Molecular Cloning and Characterization of a Plasma
Membrane-associated Sialidase Specific for Gangliosides*
Taeko
Miyagi
,
Tadashi
Wada,
Akihiro
Iwamatsu§,
Keiko
Hata,
Yuko
Yoshikawa,
Satoru
Tokuyama, and
Masashi
Sawada
From the Division of Biochemistry, Research Institute, Miyagi
Prefectural Cancer Center, Natori, Miyagi, 981-1293, Japan and
§ the Central Laboratories for Key Technology, Kirin Brewery
Co. Ltd., Yokohama 236, Japan
 |
ABSTRACT |
Gangliosides are plasma membrane
components thought to play important roles in cell surface
interactions, cell differentiation, and transmembrane signaling.
A mammalian sialidase located in plasma membranes is unique in
specifically hydrolyzing gangliosides, suggesting crucial roles in
regulation of cell surface functions. Here we describe the cloning and
expression of a cDNA for the ganglioside sialidase, isolated from a
bovine brain cDNA library based on the amino acid sequence of the
purified enzyme from bovine brain. This cDNA encodes a 428-amino
acid protein containing a putative transmembrane domain and the three
Asp boxes characteristic of sialidases and sharing 19-38% sequence
identity with other sialidases. Northern blot and polymerase chain
reaction analyses revealed a general distribution of the gene in
mammalian species, including man, and the mouse. In COS-7 cells
transiently expressing the sialidase, the activity was found to be
40-fold that of the control level with ganglioside substrates in the
presence of Triton X-100, and the hydrolysis was almost specific to
gangliosides other than GM1 and GM2, both
2
3 and
2
8 sialyl
linkages being susceptible. The major subcellular localization of the
expressed sialidase was assessed to be plasma membrane by Percoll
density gradient centrifugation of cell homogenates and by
immunofluorescence staining of the transfected COS-7 cells. Analysis of
the membrane topology by protease protection assay suggested that this
sialidase has a type I membrane orientation with its amino terminus
facing to the extracytoplasmic side and lacking a signal sequence.
 |
INTRODUCTION |
The sialidase reaction is an initial step of the degradation of
glycoproteins and gangliosides. Sialidases of mammalian origin have
been implicated not only in lysosomal catabolism but also in modulation
of functional molecules involved in many biological processes (1, 2).
However, the physiological significance and the regulation mechanisms
of desialylation remain obscure because the structure and function of
mammalian sialidases are not fully understood. Our previous studies
aimed at the biochemical characterization of mammalian sialidases
demonstrated four types in rat tissues differing in subcellular
location as well as catalytic and immunological properties:
intralysosomal (3), cytosolic (4), lysosomal membrane, and plasma
membrane (5). The multiple nature of mammalian sialidases suggests that
each form may play a unique role depending on its particular
subcellular location and catalytic properties. To elucidate the
structure and function of these low abundance proteins, cloning of the
individual genes is required. We previously cloned a rat cytosolic
sialidase gene (6), the first cDNA example of a mammalian species,
and established its involvement in differentiation of skeletal muscle
cells (7). Recently, human (8-10) and mouse (11, 12) major
histocompatibility complex (
MHC)1-related sialidases were
cloned and suggested to be primarily localized in lysosomes (13).
Membrane-associated sialidases hydrolyze gangliosides preferentially
(5, 14, 15), and those in the plasma membrane are distinct from
lysosomal membrane sialidases in acting specifically on gangliosides
(5, 16-18) residing in the same membrane. Gangliosides are thought to
play crucial roles in cell surface events, including cell
differentiation, cell-cell interactions, and transmembrane signaling
(19-21). Plasma membrane sialidases, therefore, have been considered
to participate in these phenomena through modulation of gangliosides.
