance of O-Glycosylation in the Mucin Box for Cell Surface
Expression of the CDase--
It was revealed by lectin blotting that
the mucin box of the CDase was actually glycosylated with
O-glycans (Fig. 5). To investigate the significance of
O-glycans in the mucin box for cell surface expression of
the CDase, the possible O-glycosylation sites (all Ser and
Thr residues) of the mucin box were mutated with Ala (Fig. 8A). When the mutant CDase
(Muc-Ala) was transfected into CHOP cells, a single protein band
showing 113 kDa was detected in the cell lysate. In contrast to the
133-kDa Golgi form of the wild-type CDase, the Muc-Ala 113 kDa band was
not affected when the benzyl-GalNAc, an inhibitor for
O-glycosylation, was added into the culture of CHOP cells,
indicating the mutant lost the O-glycans as expected (Fig.
8B). Although both wild-type and Muc-Ala mutant CDases were continuously released into the culture medium, the amount of secreted enzyme was significantly increased by the removal of
O-glycans from the mucin box (Fig. 8C). In
contrast, the cell surface expression of the enzyme was markedly
reduced by removal of O-glycans (Fig. 8D,
panels a versus c). It is noted,
however, that the expression of the mutant CDase was almost the same as
that of wild-type CDase (Fig. 8D, panels b
versus d). Taken together, it was concluded that
the O-glycosylation of the mucin box of the CDase helps the enzyme to localize at the surface of plasma membranes as a type II
integral membrane protein.

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Fig. 8.
Effects of mutation of possible
O-glycosylation sites on the secretion and cell
surface expression of the CDase. A, the deduced amino
acid sequences of the mucin box in a wild-type CDase and a mutant
enzyme disrupting potential O-glycosylation sites (Muc-Ala).
All underlined Ser and Thr residues were replaced by Ala as
described under "Experimental Procedures." B, Western
blotting of wild-type and Muc-Ala CDases expressed in CHOP cells. At
4 h after transfection with plasmid vector containing wild-type or
Muc-Ala CDase cDNA, the cells were treated with (+) or without ( )
5 mM benzyl-GalNAc in -MEM supplemented with 10% FBS,
and then cultured for an additional 20 h. Cell lysates were
subjected to SDS-PAGE, followed by Western blotting using
anti-myc antibody. C, time course for the
secretion of wild-type and Muc-Ala mutant CDases in CHOP cells. At
18 h after transfection, the medium was replaced with serum-free
Opti-MEM, and the cells were incubated at 37 °C for the period
indicated. The culture supernatants were employed for the determination
of neutral CDase activity using C12-NBD-Cer as a substrate and Western
blotting using anti-myc antibody. D, expression
of neutral CDase on the surface (a and c) or
inside (b and d) of CHOP cells. Cells expressing
wild-type (a and b) and Muc-Ala (c and
d) CDases were fixed, permeabilized with Triton X-100
(b and d) or not (a and c),
and stained with anti-myc antibody. The arrows
indicate the expression of CDase at plasma membrane.
|
|
Localization of Mucin Box-fused GFP--
To verify the potential
role of the mucin box per se, GFP was used as a reporting
molecule, i.e. a mucin box-fused chimera with signal/anchor
sequence (S-M-GFP) was constructed (Fig. 1F) and expressed
in HEK293 cells. Two major bands of 38 and 57 kDa were detected in cell
lysates by Western blotting using anti-GFP antibody (Fig.
9A, lane 3). The
57-kDa, but not the 38-kDa, protein was reduced in its molecular mass
by treatment with benzyl-GalNAc (data not shown), indicating it was
O-glycosylated. The fused protein was found to
distribute on the surface of HEK293 cells when stained with anti-GFP
antibody (Fig. 9B, panel b). In contrast, GFP
with signal/anchor sequence (S-GFP, Fig. 1G) was not
expressed on the cell surface (Fig. 9B, panel d),
although the intracellular expression of S-GFP was comparable to that
of S-M-GFP (Fig. 9B, panels a versus
c). Flow cytometric analysis using anti-GFP antibody also
revealed that the cell surface signal of S-M-GFP was much stronger than
that of S-GFP and mock transfectants when expressed in HEK293 cells
(Fig. 9C). These results clearly indicate that the
O-glycosylated mucin box functions as a potential signal to retain proteins on the cell surface.

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Fig. 9.
Expression of mucin box-fused GFP.
A, SDS-PAGE of the expressed proteins. HEK293 cells were
transfected with plasmid vector containing GFP (pEGFP-N2), S-GFP, or
S-M-GFP cDNA. Cell lysates were subjected to SDS-PAGE (12.5%
gels), followed by Western blotting using anti-GFP antibody. Lane
1, GFP; lane 2, S-GFP; lane 3, S-M-GFP.
