(Received for publication, February 9, 1995; and in revised form, April 21, 1995)
From the
Salivary secretions modulate taste perception. Taste buds in the
circumvallate and foliate papillae are bathed in secretions of unique
lingual salivary glands, von Ebner's glands (VEG). We have
identified a rat cDNA encoding a novel protein of 1290 amino acids,
Ebnerin, that is specifically expressed in VEG and released onto the
tongue surface along the apical region of taste buds in the clefts of
circumvallate papillae. Ebnerin possesses a putative single
transmembrane domain at the C terminus with 17 amino acids in the
cytoplasmic area. The extracellular region of Ebnerin contains a number
of repeated domains with homology to the scavenger receptor
cysteine-rich domain and to a repeated domain of bone morphogenetic
protein-I and other related proteins. Western blot analysis reveals
that Ebnerin exists in particulate and soluble forms in VEG and is
present in secretions from VEG. In situ hybridization and
immunohistochemistry demonstrate that Ebnerin is located in secretory
duct epithelial cells of VEG and is released onto the tongue surface
along the apical region of taste buds in the clefts of circumvallate
papillae. The unique structure and localization of Ebnerin suggest that
it may function as a binding protein in saliva for the regulation of
taste sensation. Saliva is the first digestive fluid secreted by the
gastrointestinal pathway and performs a variety of functions. It is
essential in the formation of small boluses of food, provides
lubrication for swallowing and speech, dissolves a number of chemicals
in food substances, and provides digestive enzymes such as amylase and
lipase (Hamosh & Scow, 1973; Field & Hand, 1987). About 90% of
saliva is produced by three major glands, the parotid, the
submaxillary, and the sublingual glands, whose secretions drain into
the oral cavity. von Ebner's glands are unique salivary glands
contained within the tongue that drain directly into the clefts of the
circumvallate and foliate papillae, which contain the major taste buds.
Secretions of von Ebner's gland directly modulate taste
perception (Gurkan & Bradley, 1988). Although von Ebner's
gland is known to secrete certain proteins, such as lipase, very little
is known of the protein composition of the gland's secretions or
the molecular mechanisms whereby they influence taste perception. In
the present study we have identified a novel von Ebner's gland
protein, designated Ebnerin, which appears to be formed in the ducts of
von Ebner's gland and released into fluid bathing the taste buds
contained in the taste papillae.
Tissue sections for
immunohistochemical studies were fixed with 4% paraformaldehyde at room
temperature for 15 min, 0.4% Triton in PBS for 30 min, and 0.3%
H
Figure 1:
Structure and deduced amino acid
sequence of Ebnerin. a, Ebnerin contains a putative
transmembrane domain (TM) of 23 amino acids (aa) and
a cytoplasmic region of 17 amino acids at the C terminus. The N
terminus and major portion of Ebnerin are extracellular and contain a
number of repeated domains. R1, R2, R3, and R6 represent the repeated domains with sequence similarity to
the SRCR domain, and R4, R5, and R7 are
repeated domains homologous to the repeat domain in bone morphogenetic
protein-I (BMP-1 domain). F3 is a region of Ebnerin
that shows homology to the zona pellucida region (ZP domain)
of related proteins (see Fig. 2). b, cDNA for Ebnerin
isolated from the taste cDNA library encodes 1290 amino acids with 15 N-linked glycosylation sites that are indicated by black
dots. A putative transmembrane domain is underlined.
Figure 2:
Alignment of amino acid sequence of
Ebnerin compared with other proteins. a, repeated domains of
Ebnerin (R1, R2, R3, and R6) show
72% amino acid sequence identity to the SRCR domain in WC1 (Wijngaard et al., 1992), 64% to CD6 (Aruffo et al., 1991), and 47% to the macrophage scavenger receptor (SCAVR) (Kodama et al., 1990). b, the
repeated domains of R4, R5, and R7 show 30%
amino acid identity to bone morphogenetic type-I (BMP-1)
protein (Fukagawa et al., 1994), 32% to metalloendopeptidase (MEPD) (Elaroussi et al., 1994), 33% to
serum-inducible protein (PS4E) (Feng & Liau, 1993), and
34% to enteropeptidase precursor (ENPR) (Matsushima et
al., 1994). c, the extracellular region near the
transmembrane domain of Ebnerin (F3) shows 12.3% and 22% amino
acid identity to the zona pellucida region of the glycoprotein in
pancreatic secretory granule membranes (GP-2) (Fukuoka et
al., 1991) and uromodulin (UROM) (Pennica et
al., 1987), respectively. Amino acids in Ebnerin that are
identical to other proteins are in bold type, and conserved
cysteine residues are indicated by asterisks above.