In fact, there are observations suggesting important biological roles
of ganglioside sialidases, although information as to what types of
ganglioside sialidase involved is not available enough. The activity
levels fluctuate consistently with cell differentiation, cell growth,
and malignant transformation. For example, a sialidase inhibitor,
2,3-dehydro-2-deoxy-N-acetylneuraminic acid, abolishes increase of a differentiation marker enzyme in human neuroblastoma cells (22, 23), and the observations by Usuki et al. (24, 25) led them to propose the participation of ganglioside sialidase in
cell growth regulation. In addition, alterations of the levels of
ganglioside sialidase expression associated with malignant transformation have been described: loss of cell
density-dependent suppression in 3T3-transformed cells (26)
and appearance of ganglioside sialidase activity in transformed cell
lines of baby hamster kidney fibroblasts (27). We previously reported
an increase of plasma membrane sialidase activity associated with
induction of anchorage-independent growth in mouse epidermal JB6 cells
exposed to phorbol esters (28). However, little is known about the
molecular mechanisms underlying such sialidase alterations. To provide
tools for their elucidation, we have focused on cloning a cDNA of
the sialidase. We recently were able to purify a ganglioside sialidase extensively from bovine brain, which is the major
ganglioside-hydrolyzing sialidase of the tissue and is located mainly
in synaptosomes (18). Using the purified enzyme protein, we have now
succeeded in cloning a membrane-associated ganglioside sialidase.
 |
EXPERIMENTAL PROCEDURES |
cDNA Cloning of the Membrane-associated
Sialidase--
Membrane-associated sialidase was purified extensively
from 5 kg of frozen bovine brain as described previously (18). The concentrated enzyme at the step of thiol-activated Sepharose column chromatography was electrophoresed on an SDS-polyacrylamide gel and
transferred to a polyvinylidene difluoride membrane. The polyvinylidene difluoride-immobilized enzyme protein was subjected successively to
reduction, S-carboxymethylation and in situ
digestion with lysylendopeptidase and endoproteinase Asp-N (29). The
digested peptides were fractionated on a reverse-phase high performance liquid chromatography column, and amino acid sequencing was performed. Based on the amino acid sequences of the four peptides derived from the
purified sialidase (see Fig. 1a), nine degenerate
oligonucleotides for both sense and antisense strands were synthesized
with deoxyinosine substitution as shown in Fig. 1b. First
strand cDNAs were synthesized from the poly(A)-rich RNA of bovine
brain using random primers and murine leukemia virus reverse
transcriptase (Superscript RNase H
, Life Technologies,
Inc.) and used as templates for PCR reactions. The PCR conditions were
as follows: denaturation at 94 °C for 30 s, annealing at
55 °C for 1 min, elongation at 72 °C for 2 min; 30 ~ 40 cycles. A fragment (482 base pairs) amplified with Ap3S and DN2A
primers was judged to be appropriate by sequencing, randomly labeled
with [32P]dCTP, and used as a probe to screen a bovine
brain
gt10 cDNA library (). Ten
positive clones were isolated by high stringency washing and sequenced
by the dideoxy chain termination method in both directions using an
AutoRead Sequencing kit (Amersham Pharmacia Biotech). Two overlapping
clones (1.5 and 2.8 kilobases) containing full-length open reading
frames were subcloned into Bluescript (pBB121 and pBB321). For 5'- and
3'-end amplification, the procedure described by Frohman et
al. (30) was employed using specific primers synthesized based on
the sequence of the positive clones.
To obtain sialidase cDNA fragments of mouse and human tissues,
cDNAs were amplified under the same conditions described above with
the primers 5'-GGACACCGGACCATGAACCCCTGTCCT-3' (sense) and 5'-CCTGGCCCCACAGC AAAAGTGGCCCA-3' (antisense) for a region in which the
amino acid sequence of the bovine sialidase is identical to that of
cytosolic sialidase.
Northern Blotting--
Total RNA was prepared from bovine brain
by the acid guanidium-phenol-chloroform extraction procedure (31), and
poly(A)-rich RNA was isolated by oligo(dT)-cellulose column
chromatography. Poly(A)-rich RNAs from human brain and skeletal muscle
were obtained from . Total RNA (15 µg)
and Poly(A)-rich RNA (5 µg) were denatured at 65 °C in a solution
of 50% (v/v) formamide, 6% (v/v) formaldehyde, and 20 mM
MOPS (pH 7.0), electrophoresed in a 1% agarose gel containing 6% (v/v) formaldehyde, and transferred to a
Hybond N+ membrane. The membrane was hybridized with
labeled cDNA probe at 42 °C in a solution containing 5 × saline/sodium phosphate/EDTA, 5 × Denhardt's, 0.5% SDS, 50%
(v/v) formamide and 50 µg/ml salmon sperm DNA, and then washed in
2 × SSC, 0.1% SDS and finally in 0.2 × SSC, 0.1% SDS at
42 °C.
