Copyright ©The Histochemical Society, Inc.

Cloning and Characterization of a Novel Animal Lectin Expressed in the Rat Sublingual Gland

Natthiya Sakulsak, Tomohiko Wakayama, Wiphawi Hipkaeo, Miyuki Yamamoto and Shoichi Iseki

Department of Histology and Embryology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan

Correspondence to: Shoichi Iseki, MD, PhD, Department of Histology and Embryology, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640, Japan. E-mail: siseki{at}med.kanazawa-u.ac.jp


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 Materials and Methods
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 Literature Cited
 
We cloned a rat gene that is expressed primarily in the sublingual gland and named the predicted 503 amino-acid protein SLAMP (sublingual acinar membrane protein). SLAMP has 63% homology with human ERGIC-53-like protein, a member of the family of animal L-type lectins. Using a cDNA probe for SLAMP mRNA and rabbit antisera against SLAMP, we examined the expression and localization of SLAMP in major rat organs and tissues. With both Northern and Western blot analyses, abundant expression of SLAMP was demonstrated predominantly in the sublingual gland, with single sizes of the mRNA and protein 1.8 kb and 50 kDa, respectively, but not in other organs or tissues, including the parotid and submandibular glands. With immunohistochemistry, SLAMP was localized to the mucous acinar cells, but not to the serous demilunes or the duct system. With immunoelectron microscopy, SLAMP was localized predominantly to regions corresponding to the ER-Golgi intermediate compartment. Besides the sublingual gland, SLAMP immunoreactivity was also demonstrated in mucous cells of the minor salivary glands in oral cavity and of Brunner's glands in the duodenum. These results suggested that rat SLAMP plays a specific role in the early secretory pathway of glycoproteins in specific types of mucous cells. (J Histochem Cytochem 53:1335–1343, 2005)

Key Words: sublingual gland • mucous cell • ERGIC • membrane protein • lectin • immunohistochemistry • rat


    Introduction
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 Introduction
 Materials and Methods
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THE MAMMALIAN SALIVARY GLANDS are composed of the major salivary glands, including the parotid, submandibular and sublingual glands, and the minor salivary glands scattered in the mucosa of oral cavity (Pinkstaff 1980Go). Of these, the sublingual gland is located close to the submandibular gland with a common secretory exit into the oral cavity. In the rodent, the secretory units of parotid gland are composed of a single type of serous acinar cells and those of the submandibular gland of a single type of seromucous acinar cells, whereas those of sublingual gland consist primarily of mucous cells forming acini or tubules that are capped by relatively few serous cells arranged as demilunes (Young and van Lennep 1978Go; Pinkstaff 1980Go). The minor salivary glands scattered in the wall of oral cavity are also composed mostly of mucous cells.

The primary component of the secretion of mucous cells is mucins, a class of high molecular-weight, highly glycosylated glycoproteins (Strous and Dekker 1992Go). The salivary mucins, derived from the submandibular and sublingual glands and the minor salivary glands, are considered to play important roles in defense against chemical and mechanical damage and microbial invasion in the oral cavity (Wu et al. 1994Go; Amerongen et al. 1995Go; Tabak 1995Go).

Whereas in the submandibular gland, numerous biologically active peptides have been isolated from both the seromucous acini and the duct system, including the secretory portion called granular ducts (Barka 1980Go), the function of sublingual gland, at least of its mucous acini, seems to be restricted to synthesis of mucins. Accordingly, the histochemical markers of sublingual gland have been confined to its specific mucin glycoprotein product that is distinguished by stainability for periodic acid Schiff and Alcian blue as well as by binding to specific plant lectins (Accili et al. 1999Go). During the analysis of a cDNA library derived from the mixture of rat submandibular and sublingual glands, we found, by chance, a cDNA clone that hybridizes with sublingual gland mRNA, but not with submandibular gland mRNA. The amino acid sequence analysis has demonstrated that the peptide encoded by this gene has high sequence homology with human ERGIC53-like protein (ERGL), a type I membrane protein belonging to the family of animal L-type lectins (Yerushalmi et al. 2001Go). Because of its highly specific expression in the mucous acinar cells of rat sublingual gland, we have named this peptide SLAMP (sublingual acinar membrane protein). The expression and localization of SLAMP in the entire rat organs and tissues have been examined at both mRNA and protein levels and at both light and electron microscopic levels.