Figure 3:
Tissue distribution of Ebnerin mRNA
analyzed by Northern blot. 20 µg of total RNA from various rat
tissues were loaded on each lane and hybridized with Ebnerin cDNA probe
under high stringency. The blot was exposed to x-ray film for 24 h. TE, tongue epithelium; VEG, von Ebner's gland; CV, circumvallate papillae; PG, parotid gland; SG, submaxillary gland; LNG, lateral nasal gland; Olf, olfactory epithelial tissues. An mRNA species of
approximately 5 kilobases was detected in VEG, circumvallate papillae,
lateral nasal gland, and olfactory epithelial
tissues.
In situ hybridization reveals
discrete localizations of Ebnerin mRNA (Fig. 4).
Autoradiographic grains are highly localized to VEG located immediately
beneath the circumvallate papillae. Higher power magnification reveals
very high levels of grains associated with the openings of the ducts of
VEG.
Figure 4:
a, In situ hybridization of
Figure 5:
Western blot analysis of Ebnerin. a, protein (100 µg/lane) extracted from a variety of
tissues was separated by electrophoresis on a 4-12%
polyacrylamide gel and transferred to a nitrocellulose filter. TE, the front region of tongue epithelium. b, Western
blot was incubated with the antibodies preabsorbed with peptide antigen
(20 µg/ml at 4 °C for 24 h). c, proteins (75
µg/lane) separated on 6% polyacrylamide gel. Pellet,
membrane preparations of VEG that were separated from supernatant by
centrifugation at 100,000
Immunohistochemistry with Ebnerin
antiserum reveals selective staining associated with VEG (Fig. 6). Staining is also evident in the clefts in apical
portions of the circumvallate papillae. Preabsorption with the
immunizing peptide abolishes immunoreactivity. High magnification
reveals staining selectively localized to the apical region of the
taste buds. Staining is also evident at the openings of the ducts of
VEG. In the VEG itself intense staining overlies the epithelium of the
ducts of the gland, whereas the acinar tissue does not stain.
Figure 6:
Immunohistochemical localization of
Ebnerin in rat tongue. Antibodies (1:200) (a) and antibodies
preabsorbed with peptide antigen (20 µg/ml) (b) were
incubated with rat tongue tissue sections containing circumvallate
papillae (CV) and VEG. Note in a the intense
immunoreactivity in the VEG, its duct opening, the cleft of
circumvallate papillae, and the surface of tongue epithelium (TE). Taste buds (TB) are located near the duct
openings of VEG (
In the present study we have identified and cloned the cDNA
for a novel protein, Ebnerin, selectively expressed and released by
VEG, which is the sole salivary gland providing secretion directly to
taste buds. Ebnerin displays substantial homology to a number of
proteins. The greatest homology lies in four repeated domains that
correspond to the scavenger receptor cysteine rich (SRCR) domain of a
variety of proteins. Some of these proteins are expressed on the
surfaces of cells involved in host defense mechanisms of the immune
system, such as T cells, B cells, and macrophages, and exemplified by
the macrophage scavenger receptor proteins. Others are secreted and
appear to participate in host defense, such as complement factor I C1r
and C1s (Journet and Tosi, 1986; Mackinnon et al., 1987),
cyclophilin C-binding protein (Friedman et al., 1993), or MAC
II-binding protein (Koths et al., 1993). Members of this
family are expressed in a wide range of organisms from mammals to
invertebrates. A prominent member of the family is a speract receptor
that occurs in sea urchin sperm and binds speract, the sperm-activating
peptide secreted by eggs and involved in the chemotaxis of sea urchin
eggs and sperm (Bleil & Wassarman, 1980; Kinloch et al.,
1992; Lepage et al., 1992). The SRCR family resembles the
immunoglobulin superfamily of proteins with multiple copies of
cysteine-rich domains occurring both in secreted and
membrane-associated proteins. Because members of the SRCR family
bind proteins and other ligands, it is possible that Ebnerin possesses
a similar function. It is unclear whether the SRCR domains would be
involved in ligand binding. They do not appear to participate in ligand
binding of the macrophage scavenger receptor (Rohrer et al.,
1990). However, almost the entire extracellular domain of the speract