B, immunocytochemical analysis of S-M-GFP and S-GFP. HEK293
cells overexpressing S-M-GFP (a and b) or S-GFP
(c and d) were fixed, and stained with anti-GFP
antibody (b and d). The panels a and
c show direct GFP fluorescence. C, flow
cytometric analysis of cell-surface GFP. HEK293 cells (3 × 105) were transfected with plasmid vector containing GFP
(pEGFP-N2), S-GFP, or S-M-GFP cDNA. At 24 h after
transfection, cells were harvested, stained with anti-GFP antibody
before a second incubation with anti-rabbit IgG-Cy3 at 4 °C, and
analyzed by flow cytometry.
|
|
Mucin Box in Neutral CDases from Mouse Tissues and Serum--
It
was found that in mouse liver 72% of neutral CDase activity was
recovered in the soluble fraction after freeze-thawing, whereas in
mouse kidney more than 90% of the activity was recovered in the
insoluble fraction (Fig.
10A). The neutral CDase in
the kidney insoluble fraction was identified as a 112-kDa protein and
that in the liver-soluble fraction as a 94-kDa protein by Western
blotting using anti-neutral CDase antibody (Fig. 10B). The
112-kDa CDase was also detected in the insoluble fraction of liver
(Fig. 10B). Interestingly, the 112-kDa insoluble CDases from
kidney and liver were stained by PNA lectin, whereas the 94-kDa soluble
CDase was not, indicating that only the CDases in the insoluble
fraction were modified with O-glycans. The
NH2-terminal sequence of the 94-kDa soluble CDase was
determined by the method described in a previous report (25). The
determined NH2-terminal sequence corresponded to
FSGYYIGVGRADCTGQVSDIN in the deduced sequence (80-100 in Fig.
3A), indicating that the protein does not possess a
signal/anchor sequence or a mucin box. Although the insoluble CDases
from both liver and kidney were O-glycosylated, the
O-glycan structure of kidney CDase seems to be somewhat
different from that of the liver enzyme. This is because sialidase
treatment significantly reduced the molecular mass and increased the
PNA reactivity of the liver CDase but had little effect on the kidney enzyme (Fig. 10B). Furthermore, we found that the
PNA-positive CDase was also present in mouse serum, which was possibly
derived from liver, because sialidase treatment of the enzyme reduced its molecular mass and increased its PNA reactivity as in the liver
CDase (Fig. 10B). These results strongly suggested that
post-translational modification of the enzyme with O-glycans
is performed in a tissue-specific manner and affects the intracellular
distribution of the CDase.

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Fig. 10.
Mucin box in neutral CDases from mouse
tissues and serum. A, solubilization by freeze-thawing
of neutral CDase from mouse liver and kidney. The preparation of the
membrane fraction and solubilization of the neutral CDase by
freeze-thawing were performed according to the method described in Ref.
25. Values are the means for duplicate determinations. B,
Western and lectin blotting of neutral CDase. Each sample was
immunoprecipitated and analyzed by Western blotting using anti-neutral
CDase antibody or lectin blotting using HRP-labeled PNA. For sialidase
treatment, the immunoprecipitants were detached from agarose beads by
addition of 10 µl of 100 mM glycine, pH 2.5. The pH of
samples was adjusted to 5.0 by addition of 500 mM sodium
acetate buffer, pH 5.0, containing 5 mM CaCl2
and 0.1% Triton X-100. The mixtures were incubated with or without 5 milliunits of sialidase (Vibrio cholera) at 37 °C for
18 h.
|
|
 |
DISCUSSION |
This study demonstrated that the neutral CDase is sorted by a
classic pathway via ER/Golgi compartments to the plasma membranes where
it is expressed as a type II integral membrane protein or alternatively
secreted out of cells after proteolytic processing of the
NH2-terminal signal/anchor sequence. This report also
clearly indicates that the fate of the protein (as a membrane protein or secretion protein) depends on the presence of a mucin box located in
the NH2-terminal region of the enzyme. Because, in contrast to the wild-type CDase, the mucin box-deleted mutant CDase does not
localize at the surface of cells and is almost entirely secreted. Furthermore, the fact that bacterial and invertebrate neutral CDases,
which lack the mucin box, are secreted without cell surface expression
may support this conclusion. In conclusion, the mucin box acquired by
the process of evolution enables the mammalian CDase to localize on the
cell surface as a type II integral membrane protein. It should be
emphasized that the mucin box-mediated cell surface localization is not
limited to the neutral CDase but occurs in general, because the mucin
box-fused chimeric GFP, but not GFP itself, with signal/anchor sequence
was found to be exclusively localized at the surfaces of HEK293 cells.
The mechanism by which the mucin box retains CDase on the cell surface
is unclear at present. However, the finding made here that replacement
of possible O-glycosylation sites (Ser/Thr residues) with
Ala greatly increased the secretion and reduced the expression of the
CDase on plasma membranes may indicate specific functions of
O-glycans, although the structures of O-glycans
remain to be elucidated.