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Animals and Tissue Preparation
Male and female Wistar rats at the age of 8 weeks were purchased from Nippon SLC, Inc. (Hamamatsu, Japan) and kept in standard laboratory conditions with free access to food and water. All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals at Kanazawa University. The animals were anesthetized with sodium pentobarbital by intraperitoneal injection, sacrificed by bleeding from the right atrium, and perfused transcardially with cold physiological saline. For Northern and Western blot analyses, 20 organs and tissues (the sublingual gland, submandibular gland, parotid gland, thymus, heart, spleen, kidney, testis, prostate, ovary, uterus, skeletal muscle, stomach, duodenum, jejunum, ileum, colon, liver, cerebrum, and cerebellum) were dissected out, frozen immediately in liquid nitrogen, and stored at –80C until use. To make tissue sections for light and electron microscopic immunohistochemistry, the animals were further perfused transcardially with 4% cold paraformaldehyde in 0.1 M PBS (pH 7.2). The organs and tissues were dissected out, further fixed in the same fixative for 4 hr, and immersed in PBS containing 30% sucrose overnight at 4C for cryoprotection. The specimens were then frozen, cut into 12-µm thick sections using a cryostat (Leica Microsystems; Wetzlar, Germany), mounted on glass or plastic slides, and air-dried.

Cloning and Sequencing of SLAMP
In the course of another study concerning the regulation of gene expression in rat salivary glands, we constructed a cDNA library from the complex of rat submandibular and sublingual glands by means of RT-PCR of the transcripts followed by cloning of the cDNA by ligation into TriplEx2 phage vector (Clontech; Palo Alto, CA). From this cDNA library, we found, by chance, a clone that does not hybridize with the total RNA from the submandibular gland separated from sublingual gland. The nucleotide sequence of this cDNA clone was determined from both strands by use of an ABI PRISM 310 genetic analyzer (Applied Biosystems; Foster City, CA).

Production of Anti-SLAMP Antisera
Polyclonal rabbit anti-SLAMP sera were raised against a synthetic peptide corresponding to the C-terminal 11 amino acids (RRQPVSPSMQA) of SLAMP deduced from its nucleotide sequence, plus an additional cysteine residue at the N terminus of the peptide. The peptide was purchased from BEX (Tokyo, Japan), conjugated to keyhole limpet hemocyanin (Nakalai Tesque; Kyoto, Japan) as described previously (Wakayama et al. 2003Go), and emulsified with 1:1 mixture of physiological saline and Freund's complete or incomplete adjuvant (Difco; Detroit, MI). The immunization was made by injecting the peptide with complete adjuvant subcutaneously into New Zealand White rabbits at 100 µg in 1 ml. Starting 3 weeks later, the booster was made every 2 weeks by injecting the peptide with incomplete adjuvant at 50 µg in 1 ml. At 3 months after the immunization, when the antibody titer exceeded 20,000-fold on ELISA using BSA-conjugated SLAMP peptide as antigen, the whole blood was collected from each rabbit and the serum was separated.

Northern Blotting
The total RNA was extracted from the frozen rat organs and tissues with the guanidine-phenol-chloroform method using a commercial solution (TRI reagent; Sigma-Aldrich Co., St Louis, MO). Twenty-µg aliquots of the RNA samples were denatured by glyoxal and electrophoresed in 1% agarose gel as described previously (Hipkaeo et al. 2004Go). As molecular size marker, RNA ladder (Life Technologies, Inc.; Rockville, MD) was used. The samples were then blotted onto nylon membranes (Pall BioSupport; East Hills, NY) and cross-linked by ultraviolet irradiation. For hybridization probe, the entire SLAMP cDNA, 1588 bp in length, was amplified by PCR and labeled with [{alpha}-32P]dCTP (Dupont; Wilmington, DE) using a Megaprime DNA labeling system (Amersham Pharmacia Biotech; Uppsala, Sweden). The membranes were first prehybridized at 65C for 2 hr in 1 M NaCl, 50 mM Tris-HCl (pH 7.5), 10 x Denhardt's solution, 0.1% Sarkosyl, 10 mM EDTA, and 250 µg/ml denatured salmon sperm DNA, and then hybridized in the same solution with 32P-labeled SLAMP cDNA probe. After incubation at 65C overnight, the membranes were washed extensively in 6 x SSC (1 x SSC contains 150 mM sodium chloride and 15 mM sodium citrate, pH 7.0) containing 0.1% Sarkosyl at 65C. They were then exposed to Kodak BIOMAX MS film (Kodak; Rochester, NY) with intensifying screen at –80C for autoradiography.