receptor comprises SRCR domains, which presumably mediate binding to
speract secreted by eggs (Kinloch et al., 1992; Lepage et
al., 1992). Interestingly, the C-terminal region of Ebnerin
displays modest homology to the zona pellucida protein, which is
released by eggs and interacts with the sperm outer membrane. It is
unclear whether the coincidence of homology with egg and sperm
signaling proteins in the Ebnerin sequence has physiological relevance. Ebnerin also possesses three repeated domains that display
30-35% amino acid identity to domains that are held in common by
bone morphogenetic protein-I, complement I receptor, and the Drosophila
protein Tolloid, which determines dorsal-ventral patterning (Wozney et al., 1988; Shimell et al., 1991). The repeated
domain that is common to these proteins and Ebnerin is thought to play
a role in ligand binding (Shimell et al., 1991). Despite
its putative transmembrane domain, Ebnerin exists mainly as a soluble
protein. The putative transmembrane domain may participate in
translocation of Ebnerin to the plasma membrane where the cleavage of
this domain would occur in analogy to other membrane-anchored secretory
proteins such as granule secretory proteins and uromodulin (Rindler et al., 1990; Fukuoka et al., 1991; Hoops &
Rindler, 1991). The cleavage might occur through
glycophosphatidylinositol-anchored regions or at a lysine-lysine
bond. In such a cleavage the very short cytoplasmic and transmembrane
domains would be removed, giving rise to a smaller protein, consistent
with Western blots in which proteins that are collected from fluids on
the apical region of circumvallate papillae are smaller than in VEG. The selective localization of Ebnerin in cells of the ducts of VEG
but not in the acinar cells distinguishes Ebnerin from lipase, mucin,
and other proteins secreted by the acinar cells of salivary glands
(Hamosh & Scow, 1973; Field & Hand, 1987). It is unclear
whether Ebnerin primarily binds to soluble proteins or other tastants
or preferentially binds directly to surface proteins of the taste buds.
One protein that might interact with Ebnerin is the von Ebner's
gland protein that is also a secretory protein specifically expressed
in von Ebner's gland and whose sequence resembles that of the
odorant-binding protein (Schmale et al., 1990; Pevsner et
al., 1988). Growth hormone is secreted by the parotid and
submaxillary salivary glands, (Amano et al., 1993;
Humphreys-beher et al., 1994), but this possibility has not
been investigated for VEG. The structural similarity of Ebnerin to
binding proteins for growth hormone such as bone morphogenetic
protein-I (Fukagawa et al., 1994) and transforming growth
factor
Polymerase Chain Reaction (PCR)
Circumvallate papillae
containing taste buds and tongue epithelium were dissected as described
previously (Hwang et al., 1990), and RNA was extracted from
the circumvallate papillae. 10 µg of total RNA were reverse
transcribed using 1 µg of oligo(dT) as primer. 1 µl of the
reverse transcription mix was used in a 25-µl PCR containing 1.5
mM MgCland Northern Analysis
, 400 nM primers, 200 µM dNTP, and 0.5 unit of Taq polymerase (Boehringer
Mannheim). To search for genes associated with salty taste, degenerate
primers based on the published sequences of the amiloride-sensitive
sodium channel (Canessa et al., 1993) were used to amplify
mRNA from rat circumvallate papillae. PCR conditions were 35 cycles of
1 min at 95 °C, 2 min at 45 °C or 65 °C, and 1 min at 72
°C. PCR products were then subcloned into Bluescript vector and
sequenced. The PCR primers used in this study were the forward primer
(GGACAGAATTCGNGGNAA(T/C)TA(T/C)GGNGA(T/C)TG) and the reverse primer
(GATCCACTCGAGNGA(T/C)TTNACNGANGGCCA), which correspond, respectively,
to S
LGGNYGDC
and
W
PSVKSQ
of the amiloride-sensitive sodium
channel (Canessa et al., 1993). Total RNA (20 µg) was
prepared from various rat tissues, fractionated on 1%
agarose/formaldehyde gels, and blotted onto a nitrocellulose membrane.
[
P]dCTP-labeled PCR product (400 base pairs) was
used as a probe. The blot was hybridized in 50% formamide and 5
SSC hybridization buffer at 42 °C and washed with 0.1
SSC
at 65 °C. The blot was then exposed to x-ray film for 1 day at
-70 °C.