Expression of the Membrane Sialidase cDNA in COS-7
Cells--
A sialidase expression plasmid (pMEmSD) was constructed by
subcloning the sequence containing the open reading frame, amplified by
PCR at an annealing temperature at 60 °C using pBB121 as template, into the EcoRI site of pME18S bearing the SR
promotor.
The tag-epitoped sialidase expression vector, pMEmSD-HSVTag, was
constructed by incorporating the nucleotides coding for HSV tag
(SQPQLAPQDPQD, Novagen) into the 3' end of the full-length sialidase
cDNA in the pME18S vector.
The expression plasmid was transfected into COS-7 cells by
electroporation. After 48 h of growth in cell culture, cells
(5 × 107) were collected, disrupted by sonication in
9 volumes of phosphate-buffered saline containing 1 mM
EDTA, 1 mM dithiothreitol, and 0.5 mM
phenylmethylsulfonyl fluoride, centrifuged at 1000 × g
for 10 min, and additionally at 100,000 × g for 1 h, and the resulting pellet was used for sialidase activity assay. The
reaction mixture contained 50 nmol of substrate as bound sialic acid,
0.2 mg of bovine serum albumin, 10 µmol of sodium acetate (pH 4.6)
and 0.2 mg of Triton X-100. After incubation at 37 °C for 10-30
min, the sialic acid released was determined by the thiobarbituric acid
method as described elsewhere (4). Sialidase activity toward
4-methylumbelliferyl-neuraminic acid (4MU-Neu5Ac) was assayed by
spectro-fluorometrical measurement of 4MU released (4). One unit of
sialidase was defined as the amount of enzyme that catalyzed the
release of 1 nmol of sialic acid/h.
Percoll density gradient centrifugation of the sialidase in
transfected cells was conducted as follows: 1 ml of cell homogenate (1,000 × g supernatant) was applied on top of 16 ml of
40% Percoll (in 0.25 M sucrose) underlaid with 1 ml of 2.5 M sucrose and centrifuged at 48,000 × g
for 45 min. Fractions of 300 µl were collected and assayed for
sialidase, N-acetyl-
-hexosaminidase and 5'-nucleotidase activities (5).
For immunofluorescence staining, transfected cells expressing HSV
epitope-tagged sialidase were fixed with 4% (w/v) paraformaldehyde for
10 min, permeabilized with 0.2% (w/v) Triton X-100 in
phosphate-buffered saline for 2 min, and immunostained with HSV tag
anti-monoclonal antibody (Novagen). As a control, cells transfected
with pMEcSD-HSVtag, in which the open reading frame of membrane
sialidase was replaced by that of cytosolic sialidase (6), were stained
in the same manner. To test antibody binding to the cell surface,
non-permeabilized cells transfected with pMEmSD-HSVTag or pMEmSD were
stained with HSV tag monoclonal or anti-sialidase peptide antibodies,
respectively. The anti-peptide antibody was prepared by immunizing
rabbits with keyhole limpet hemocyanin-coupled oligopeptides
corresponding to amino acid residues 109-128, according to standard
procedures. The specific antibody was affinity purified from the sera
of immunized animals using immobilized peptides. Fluorescein
isothiocyanate-conjugated anti-mouse (Tago) and anti-rabbit (Bio-Rad)
IgG Fab fragments were used as secondary antibodies.