Western Blotting
The frozen mouse organs and tissues were homogenized in a lysis buffer composed of 1% Nonidet P40, 0.5% sodium deoxycholate, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and the proteinase inhibitor cocktail (Roche; Mannheim, Germany). After centrifugation at 3000 rpm, the supernatants were examined for the protein concentration using a BCA protein assay kit (Pierce; Rockford, IL) and used as cell lysates. Aliquots of cell lysates at 20 µg protein/lane were separated by electrophoresis in 15% polyacrylamide gel in the presence of 0.1% SDS and then transferred to PVDF membranes (BioRad Laboratories; Hercules, CA). After treatment with 4% nonfat skimmed milk in PBS, the membranes were incubated with anti-SLAMP antisera at 1:20,000 dilutions for 1 hr at room temperature. After washing, the membranes were incubated with horseradish peroxidase–conjugated anti-rabbit IgG antibody (Dako; Glostrup, Denmark) at 1:3000 dilutions for 1 hr. The immunoreaction was detected with X-ray films (Kodak X-OMAT AR) after treatment of the membranes with the chemiluminescence kit ECL-plus (Amersham Pharmacia Biotech).

Immunohistochemistry
For light microscopic immunohistochemistry, the cryostat sections mounted on glass slides were treated successively with 0.3% Tween 20 in PBS for 1 hr for cell permeabilization, 0.3% H2O2 in methanol for 10 min to inhibit intrinsic peroxidase activity, and 3% normal swine sera for 30 min to prevent nonspecific antibody binding. They were then incubated overnight at room temperature with anti-SLAMP antisera at 1:2000 dilutions in PBS. To confirm the specificity of the immunoreaction, the antisera were absorbed with the synthetic SLAMP peptide used for the immunization at 100 µg/ml for 1 hr at 4C before use. After washing with PBS, the sites of immunoreaction were visualized by incubating the sections successively with biotinylated anti-rabbit IgG antibody (Vector Laboratories; Burlingame, CA) at 1:200 dilutions for 1 hr, horseradish peroxidase-conjugated streptavidin (Dako) at 1:300 dilutions for 1 hr, and 0.01% diaminobenzidine tetrahydrochloride in the presence of 0.02% H2O2 in 50 mM Tris-HCl, pH 7.5 for ~10 min. The sections with or without counterstaining with hematoxylin were subjected to observation under an Olympus BX50 microscope (Olympus; Tokyo, Japan).

In some sections, the fluorescent double-immunostaining for SLAMP and GM130 or BiP/GRP78 was performed. The sections were incubated with the mixture of rabbit anti-SLAMP antisera (1:2000 dilution) and mouse anti-GM130 antibody or mouse anti- BiP/GRP78 antibody (2 µg/ml, BD Biosciences; San Jose, CA) overnight at room temperature. After washing with PBS, the sections were incubated with the mixture of Alexa Fluor 594-labeled anti-rabbit IgG and Alexa Fluor 488-labeled anti-mouse IgG antibodies (1:400 dilution, Molecular Probes; Eugene, OR) for 1 hr. They were mounted in glycerol and subjected to examination first with a fluorescent microscope (Olympus BX50/BX-FLA) using green emission for Alexa Fluor 594 and blue emission for Alexa Fluor 488, and then with a confocal laser scanning microscope (Carl Zeiss LMS5 PASCAL).