A diverse range of membrane proteins of type I or type II topology are
occasionally released from the lipid bilayer by proteolysis catalyzed
by a group of enzymes referred to as secretases (30). The proteolytic
processing of membrane-bound proteins by secretases is well
characterized in amyloid precursor protein (31), a type I integral
membrane protein, and a Golgi-resident sialyltransferase (32), a type
II integral membrane protein. The latter is cleaved in its
membrane-anchoring region by
-secretase and secreted out of the cell
(32). Thus, it is possible that the neutral CDase is detached from
cells after processing of the NH2-terminal signal/anchor sequence by secretases. We indicate the possibility that the mucin box
interferes with the action of secretases resulting in the generation of
the type II membrane-bound CDase, because the mucin box exists very
close to the possible cleavage site of the signal/anchor sequence.
However, the molecular mechanism for proteolytic processing of the
CDase, including the participation of secretases, remains to be
clarified. Another possibility is that the mucin box is a potential
signal for the sorting of proteins to plasma membranes without
processing of the signal/anchor sequence, although the counterparts for
the recognition of O-glycans have yet been characterized.
There have been few reports on the role of the
O-glycosylation of secretory proteins. It has been reported
that O-glycosylation of the COOH-terminal tandem-repeated
sequences regulates the secretion of rat pancreatic bile
salt-dependent lipase. The O-glycosylation of
the enzyme concealed the Pro-, Glu-, Ser-, and Thr-rich domain (PEST
region), which is commonly present in rapidly degraded proteins, resulting in delivery to a secretion instead of a degradation pathway
(33). In polarized cells, the O-glycosylation was also found
to be required for apical sorting of sucrase isomaltase (34) and the
neurotrophin receptor (35). It was also reported that a mucin-like
domain of enteropeptidase directs apical targeting in Madin-Darby
canine kidney cells (36). The neutral CDase was localized at the apical
membranes of proximal tubules, distal tubules, and collecting ducts in
rat kidney (17) and thus the possible functions of O-glycans
in apical sorting should be examined in this CDase.
In contrast to our observation, human neutral CDase has been reported
to be expressed in mitochondria when overexpressed in HEK293 and MCF7
cells (18). We found that the deduced NH2-terminal sequence
of the human CDase lacks 19 amino acid residues in comparison with
those of mouse and rat CDases (16-18). This may result in the
generation of an incomplete signal/anchor sequence for ER targeting,
allowing the human CDase to target mitochondria instead of the lumen
side of the ER.
Recently, evidence has emerged that the outer leaflet of the plasma
membrane is a site of SM metabolism; SM is abundant here particularly
in lipid microdomain rafts (37), and sphingomyelinase as well as CDase
is actively secreted from endothelial cells (23, 38). Furthermore, acid
sphingomyelinase was reported to be secreted and helped to metabolize
SM in oxidized lipoproteins (39), and CD95 induces the translocation of
acid sphingomyelinase onto the cell surface, resulting in the
generation of extracellularly orientated Cer (40). The neutral CDase is
expressed at the plasma membrane as a type II membrane protein, the
catalytic domain of which is located on the extracellular side, or is
secreted out of the cell. Importantly, the generation of S1P in the
extracellular milieu, which occurs due to the export of the Sph kinase,
was reported (41). Very recently, it was found that phorbol
12-myristate 13-acetate induced the protein kinase
C-dependent translocation of Sph kinase to the plasma
membrane, resulting in the extracellular release of S1P (42). Taken
together, all metabolic enzymes required for hydrolysis of SM to
generate S1P could be present at the outer leaflet of the plasma
membrane or in extra milieu, allowing for the formation of an
alternative pathway from SM to S1P. In this context, it is important to
note that the neutral CDase was actually detected in mouse serum, which
may indicate an important role in vascular biology, i.e.
regulating the extracellular content of Cer, Sph, and possibly S1P, all
of which may function in autocrine/paracrine signaling.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. J. W. Dennis and
Dr. K. Nara for the gift of CHOP cells. We also thank Dr. S. Tanaka for
the gift of anti-Rab6 antibody. Thanks are also due to Dr. N. Okino and
H. Monjusho in our laboratory for supplying the sequence data of the
slime mold CDase.
 |
FOOTNOTES |
*
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.
¶
To whom correspondence should be addressed. Tel.:
81-92-642-2900; Fax: 81-92-642-2907; E-mail:
makotoi@agr.kyushu-u.ac.jp.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M207932200
 |
ABBREVIATIONS |
The abbreviations used are:
Cer, ceramide;
CDase, ceramidase;
FBS, fetal bovine serum;
GFP, green fluorescent
protein;
HRP, horseradish peroxidase;
NBD, 4-nitrobenzo-2-oxa-1,3-diazole;
PBS, phosphate-buffered saline;
PNA, peanut agglutinin;
SM, sphingomyelin;
Sph, sphingosine;
S1P, sphingosine 1-phosphate;
CHOP cells, Chinese hamster ovary cells that
express polyoma LT antigen;
DMEM, Dulbecco's modified Eagle's medium;
ER, endoplasmic reticulum.
 |
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