To determine the membrane topology of the sialidase, a proteinase K
protection assay was performed. Transfected cells (5 × 107) harboring pMEmSD-HSVTag or pMEmSD were suspended in 9 volumes of 0.25 M sucrose containing 10 mM
Tris-HCl, 1 mM dithiothreitol, 1 mM EDTA, and
0.5 mM phenylmethylsulfonyl fluoride and disrupted by 10 passages through a 22-gauge needle. The vesicles obtained were
subjected to proteolysis with proteinase K (1 mg/ml) for 3 h at
0 °C in the presence or absence of 1% Triton X-100. After stopping
the reaction with 5 mM phenylmethylsulfonyl fluoride, the
mixture was centrifuged at 10,000 × g for 20 min, and
the resulting pellets were dissolved in 300 µl of SDS-PAGE sample buffer. Expressed sialidase proteins were then analyzed by Western blotting using HSV tag monoclonal or anti-sialidase peptide antibodies followed by exposure to alkaline phosphatase-conjugated anti-mouse or
anti-rabbit IgG Fab fragments, respectively. The presence of N-linked glycosylation in the expressed sialidase was
assessed by tunicamycin and N-glycosidase treatments.
Tunicamycin was added into the culture medium at 2.5 µg/ml at 28 h prior to harvesting. For N-glycosidase sensitivity test,
homogenates (30 µg of protein) of the transfected cells were
incubated with 1 milliunit of N-glycosidase F
(Flavobacterium meningosepticum, Takara, Japan) at 37 °C
overnight under denaturation conditions as recommended by the supplier. Fetuin (25 µg) was treated simultaneously as a control glycoprotein.
 |
RESULTS |
The PVDF-immobilized enzyme protein was digested with
lysylendopeptidase followed by endoproteinase and microsequenced. Based on the four peptides thus obtained (Fig.
1, top), degenerate
oligonucleotides for sense and antisense primers were prepared (Fig. 1,
bottom) and used in the PCR reaction with bovine brain
random-primed cDNA as template. An amplified cDNA fragment (482 base pairs) produced with Ap3S and DN2A primers was identified as a
candidate because the deduced amino acid sequence contained two Asp
boxes (32) and demonstrated 30% identity with cytosolic sialidase
unexpectedly. With this cDNA as a probe, we screened a bovine brain
gt10 library (2 × 106 plaques) and obtained ten
positive clones. Two overlapping clones were found to contain the
entire open reading frame, and the 5'-end amplification by PCR resulted
in further extension at the 5'-end of 83 nucleotides. The longest
positive clone (pBB321) and the amplified cDNA with an extended
5'-end were used to generate a cDNA (Fig.
2a) of 2898 nucleotides having
an extended 5'-end of 258 nucleotides with a putative initiating
methionine in perfect agreement with the consensus for eukaryotic
genes. We predict that the methionine codon at nucleotides 259-261 is
the initiation site rather than the residue at nucleotides 229-231
because of the existence of a clone different in the 5'-portion
upstream of nucleotide 249. None of the positive clones were found to
possess a polyadenylation signal but ATTTA motifs were present in the 3'-untranslated region, thought to be involved in destabilization of
mRNAs coding for proto-oncogenes and cytokines and therefore in the
regulation of gene expression during cell growth and differentiation (33). Despite repeated attempts, amplification of the 3'-end portion of
the gene by PCR was unsuccessful. The deduced protein has 428 amino
acids, with a molecular mass of 47,916 Da, that includes the four
sequenced peptides, three Asp boxes, and one potential glycosylation
site. Comparison of the primary sequence with those of other mammalian
and bacterial sialidases revealed 19-38% sequence identities: the
highest degree of homology (38%) was found with rat (6) and hamster
(34) cytosolic sialidases, and 24, 21, and 19% with human MHC-related
(8-10), Clostridium perfringens (35), and Salmonella
typhimurium (36) sialidases, respectively. Searches for primary
sequence homology in protein data bases showed no significant
similarities to other proteins, but interestingly, one region of the
sialidase (59-103 aa) contained 35.6% identity to a region (291-335
aa) of the mouse embryonic growth factor (GDF1) (37), a member of the
TGF-
superfamily. A hydropathy plot generated by the method of Hopp
and Wood (38) suggested that the sialidase has a hydrophobic segment of
21 amino acids (174-194 aa) and no signal sequence as shown in Fig.
