ARTICLE |
Correspondence to: Lily Mirels, Dept. of Molecular and Cell Biology, Div. of Genetics, 401 Barker Hall #3204, University of California, Berkeley, CA 94720-3204..
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Summary |
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Gross cystic disease fluid protein-15 (GCDFP-15)/prolactin-inducible protein (PIP) is present at moderate levels in human submandibular and sublingual glands and is barely detectable in human parotid gland. The rodent homologue, PIP, has previously been identified in adult submandibular and lacrimal glands. Here we present the molecular characterization of rat PIP and show that this protein is a product of neonatal and adult rat submandibular, sublingual, and parotid glands. cDNA clones encoding rat PIP were isolated and sequenced. The deduced amino acid sequence of rat PIP shows 56% overall identity and 80% similarity with mouse PIP. By SDS-PAGE, secreted rat PIP has an apparent Mr of 17,000, with a minor proportion present as Mr 2022,000 N-glycosylated forms. PIP was localized in rat salivary glands by immunogold silver staining. PIP was identified in acinar cells of developing and mature submandibular and parotid glands and at very low levels in sublingual gland serous demilunes. Typically, rat submandibular gland secretory proteins are produced by either acinar cell progenitors (Type III cells) or mature acinar cells. The expression pattern observed for PIP is similar to that previously reported for salivary peroxidase, an important component of nonimmune mucosal defense.
(J Histochem Cytochem 46:10611071, 1998)
Key Words: submandibular gland, sublingual gland, parotid gland, prolactin-inducible protein, GCDFP-15, rat, development
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Introduction |
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Gross cystic disease fluid protein-15 (GCDFP-15) was first identified as a major component of breast cyst fluid and is also present in human milk and saliva (
By cDNA (
Several functions for PIP have been suggested. PIP binds to many proteins, including fibrinogen (
PIP gene expresion is hormonally regulated in human breast cancer cells. PIP expression is increased by prolactin and steroid hormones, with dihydrotestosterone the most effective (
cDNA clones for the mouse homologue of PIP have been identified (
Our laboratories have studied gene expression in developing rodent salivary glands. Neonatal rat submandibular glands contain acinar cell progenitors that produce the developmentally regulated secretory proteins parotid secretory protein (PSP), SMGB (glycoforms SMGB1 and SMGB2), and common salivary protein-1 (CSP-1). None of these proteins is present in the seromucous acinar cells of the adult gland, but each is a product of the parotid gland during postnatal development and of the sublingual serous demilunes (
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Materials and Methods |
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Materials
All molecular biological reagents were purchased from New England Biolabs (Beverly, MA) or from BoehringerMannheim (Indianapolis, IN), unless otherwise specified. Radioactive nucleotides [-32P]-dCTP, 6000 Ci/mmol, and [
-35S]-dATP (10001500 Ci/mmol, Sequenase grade) were obtained from New England Nuclear (Boston, MA).
Animals
Care and manipulation of animals were performed according to the guidelines of the Animal Care and Use Committees, University of California, Berkeley, and University of Connecticut Health Center. Adult male and untimed (Figure 2 and Figure 3) or timed (Figure 4) pregnant female SpragueDawley rats were purchased from Harlan (Indianapolis, IN). Pregnant female rats were observed daily at 1700 hr; the first day pups were seen was designated as Day 0. For harvesting of organs, rats were anesthetized with Metofane and sacrificed by cardiac puncture. Secretion products were collected in vitro as described in
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RNA Preparation and Northern Blot Analysis
Organs to be used for RNA preparation were frozen immediately in liquid nitrogen. RNA was prepared according to the method of
Screening of cDNA Libraries
The adult male Wistar rat submandibular gland cDNA library was synthesized and propagated in plasmid pBR322 and screened by filter colony hybridization as described previously (
A 5-day-old SpragueDawley rat submandibular gland cDNA library was prepared in the vector ZapII and screened under high stringency conditions using 32P labeled adult submandibular gland cDNA clone gIIG4 as previously described (
DNA Sequence Analysis and 5' Race
Clones gIIG4 and zIIG4 were sequenced completely on both strands using a Sequenase version 2.0 kit according to instructions provided by the manufacturer (Amersham; Arlington Heights, IL). Plasmids were sequenced from either end using T3 and T7 (zIIG4) or SP6 and T7 (gIIG4) primers, and bidirectionally from the Hind III site (BP 222 in Figure 1A) after subcloning HindIII fragments into pBSKS-. The remainder of the sequence was determined using primers GGTTGACATTGTTAAGGA (BP 355372 in Figure 1) and CTTTGTTAATTCACGTA (complement of BP 446462 in Figure 1A) synthesized at Operon Technologies (Richmond, CA). The deduced amino acid sequence from clones gIIG4 and zIIG4 was compared with the protein sequence databases of the National Center for Biotechnology Information using the program BlastP. Rat, mouse, and human PIP sequences were aligned (Figure 1B), using ClustalW 1.7, via the web site of the Human Genome Center Baylor College of Medicine (Houston, TX). The GenBank accession number of rat PIP is AF054270.
5' ends of IIG4 transcripts from 5-day submandibular gland, adult submandibular gland, and adult parotid gland poly (A)+ RNA were cloned using a Marathon cDNA Amplification Kit (Clontech; Palo Alto, CA) as specified by the manufacturer. The IIG4-specific primer was TCCGGGATCCAGGGTGGTAGGATGATCATTGCAG. The first 24 nucleotides are complementary to BP 286309 of Figure 1; the remainder provide a Bam HI site for subcloning purposes. 5' RACE products were cut with Not I and Bam HI, directionally cloned into pBSKS-, and sequenced.
