Cloning and Characterization of a Novel Animal Lectin Expressed in the Rat Sublingual Gland
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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Summary |
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Key Words: sublingual gland mucous cell ERGIC membrane protein lectin immunohistochemistry rat
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Introduction |
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The primary component of the secretion of mucous cells is mucins, a class of high molecular-weight, highly glycosylated glycoproteins (Strous and Dekker 1992). 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. 1994
; Amerongen et al. 1995
; Tabak 1995
).
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 1980), 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. 1999
). 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. 2001
). 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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Materials and Methods |
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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. 2003), 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. 2004). 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 [
-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 peroxidaseconjugated 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. 1992) 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).
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Results |
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Discussion |
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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. 1991; Wolff et al. 2002
). 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 1972
; Strous and Dekker 1992
). 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. 1994
; Amerongen et al. 1995
; Tabak 1995
). Mucins are also known to contain N-linked oligosaccharide chains attached to an asparaginyl residue of the peptide backbone (Strous and Dekker 1992
). 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. 2000
). 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 1975
; Slomiany and Slomiany 1978
), a characteristic distinct from that of other mucin types, including rat submandibular mucin (Tabak et al. 1985
). 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. 1999
). The secretory units of rat Brunner's glands consist predominantly of mucin-producing cells (reviewed in Krause 2000
). 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. 1982
). 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.
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Acknowledgments |
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This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (SI).
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Footnotes |
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