2b. The hydrophobic stretch divides the sialidase sequence
into two parts and can be assumed to be a transmembrane segment flanked by a positively charged residue on the carboxyl-terminal side. Like
viral and bacterial sialidases, this enzyme has a high content of
cysteine residues (21 cysteines) and
-sheet structures. S. typhimurium sialidase, whose three-dimensional structure has been determined by x-ray crystallography (39), was used to investigate active site residues of our bovine sialidase. Alignment of the two
sequences using the mode described by Milner et al. (9) revealed a strikingly similar spatial arrangement of the catalytic residues: 8 of the 13 active site residues are conserved in the bovine
sialidase. Two of the three active residues involved in binding the
acetyl group of sialic acid (Asp-62 and Asp-100) and three of the four
residues forming the hydrophobic pocket (Trp-121, Trp-128, and Leu-175
in the bacterial sialidase) demonstrated replacement by Glu-51, Asn-88,
Val-107, Arg-114, and Gly-162, respectively, in the bovine enzyme. The
three active site residues in the hydrophobic pocket are identical to
the corresponding residues in the rat cytosolic sialidase, and all four
residues differ from those of human MHC-sialidase, probably reflecting
differences in substrate specificity.

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Fig. 1.
Peptide and synthetic oligonucleotide
sequences. Top, peptide amino acid sequence data for
the bovine brain ganglioside sialidase. Polyvinylidene
difluoride-immobilized enzyme proteins were digested with
endoproteinase (DN) and lysylendopeptidase (AP)
and microsequenced. Amino acids in small letters are
tentative identifications. Bottom, degenerate
oligonucleotide primers for sense (S) and antisense
(AS) directions prepared based on the peptide
sequences.
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Fig. 2.
Nucleotide and deduced amino acid sequences
of the bovine ganglioside sialidase. a, The predicted amino
acid sequence is shown by the single letter amino acid code under the
nucleotide sequence. The positions of the four peptide sequences
obtained from the purified enzyme are underlined. The Asp boxes are
boxed. The putative transmembrane domain is double
underlined and an asterisk indicates the
N-glycosylation site. ATTTA motifs in the 3' untranslated
region are bold faced. b, hydropathy profile of
the ganglioside sialidase. The hydrophilicity profile of the sialidase
was determined using the method of Hopp and Woods (34). This sequence
will appear in the DDBJ, EBI, and GenBankTM nucleotide
sequence data bases with accession number AB008184.
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Northern blot analysis of bovine brain revealed an approximate 7.5-kb
length of the mRNA using a cDNA covering the entire coding
sequence as a probe (Fig. 3a),
indicating a long stretch (5.8 kilobases) of the gene at the
3'-untranslated region. The same size transcripts were found to be
present in human skeletal muscle and brain. Analysis of the partial
sequences obtained by PCR reaction for the human and mouse sialidase
genes, as shown in Fig. 3b, demonstrated extensive sequence
identity to the bovine gene. In this region, the four active site
residues forming the hydrophobic pocket, as aligned with S. typhimurium sialidase, were found, and their sequences were
all identical. In fact, the primary sequence of the corresponding human
gene displayed an 83% overall identity (data not shown).

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Fig. 3.
General distribution of the ganglioside
sialidase gene in mammalian cells. a, Northern blot
analysis of the sialidase mRNA in bovine brain and human tissues.
Total RNA (15 µg) and poly(A)-rich RNA (5 µg) from bovine brain in
lanes 1 and 2, and poly(A)-rich RNA (5 µg) from
human skeletal muscle and brain in lanes 3 and 4,
respectively, were electrophoresed and hybridized. RNA sizes in
kilobases were determined relative to a RNA ladder. b,
alignment of amino acids 83-143 of the bovine brain cDNA with the
fragments obtained by amplification of the sialidase cDNA from
human and mouse brains. Long arrows indicate the regions for
primers. Residues that are identical are boxed.
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To confirm that the isolated cDNA clone encodes the
ganglioside-hydrolyzing sialidase, plasmids (pMEmSD) were constructed by introduction of the cDNA into a eukaryotic expression vector, pME18S, containing the SR
promotor. COS-7 cells transiently
transfected with pMEmSD showed an over 40-fold increase in sialidase
activity toward gangliosides in the presence of 0.1% Triton X-100
using cell homogenates and particulates as enzyme sources in comparison with untransfected cells or with vector-transfected cells, while the
activity level toward 4MU-NeuAc, a synthetic substrate, was not changed
(Fig. 4a). The sialidase acted
preferentially on gangliosides other than GM1 and GM2 and thus on both
sialyl linkages of
2
3 and
2
8, but hardly on
sialo-glycoproteins and -oligosaccharides, as demonstrated previously
(5, 23) with the purified enzyme from bovine brain (Fig.