For electron microscopic immunocytochemistry, a preembedding method using the immuno-nanogold probe and silver enhancement (Burry et al. 1992Go) was performed. The cryostat sections on plastic slides, after successive pretreatments with 0.3% Tween 20 and 3% normal swine serum, were incubated with anti-SLAMP antisera at 1:1000 dilution overnight at room temperature. They were then washed in PBS and incubated with goat anti-rabbit Fab' conjugated to 1.4-nm nanogold (Nanoprobes; Stony Brook, NY) at 1:100 dilution in PBS plus 0.5% BSA for 1 hr. After washing with PBS-BSA, the sections were postfixed in 1% glutaraldehyde in PBS for 10 min, washed thoroughly in distilled water (DW), and subjected to development with HQ silver enhancement solution (Nanoprobes) for 5 min in the dark room. They were then washed in DW and postfixed with 0.5% osmium tetroxide for 15 min. After washing and subsequent dipping in DW overnight at 4C, the sections were stained with 2% uranyl acetate, washed in DW, dehydrated in ethanol series, and embedded in an epoxy resin based on Glicidether 100 (Selva Feinbiochemica GmbH and Co; Heidelberg, Germany). Ultrathin sections were made and examined with an H-700 electron microscope (Hitachi High-technologies Co.; Tokyo, Japan).


    Results
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 Materials and Methods
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 Literature Cited
 
The Structure of SLAMP
Figure 1 illustrates the sequence of 1588 nucleotides for SLAMP cDNA and the predicted sequence of 503 amino acids encoded by the open reading frame of this cDNA (GenBank; AB188302, NM_001012465). The amino acids (aa) 1-28 contain many hydrophobic ones and represent the signal peptide. The aa 438-460 also contain many hydrophobic ones and is a strong candidate for the transmembrane domain, suggesting that SLAMP is a type I membrane protein. There is a potential N-glycosylation site at aa 76-78. The aa 29-503 of SLAMP show 63% sequence homology with human ERGL (GenBank; NP_068591; Yerushalmi et al. 2001Go). In the GenBank peptide database, there are also sequences registered as rat and mouse ERGL (XP_217166 and NP_954692, respectively). The aa 23-503 of SLAMP have 95% homology with rat ERGL, but the latter lacks the first methionine of SLAMP and instead has 128 extra N-terminal amino acids that have no homology with SLAMP. Also, aa 23-357 of SLAMP have 83% homology with mouse ERGL, but the latter lacks the C-terminal sequence corresponding to aa 358-503 of SLAMP. In this regard, we have recently cloned a mouse gene with the predicted amino acid sequence 79% homologous with that of the entire length of rat SLAMP, including its C terminus, and have registered this gene as mouse SLAMP (GenBank; AB211986). SLAMP also has 33% homology at aa 30-471 with p58, the rat homolog of ERGIC-53 (GenBank; NP_446338; Lahtinen et al. 1996Go).



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Figure 1

The nucleotide sequence of sublingual acinar membrane protein (SLAMP) cDNA with the predicted amino acid sequence of SLAMP. SLAMP has 503 amino acids and contains a signal peptide (shown in italic), a putative transmembrane domain (shown with underline), and a potential N-glycosylation site (shown in italic with underline). *, peptide termination. GenBank accession: AB188302, NM_001012465.

 
Expression of SLAMP in Rat Tissues
In Northern blot analysis, the SLAMP mRNA forming a single 1.8-kB band was detected predominantly in the sublingual gland out of 20 major rat organs and tissues (Figure 2A). Also, in Western blot analysis, the SLAMP immunoreactivity forming a single 50-kDa band, which corresponded to the predicted molecular weight of 503 amino acids of SLAMP, was detected predominantly in the sublingual gland (Figure 2B).