Constructing and Screening of a Rat Circumvallate
Papillae cDNA Library
A rat taste cDNA library was constructed
as described previously (Li et al., 1994). Total RNA was
extracted from the circumvallate papillae of 500 rats.
Poly(A) RNA was prepared by passing RNA through an
oligo(dT) column twice. About 5 µg of poly(A
) RNA
were converted to cDNA for construction of the cDNA library using a
Lambda Zap vector cDNA library synthesis kit (Stratagene). The library
consists of 1.5
10
independent clones with an
average insert size about 1.2 kilobase pairs. The cDNA clone generated
by PCR was labeled using a random primer kit and
[
P]dCTP and was used to screen a rat
circumvallate papillae cDNA library under high stringency (50%
formamide and 5
SSC at 42 °C for hybridization and 1
SSC at 55 °C for a final wash). Positive clones were
purified, and cDNA was prepared for restriction mapping and sequence
analysis.
In Situ Hybridization
cRNA probes were made for in situ hybridization on rat tongue tissue sections. A PCR
clone in a Bluescript vector containing a 400-base pair insert was used
to generate antisense RNA probes with [P]UTP
labeling (DuPont NEN). Sense RNA probes provided negative controls.
Tissue sections (16 µm) were fixed in 4% paraformaldehyde, rinsed
in phosphate-buffered saline, and digested with 10 µg/ml proteinase
K at 37 °C for 30 min. Sections were then rinsed in 0.1 M triethanolamine, acetylated in 0.25% acetic anhydride for 10 min,
and dehydrated in a graded series of ethanol solutions. Hybridization
was performed with 10
cpm/100 µl of probe in 50%
formamide, 10% dextran sulfate, 0.3 M MgCl
, 10
mM Tris (pH 8), 1
Denhardt's solution, 0.5 mg/ml
tRNA, and 10 mM DTT overnight at 55 °C. Excess cRNA probe
was removed by digestion with RNase A (20 µg/ml) for 30 min and
washed at a final stringency of 0.1
SSC at 60 °C for 30
min. Slides were hand-dipped in Kodak NTB2 emulsion, exposed for
3-4 days at 4 °C, developed, and stained with Giemsa stain
(Sigma).
Immunoblot and Immunohistochemistry
A peptide
corresponding to a partial amino sequences of Ebnerin
(FTTDHSVTRRGFRADYYS
) was synthesized with
the addition of a lysine on the N terminus to facilitate conjugating to
bovine serum albumin with glutaraldehyde. The conjugated antigen was
injected into rabbits, and antisera were purified by an Affi-Gel 15
(Bio-Rad) affinity column following standard procedures (Harlow &
Lane, 1988). The purified antiserum contains 12 µg/ml antibody. In
Western blot analysis, a 1:100 dilution of purified antiserum was used.
Protein samples were prepared from various tissues homogenized in PBS
buffer containing proteinase inhibitors (1 mM pepstatin A, 0.1
mM aprotinin, 0.1 mM phenylmethylsulfonyl fluoride,
and 10 mM leupeptin). Protein samples (100 µg) were
denatured in SDS sample buffer at 100 °C for 5 min before loading
onto a SDS gel. The secretions from VEG were collected by gently
removing the mucous materials on the apical region of circumvallate
papillae from 5 rats using a razor blade, and half of the collected
materials were loaded onto a 6% SDS gel. Fractionated proteins were
transferred onto nitrocellulose membranes, and blots were blocked in
PBS with 5% nonfat dry milk. Blots were then incubated with a 1:200
dilution antibody in the same buffer overnight at 4 °C. Enhanced
chemiluminescence (ECL) assay was used to detect stained proteins
following instructions from the kit (Amersham).
O
in methanol for 15 min with washes in PBS
following each treatment. Sections were then blocked in 5% normal goat
serum in PBS for 1 h and incubated with 1:200 diluted antibody in PBS
for 24 h at 4 °C. ABC and DAB detecting kits (Vector) were used to
visualize immunoreactivity signals in the tissue sections.