4b).

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Fig. 4.
Expression of the ganglioside sialidase in
COS-7 cells. a, sialidase activity in cells transfected
with pMEmSD was assayed using gangliosides or 4-MUNeuAc as substrates.
The values are means ± S.D. of five independent experiments.
b, substrate specificity of the sialidase expressed in COS-7
cells. Sialidase activities toward various sialo-conjugates were
examined in the particulate fractions and expressed as the percentage
of sialic acid released relative to the desialylation of GD3.
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When the transfected cell homogenates were subjected to Percoll density
gradient centrifugation, most sialidase activity toward gangliosides in
the presence of Triton X-100 co-migrated with 5'-nucleotidase, a plasma
membrane marker enzyme, but little was detected in the fractions
corresponding to lysosomes (Fig.
5a). To confirm the
association with the plasma membrane, transfected cells with HSV
epitope-tagged sialidase genes were fixed, permeabilized, and
immunostained with anti-HSV antibody (Novagen) and analyzed by confocal
microscopy (Fig. 5b). Strong surface staining was detected
in cells expressing the sialidase, whereas transfection of the tagged
cytosolic sialidase gene resulted in cytosolic and nuclear expression,
as demonstrated previously in skeletal muscle cells by electron
microscopy (40), indicating no interference of the HSV tag with the
sialidase expression.

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Fig. 5.
Plasma membrane localization and membrane
topology of the ganglioside sialidase. a, Percoll
density gradient centrifugation of the sialidase in transfected cells
harboring pMEmSD. The enzyme activities of each fraction were
determined for ganglioside sialidase, 5'-nucleotidase, and
N-acetyl- -hexosaminidase. b, immunofluorescent
staining of COS-7 cells expressing epitope-tagged sialidases. Cells
transfected with the pME18S vector alone, pMEmSD-HSVTag, or
pMEcSD-HSVTag were cultured in Lab-Tek Chamber Slides (Nunc). After
48 h, the cells were fixed, permeabilized, and stained using an
HSV tag monoclonal antibody, and analyzed with a LSM 310 Zeiss laser
microscope. c, membrane topology of the ganglioside
sialidase. COS-7 cells were transfected with pMEmSD-HSVTag (lanes
1-5) or pMEmSD (lanes 6-9), and the proteins
expressed were run on a 10% SDS-PAGE gel, transferred to Hybond C
(Amersham Pharmacia Biotech), and Western blotted with anti-HSVTag
monoclonal antibody or anti-sialidase peptide antibodies, respectively.
The homogenates (10 µg) from the cells grown in the absence
(lanes 1 and 3) or presence (lane 2)
of tunicamycin 28 h before harvesting were analyzed by Western
blotting. In lane 3, the homogenates were treated by
N-glycosidase F as described under "Experimental
Procedures" prior to SDS-PAGE. Membrane vesicles prepared from
transfectants were subjected to proteinase K protection assay as
described under "Experimental Procedures." The vesicles were
treated with buffer alone (lanes 4 and 6) or
proteinase K in the absence (lanes 5, 7, and
8) or presence (lane 9) of 1% Triton X-100. The
samples, 5 µl in lanes 4 and 6 and 20 µl in
lanes 5 and 7-9 were run on an SDS-PAGE gel and
Western blotted. The molecular masses of the standard proteins are
shown on the left.