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Figure 2

Northern blot analysis (A) and Western blot analysis (B) for sublingual acinar membrane protein (SLAMP) expression in the sublingual gland (Lane 1), submandibular gland (2), parotid gland (3), thymus (4), heart (5), spleen (6), kidney (7), testis (8), prostate (9), ovary (10), uterus (11), skeletal muscle (12), stomach (13), duodenum (14), jejunum (15), ileum (16), colon (17), liver (18), cerebrum (19), and cerebellum (20). (A) The total RNA samples were electrophoresed and stained for rRNA with ethidium bromide (lower panel) and then blotted and hybridized with SLAMP cDNA probe (upper panel). The single 1.8-kb mRNA band is detected only in the sublingual gland. (B) The protein samples were electrophoresed, blotted, and immunostained with anti-SLAMP antisera (upper panel) or anti-tubulin antibody as control (lower panel). The single 50-kDa protein band is detected only in the sublingual gland.

 
Localization of SLAMP in Rat Tissues
On light microscopic immunohistochemistry, intense SLAMP immunoreactivity was demonstrated by diaminobenzidine tetrahydrochloride staining in the sublingual gland, but was absent in the neighboring submandibular gland (Figure 3A). The parotid gland was also immunonegative (Figure 3D). In the minor salivary glands scattered in the oral cavity, the mucous glands were immunopositive, but the serous glands represented by von Ebner's gland was negative (Figure 3E). Besides the salivary glands, anti-SLAMP antisera immunostained only Brunner's glands in the duodenum (Figure 3F), but no other mucous or serous glands, including the surface mucous epithelium, fundic, and pyloric glands in the stomach, goblet cells in the small and large intestines, and goblet cells and tracheal glands in the respiratory tract (data not shown). The control sections incubated with the anti-SLAMP antisera preabsorbed with SLAMP peptide showed no immunostaining in any cellular or extracellular component (Figure 3B).



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Figure 3

Immunohistochemical localization of sublingual acinar membrane protein (SLAMP) in the rat tissues. Cryostat sections of the sublingual and submandibular glands (A,B), sublingual gland (C), parotid gland (D), tongue (E), and duodenum (F) were immunostained with anti-SLAMP antisera (A,C,D,E,F) or the antisera preabsorbed with SLAMP (B) and shown without (A–F) or with (C) counterstaining with hematoxylin. The immunoreactivity is localized exclusively to the sublingual gland (SL), mucous minor salivary glands (MM), and Brunner's gland (B) and not detected in any other cell or structure either shown or not shown here. (B) No immunoreactivity is obtained with the preabsorbed antisera. (C) Mucous acinar cells (M) are immunopositive, but serous acinar cells (S) and duct cells (D) are negative. SM, submandibular gland; P, parotid gland; CP, circumvallate papilla, vE, von Ebner's glands, V, intestinal villi. Bars: A–F = 200 µm; C = and 50 µm.

 
In the sublingual gland, SLAMP immunoreactivity was present in the acini, but absent in the duct system (Figures 3A and 3C). In the acini, the mucous cells were immunopositive, whereas the serous cells, as distinguished by cytoplasmic hematoxylin staining, were negative (Figure 3C). In the mucous cells, the immunoreactivity appeared to be absent in the secretory granules, but present mainly in the basolateral cell regions. However, these regions were so thin that it was difficult to localize the immunoreactivity to the plasma membrane, cytosol, or any cytoplasmic organelle. Therefore, we performed fluorescent double-immunostaining to compare the localization of SLAMP with that of GM130, a cis-Golgi marker (Nakamura et al. 1995Go), and of BiP/GRP78, an endoplasmic reticulum (ER) marker (Munro and Pelham 1986Go). In the mucous acinar cells, both markers partially overlapped with SLAMP, but neither marker completely overlapped with SLAMP, as demonstrated by both fluorescent microscopy and confocal laser scanning microscopy (Figures 4A–4D). The extent of colocalization with SLAMP was greater for GM130 than for BiP/GRP78. Again, it was confirmed that SLAMP immunoreactivity is absent in the duct epithelial cells or serous acinar cells. These results suggested that SLAMP is primarily localized to the cis-end portions of Golgi apparatus and the peripheral layers of ER adjacent to the Golgi apparatus in mucous acinar cells.



