©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning of Ebnerin, a von Ebners Gland Protein Associated with Taste Buds (*)

(Received for publication, February 9, 1995; and in revised form, April 21, 1995)

Xiao-Jiang Li Solomon H. Snyder

From the From The Johns Hopkins University School of Medicine, Department of Neuroscience, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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.


MATERIALS AND METHODS

Polymerase Chain Reaction (PCR)and Northern Analysis

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 MgCl, 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 SLGGNYGDC and WPSVKSQ 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).

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% HO 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.


RESULTS

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).


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.



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.


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 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)


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 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.



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 (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).




DISCUSSION

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 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: PCR, polymerase chain reaction; VEG, von Ebner's gland; SRCR, scavenger receptor cysteine-rich; PBS, phosphate-buffered saline.


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