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We then investigated whether the sialidase is a transmembrane protein
and how it is oriented using an antibody to the HSV peptide fused at
the carboxyl terminus and an anti-peptide antibody to the oligopeptide
corresponding to amino acid residues 109-129 upstream of the putative
transmembrane domain. Cells expressing pMEmSD-HSV exhibited the same
degree of sialidase activity as pMEmSD-transfected cells. As shown in
Fig. 5c, the overexpressed sialidase was recognized
specifically as a diffuse band of 48-54 kDa on Western blotting with
anti-HSV (Fig. 5c, lanes 1 and 4) and
anti-peptide (lane 6) antibodies. The presence of
tunicamycin in cell medium gave a little change in mobility of the
recombinant protein on Western blots (lane 2), but
incubation of the homogenates with N-glycosidase resulted in
no change under the condition where a control glycoprotein enhanced
sufficiently its mobility (lane 3), indicating little
possibility of the presence of N-glycosylation. Prepared
membrane vesicles were digested with proteinase K, and the products
were analyzed by Western blotting. An approximately 22-kDa fragment
(lower arrow) with its degradated forms and an unprocessed
form (upper arrow) was detected with the anti-peptide antibody (lanes 7 and 8), which recognizes the
peptide on the amino-terminal side of the sialidase, but no protection
was apparent with the HSV epitope-tagged sialidase (lane 5).
The fragment unaffected by proteinase K treatment corresponds to the
amino terminus and a part of the membrane-spanning domain region, being
completely degradated with proteinase K in the presence of Triton X-100
(lane 9). This expressed sialidase, therefore, appears to be
a transmembrane protein with an extracytoplasmic amino terminus.
Support for this conclusion was provided by immunostaining of
non-permeabilized transfected cells. The anti-sialidase peptide
antibody gave rise to a cell surface staining, even if low staining
intensity (data not shown), whereas staining was hardly observed
without permeablization using anti-HSV antibody. The prediction for
transmembrane proteins by the Phdtopology (41) and Tmpred (42) programs
is consistent with these results. Fusion protein expression plasmids at
the amino terminus demonstrated decreased sialidase activity, and introduction of the constructs with prokaryote expression vectors into
E. coli gave no sialidase activity. Taken together, these results strongly indicate that the ganglioside sialidase is associated with plasma membrane and suggest that it is an atypical type I membrane
protein lacking a signal sequence and exposing the amino terminus to
the extracytoplasmic side.
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DISCUSSION |
Two types of mammalian sialidase, whose major locations are the
cytosol (6, 33) and lysosomes (8-12), have been cloned to date. Their
primary sequences are not very similar but contain several conserved
sequences including ASP boxes characteristic of sialidases. The plasma
membrane-associated sialidase has been found to be clearly distinct
from cytosolic and lysosomal sialidases in enzymatic properties,
especially in its strict substrate specificity with hydrolysis of
gangliosides but not glycoproteins or oligosaccharides to any great
extent (5, 17, 18). In the present study, we have cloned a bovine
cDNA encoding a ganglioside sialidase associated mainly with the
plasma membrane, which is considered to be the major sialidase for
ganglioside hydrolysis in bovine brain. Like other sialidases, the
primary sequence contained several consensus amino acid sequences,
including some putative active site amino acid residues and ASP boxes
that are conserved in bacterial sialidases, suggesting a structure
related to microbial counterparts and supporting the idea that
mammalian and microbial sialidases have a common phylogenic origin
(43). The primary structure of the sialidase gene showed the highest
homology (38%) to rat and hamster cytosolic sialidases among
sialidases so far cloned and less similarity (18%) to human and mouse
lysosomal sialidases. This is in line with the differences in substrate
specificity of these three types because the former two can efficiently
hydrolyze gangliosides to be poor substrates for MHC-related lysosomal
sialidase (9). Although the similarities in partial sequence and
structure indicate that the three mammalian sialidases share a common
ancestor with bacterial sialidases, there must be some sequences
responsible for the substrate specificities, active site residues in
the hydrophobic pocket presumably being involved because of their
difference from those of MHC-related sialidase as well as bacterial
sialidases. Interestingly, the region containing these residues was
predicted to be on the extracellular side of the plasma membrane by
protease protection assays. Sequences for subcellular location are
characteristic for each sialidase: the ganglioside sialidase possesses
a putative transmembrane domain, whereas no such sequences exist for
cytosolic and lysosomal sialidases, which, respectively, have a nuclear translocation signal and a lysosomal targeting sequence.