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Figure 4

Light micrographs of the sublingual gland double-immunostained for sublingual acinar membrane protein (SLAMP) and GM130 (A,B) or SLAMP and BiP/GRP78 (C,D), shown with fluorescent microscopy (A,C) and confocal laser scanning microscopy (B,D). SLAMP immunoreactivity (red) is present in mucous cells (M) but absent in the serous cells (S) or duct cells (D). (A,B) SLAMP (red) and GM130 (green) immunoreactivities are moderately overlapped (yellow) in the mucous cells. (C,D) SLAMP (red) and BiP/GRP78 (green) immunoreactivities are only slightly overlapped (yellow) in mucous cells. Bars: A,C = 100 µm; B,D = 20 µm.

 
The subcellular localization of SLAMP was further examined with immunoelectron microscopy. With lower magnification, the silver grains representing the signal for SLAMP immunoreactivity were scarce in the serous demilune cells, but abundant in the mucous cells, where they appeared to be localized not to the plasma membrane, but to restricted cytoplasmic regions surrounding the secretory granules (Figure 5). With higher magnification, the strong signal appeared to be mostly concentrated on the cisternal membrane structure located between the ER and Golgi apparatus (Figure 5, inset). From the literature, this structure is likely to represent the ER-Golgi intermediate compartment (ERGIC) (Hauri et al. 2000Go). A weak signal was also present all over the ER, with similar intensity as in the ER of serous demilune cells.



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Figure 5

Electron micrographs of the sublingual acinus immunostained with anti–sublingual acinar membrane protein antisera using the preembedding method. In the mucous cell (M), many silver grains representing the immunoreactivity are concentrated on restricted portions of the cytoplasm, but not detected on the plasma membrane or in the mucous granules. A much lower number of silver grains is scattered over the endoplasmic reticulum (ER) in both the mucous cell and serous demilune cell (S). Bar = 1 µm (inset). With higher magnification, the silver grains are concentrated on the membrane structure located between the ER (ER) and Golgi apparatus (G). Bar = 200 nm.

 

    Discussion
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 Summary
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 Materials and Methods
 Results
 Discussion
 Literature Cited
 
In the present study, we have cloned and characterized a novel rat gene that is expressed primarily in the sublingual gland. The predicted protein, designated as SLAMP, has high sequence homology with human ERGL (Yerushalmi et al. 2001Go), a member of the animal L-type lectins consisting of a carbohydrate recognition domain similar to that of plant L-type lectins, a transmembrane domain, and a short cytoplasmic domain. The family of animal L-type lectins includes human ERGIC-53 (p58 in rat) (Saraste et al. 1987Go; Schweizer et al. 1988Go), VIP36 (Fiedler et al. 1994Go), ERGL, and VIPL (Nufer et al. 2003Go). ERGIC-53, the most investigated of the family, occurs ubiquitously in all cells, where it is localized predominantly to ERGIC of the internal membrane system. ERGIC-53 with its carbohydrate recognition domain binds the mannose residues of correctly folded glycoproteins in the ER. Furthermore, ERGIC-53 with its cytoplasmic domain binds COPII, which coats vesicles mediating ER-to-ERGIC transport, and COPI, which coats vesicles mediating Golgi/ERGIC-to-ER transport, thus playing a role in the sorting and traffic of glycoproteins in their early secretory pathway (reviewed in Hauri et al. 2000Go). However, experimental approaches have demonstrated that ERGIC-53 may function as cargo receptor only for a limited number of glycoproteins, including coagulation factors V and VIII, cathepsin C, and cathepsin Z. This raises the possibility that there are many other members of L-type lectins functioning in cell type– and protein-specific manners. VIP36, which is located not only on ERGIC but also on Golgi and plasma membranes and may be responsible for glycoprotein transport to plasma membrane (Hara-Kuge et al. 1999Go), also occurs ubiquitously in all cells. In contrast, the expression of human ERGL mRNA is confined to the normal and neoplastic prostate cells, as well as the spleen, salivary glands, cardiac atrium, and selective cells of the central nervous system, although the subcellular localization and the physiological role of ERGL are unknown (Yerushalmi et al. 2001Go). The abundant expression of SLAMP, as revealed by Northern and Western blot analyses in the present study, is restricted primarily to the sublingual gland and not detected in other tissues where human ERGL has been reported to occur. Therefore, even if SLAMP may represent the rat homolog of ERGL, its physiological role seems to be distinct from that of human ERGL.