Molecular Cloning of Ebnerin
In a search for
taste bud-associated proteins linked to salty taste perception, we
conducted PCR analysis employing primers based on the sequence of the
amiloride-sensitive sodium channel, which is involved in salty taste
perception (Heck et al., 1984; Schiffman et al.,
1983). We identified a novel PCR product unrelated to the
amiloride-sensitive channel. Initial Northern blots of this PCR product
revealed selective enrichment in von Ebner's gland. We employed
the PCR product to screen a cDNA library derived from circumvallate
papillae containing taste buds and obtained a cDNA of 4267 base pairs
that encodes a protein of 1290 amino acids that is designated Ebnerin (Fig. 1). Ebnerin displays sequence homology to a variety of
proteins (Fig. 1a and 2). It possesses four repeat
domains with 50-70% amino acid identity to various members of the
scavenger receptor protein family, which binds a variety of proteins
and peptides (Kodama et al., 1990; Rohrer et al.,
1990). There also are three repeat domains with about 30-35%
amino acid identity to bone morphogenetic type-I protein (Fukagawa et al., 1994), transforming growth factor receptor type
III (Lopez-Casillas et al., 1991; Wang et al., 1991;
Moren et al., 1992), and related proteins (Wozney et
al., 1988; Elaroussi & DeLuca, 1994; Feng & Liau, 1993;
Fukagawa et al., 1994). In the C-terminal area of Ebnerin, a
305-amino-acid domain displays 12-22% amino acid identity to the
zona pellucida region in a sperm-binding protein of eggs (Lepage et
al., 1992) and in uromodulin (Pennica et al., 1987) and
to the glycoprotein of zymogen granule membranes (Fukuoka et
al., 1991). Ebnerin possesses a single 23-amino-acid transmembrane
region with a very short, 17-amino-acid presumed intracellular
C-terminal area. There are 15 N-linked glycosylation sites in
the putative extracellular domain (Fig. 1b).
Localization of Ebnerin mRNA
We conducted Northern
blot analysis of Ebnerin in a variety of tissues (Fig. 3). The
most prominent expression of Ebnerin mRNA occurs in VEG with an intense
band at about 4.5-5 kilobase pairs. Circumvallate papillae tissue
displays a similarly intense band of the same size. Because
circumvallate papillae tissue contains VEG tissue, this band probably
derives from contaminating VEG material. Tissue of the olfactory
epithelium and the lateral nasal gland displays less intense bands that
are slightly smaller than those in VEG. We fail to detect mRNA
expression in any other tissue examined including epithelium from the
frontal region of the tongue, tongue tissue that is devoid of VEG, or
in parotid gland, submaxillary gland, liver, lung, kidney, colon,
testes, brain, and spleen.
P-Ebnerin cRNA probe on
tongue in saggital section (
10). Hybridization signals are
located in the VEG. CV, circumvallate papillae. b,
bright field image of coronal section of circumvallate papillae
(
200). TB, taste buds; DO, duct opening of von
Ebner's gland. c, dark image of same section as in Fig. 4b. Note that the hybridization signals are
confined to the duct opening in VEG.
Localization of Ebnerin Protein
We developed an
antiserum to an 18-amino-acid peptide in the repeat domain that is
homologous to bone morphogenetic protein-I. Western blot analysis
reveals a single immunoreactive band of 210 kDa that occurs only in
VEG. HEK293 cells transfected with cDNA for Ebnerin display a band of
about 170 kDa, whereas untransfected cells have no immunoreactivity (Fig. 5a). The fact that native Ebnerin is larger than
transfected protein could be due to N-glycosylation of protein
in VEG or an additional N-terminal peptide not revealed by cDNA
cloning. We detect no immunoreactivity in tongue tissue lacking VEG or
in kidney, lung, brain, or colon. Preabsorption with the immunizing
peptide eliminates immunoreactivity (Fig. 5b). Western
blots also reveal more intensive immunoreactivity in soluble proteins
than the particulate form in VEG. The molecular mass of protein in
secretions from VEG is about 4-5 kDa smaller than in VEG (Fig. 5c)
g for 60 min; Secretion, protein in secreted fluids from VEG. The blots were
incubated with affinity-purified antiserum to Ebnerin (1:200), and
immunostained proteins were visualized by the ECL
method.
100). c, higher magnification of
circumvallate papillae section immunostained for Ebnerin antibodies.
The intense immunoreactivity is in the cleft of circumvallate papillae
where taste buds are located (
400). d, higher
magnification of the VEG section immunostained for Ebnerin. Note that
intense immunoreactivity is confined to the epithelial cells of
secretory ducts (SD) and is absent from secretory acini (SA) (
400).
receptor III (Lopez-Casillas, 1991; Wang et al.,
1991; Moren et al., 1992) suggests that it might serve as a
carrier for putative growth factors produced by VEG.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.