COS-7 cells transiently transfected with the cDNA showed increased
sialidase activity toward ganglioside substrate in the presence of
Triton X-100 in the assays. This is consistent with observations using
cell homogenates as the enzyme source by Lieser et al. (44)
that plasma membrane sialidase is stimulated by non-ionic detergents,
suggesting that the increased activity was because of plasma membrane
sialidase. The fact that the substrate specificity of the expressed
sialidase toward gangliosides was similar to that of the purified
enzyme from bovine brain provides further support that the cDNA
encodes the sialidase protein.
The subcellular localization of this sialidase was determined to be the
plasma membrane by sucrose density gradient centrifugation of cell
homogenates and by immunofluorescence staining of the transfected
cells. Immunostaining was able to demonstrate differences in
subcellular localization between the ganglioside sialidase and rat
cytosolic sialidase used as a control, precluding an effect of the HSV
peptide on expression of these genes because of the consistency with
our previous data for anti-peptide antibody specific to the cytosolic
sialidase (40). Examination of the transmembrane topology of the
sialidase by protease protection assays provided evidence for an
extracytoplasmic side at the amino terminus, implying that a single
putative N-glycosylation site resides unusually in the
cytoplasm. This raised a question whether the
N-glycosylation site is functional on the sialidase protein
expressed in COS-7 cells because our previous data, that
RCA120-lectin binds the sialidase fraction at a
purification step from bovine brain particulates, have suggested that
the sialidase may be an N-glycosylated protein (18).
N-glycosidase treatment, however, resulted in no change in
the size of the protein under appropriate conditions, indicating that
this protein does not undergo N-glycosylation although it seems to be inconsistent with the tunicamycin results. Tunicamycin treatment in this case may result in changes in other
post-translational modification. If it can be concluded that the
sialidase is not N-glycosylated, it raises a question about
the RCA-lectin binding ability of the brain enzyme. One explanation for
this may be that the enzyme is bound to the lectin indirectly through
the mediation of a/some protein(s) having the ability, which COS-7
cells do not express. As the alignment of the sequence suggested that
possible active site residues are distributed all over the molecule, it is not clear whether this sialidase is catalytic in the
membrane-anchored state. Rather, it is likely that the catalysis would
occur in the cytoplasmic site of the membrane. There are two
possibilities. First, like lectins, the sialidase could bind
ganglioside substrates at the cell surface by its extracytoplasmic
region covering 7 of 13 active site residues, become internalized into
some intracellular compartment such as endosomes, and then catalyze
desialylation reaction. Alternatively, it may encounter ganglioside
substrates on the cytoplasmic site of the membrane where an appropriate
conformation necessary for catalytic activity is maintained, as
suggested by the reports on gangliosides present in cytoplasmic site
such as cryptic GM3 hidden from the cell surface (45, 46). It is quite possible that the above two possibilities could occur concurrently. These hypotheses may explain why the sialidase possesses an acidic optimum pH. In any case, the functional site of the sialidase is
probably not only the plasma membrane but also some intracellular components. Because gangliosides are known to influence signal transduction processes (19, 20), this sialidase may be involved by
their modulation (47). The sialidase may itself be regulated by
phosphorylation of sites in its cytoplasmic portion which could be
targeted by kinases, including tyrosine kinase and protein kinase C. The sialidase might be a modulator of diverse phenomena such as cell
differentiation, cell growth, and malignant transformation as described
above. The presently described cDNA should find application as a
useful tool for elucidation of the underlying mechanisms.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Kazuo Maruyama, Tokyo Medical
and Dental University, for kindly providing the pME18S vector. We also
appreciate the skillful technical assistance of Setsuko Moriya.
 |
FOOTNOTES |
*
This work was supported in part by The Naito Foundation and
by a Grant-in-Aid for Scientific Research on Priority Areas from the
Ministry of Education, Science, Sports and Culture of 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB008184.
To whom correspondence should be addressed. Tel.:
81-22-384-3151(ext. 910); Fax: 81-22-381-1195; E-mail:
tmiyagi{at}mcc.pref.miyagi.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
MHC, major
histocompatibility complex;
MOPS, 4-morpholinepropanesulfonic acid;
4MU-NeuAc, 4-methylumbelliferyl-neuraminic acid;
PCR, polymerase
chain reaction;
HSV, Herpes Simplex virus;
aa, amino acid(s);
PAGE, polyacrylamide gel electrophoresis.
 |
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