The present immunohistochemical study at both light and electron microscopic levels has demonstrated that rat SLAMP is localized predominantly to the ERGIC in the mucous acinar cell of sublingual gland, suggesting that SLAMP may function in a similar way as ERGIC-53 in the early secretory pathway in this specific cell type. Several secretory proteins has been known to be produced by the sublingual gland, including neonatal submandibular gland secretory protein B, parotid secretory protein, common salivary protein 1, submandibular gland protein D, and sublingual mucin (Ball et al. 1991Go; Wolff et al. 2002Go). However, of these, only sublingual mucin is the specific product of mucous acinar cells, whereas the other proteins are mainly produced in serous demilune cells and immature acinar cells, although submandibular gland protein D is also produced in mucous acinar cells. Mucins are high molecular-weight glycoproteins characterized by greater than 50% carbohydrate content attached as O-glycosidically linked oligosaccharides to serine and threonine residues of a linear polypeptide backbone (Pigman 1972Go; Strous and Dekker 1992Go). The salivary mucins, produced by the sublingual, submandibular, and minor salivary glands, may be of primary importance in defense against chemical and mechanical damage and microbial invasion in the oral cavity. In addition, they may be involved in the processes specific for the oral cavity, such as mastication, speaking, and food bolus formation (Wu et al. 1994Go; Amerongen et al. 1995Go; Tabak 1995Go). Mucins are also known to contain N-linked oligosaccharide chains attached to an asparaginyl residue of the peptide backbone (Strous and Dekker 1992Go). In contrast to O-glycosylation that proceeds in the Golgi apparatus, N-glycosylation is common to all glycoproteins and takes place mainly in the ER. N-glycosylation is considered to promote proper folding of the glycoprotein precursors, which is required for protein translocation by vesicular transport from ER to Golgi apparatus, a process that may be promoted by the mannose-binding lectin, ERGIC-53 (Hauri et al. 2000Go). If the role of SLAMP is similar to that of ERGIC-53, it may be associated with the earlier processes of mucin formation and secretion, although the possibility is not ruled out that some sublingual-specific glycoprotein other than mucin is the target of SLAMP. In vitro experimental approaches will be required to clarify this issue.

The present immunohistochemical results demonstrated that SLAMP is expressed only in limited mucous cell populations, namely, the mucous acinar cells of sublingual and minor salivary glands and of duodenal Brunner's glands. The discrepancy between this and the result of Northern and Western blotting, which failed to detect SLAMP expression in the duodenum, may be interpreted by the fact that Brunner's glands of rats extend only a few millimeters distal to the pylorus and thus occupy only a small part of the total volume of the present duodenal samples, which are ~1.5 cm in length from the pylorus. Other mucous cell populations in the submandibular gland, stomach, intestines, and respiratory tract are devoid of SLAMP expression. Such tissue and cell specificity of SLAMP expression may represent the mucin-type specificity of the function of SLAMP. Consistent with this notion, rat sublingual mucin is known to have complicated oligosaccharide chains rich in peripheral acetylated sialic acid residues (Moschera and Pigman 1975Go; Slomiany and Slomiany 1978Go), a characteristic distinct from that of other mucin types, including rat submandibular mucin (Tabak et al. 1985Go). Such a difference in structure of mucin glycoproteins is also reflected in different stainability of mucous granule contents between the sublingual and submandibular glands for plant lectins (Accili et al. 1999Go). The secretory units of rat Brunner's glands consist predominantly of mucin-producing cells (reviewed in Krause 2000Go). Despite the present result suggesting SLAMP expression in Brunner's gland, the structure of mucin glycoprotein from rat Brunner's gland has no similarity to that of rat sublingual mucin (Smits et al. 1982Go). Investigation of the possible common features in the early secretary pathway between the sublingual and Brunner's glands may shed light on the physiological role of SLAMP.


    Acknowledgments
 
We wish to thank S. Yamazaki for technical assistance.

This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (SI).


    Footnotes
 
Received for publication January 6, 2005; accepted May 4, 2005


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

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