Fusion Protein and Antibody Preparation
A glutathione-S-transferaseIIG4 fusion protein was prepared using the vector pGEX-KG (
The GSTIIG4 fusion protein was prepared by a modification of the method previously described (
A rabbit anti-GSTIIG4 antiserum was raised against this fusion protein at Pocono Farms (Canadensis, PA) according to their standard fusion protein protocol (see
Anti-GSTIIG4 antibodies were isolated by incubation of rabbit antiserum with the prepared Reacti-Gel beads overnight at 4C. The beads were washed with 120 mM NaCl, 50 mM Tris-HCl, pH 8.0, 0.5% NP 40 (Buffer B), followed by 1 M LiCl, 50 mM Tris-HCl, pH 8.0, 0.5% NP40, again with Buffer B, and finally with PBS. Bound antibodies were eluted with with 50 mM glycine, 150 mM NaCl, pH 2.5, and fractions were immediately neutralized with 1 M Tris-HCl. Fractions containing antibody were identified by SDS-PAGE and brought to 10 µg/ml with RIA grade BSA.
SDS-PAGE and Western Blot Analysis
SDS-PAGE was performed according to the method of
PNGase F was obtained from New England Biolabs, and digestion of secretion product was performed overnight at 37C as directed by the manufacturer. Digestions contained 9 µg total secretion product and 1000 U of PNGase F in a final volume of 60 µl. Controls were prepared identically to PNGase F-treated samples but without addition of enzyme.
ImmunogoldSilver Staining
For light microscopic immunocytochemistry, developing and adult salivary glands were collected from anesthetized rats and fixed in cold 4% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 1624 hr. The tissues were dehydrated in cold methanol solutions, embedded in LR Gold resin (London Resin; Basingstoke, UK), and polymerized under UV light (365 nm) at -20C. One-micrometer sections were cut with glass knives, collected on SuperFrost Plus slides (Fisher Scientific; Springfield NJ), and dried at RT. Nonspecific binding was blocked with 1% bovine serum albumin (BSA)5% normal goat serum (NGS) in PBS. Then the sections were incubated with the primary antibody diluted in the same solution for 90 min at RT. The sections were rinsed with PBS, blocked again with 1% BSA5% NGS, and then incubated for 60 min with goat anti-rabbit IgG labeled with 5-nm-diameter gold particles (Amersham) diluted in 1% BSA in PBS. After rinsing with PBS and distilled water, silver enhancement (British BioCell; Cardiff, UK) was used to visualize the bound gold particles. The sections were rinsed in tapwater, stained with 1% azure II and 1% methylene blue in 1% sodium borate, and coverslips were mounted with DPX. The sections were observed and photographed with brightfield illumination.
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Results |
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To identify cDNA clones encoding protein(s) expressed in both neonatal and adult submandibular glands, an adult submandibular gland cDNA library was screened with first-strand cDNA synthesized using poly(A)+ RNA from 1-day-old rat submandibular gland as a template. From this very low complexity library (495 clones), several clones that hybridized to the first-strand cDNA were selected. Clones were tested by use as probes for Northern blot analysis of RNA from neonatal and adult submandibular gland. Of the clones identified in this screen, four corresponded to low-abundance transcripts present in neonatal and adult gland and were thought unlikely to encode major secretory products. One clone encoded neonatal submandibular gland protein CSP-1 (
IIG4 Is Homologous to Human PIP/GCDFP-15/EP-GP/SABP/gp 17
The nucleotide and deduced amino acid sequences determined from adult submandibular gland clone gIIG4 are presented in Figure 1A, nucleotides 9558. Comparison of the 146 amino acid protein encoded by this clone with the National Center for Biotechnology Information protein sequence databases revealed that the IIG4 protein is the rat homologue of the human prolactin-inducible protein (PIP) (also identified as gross cystic disease fluid protein-15, secretory actin binding protein, gp 17, and extraparotid glycoprotein). The mouse homologue (
The identity of the neonatal submandibular gland transcripts that hybridize to clone gIIG4 was confirmed by isolating a PIP cDNA from a 5-day-old rat submandibular gland cDNA library. DNA sequence analysis demonstrated that this clone (zIIG4) was identical to gIIG4 but was 20 nucleotides shorter at the 5' end. To attempt to determine further 5' untranslated sequence, the 5' ends of PIP transcripts were amplified as described in Materials and Methods. A total of 18 5' RACE products, derived from 5-day submandibular, adult submandibular, and adult parotid glands, was sequenced. An additional eight nucleotides (Figure 1A, BP 18) were determined in this manner. The sequences of the 5' RACE products were identical to that shown in Figure 1A through BP 286, with the exception that at BP 52, six of 18 clones contained G rather than C. The fact that this identical substitution was found in one third of the clones analyzed suggests that it represents a genuine polymorphism, although one that would not alter the amino acid sequence of the PIP protein.
Expression Patterns of PIP in Developing Salivary Glands
A Northern blot in which the gIIG4 cDNA was hybridized to RNA from submandibular glands of neonatal rats aged 030 days and from those adult male and female rats is shown in Figure 2A. In all samples, the probe hybridized to an approximately 650-NT transcript present in the glands of adult males and females at similar levels. Liver RNA was used as a negative control. The levels of PIP transcript were similar from Days 0 to 7, then increased between Days 7 and 14 to a level that appeared to be relatively constant throughout adulthood. The apparent decrease in PIP transcripts on Day 3 seen in Figure 2A was not seen consistently. As previously described (
Northern blot analysis of RNA from sublingual gland gave inconclusive results. Hybridization was observed at low levels in some samples from two developmental series. It was not possible with this method to distinguish between the presence of low levels of genuine sublingual PIP transcripts and minor contamination of some sublingual gland dissections with submandibular gland. The synthesis of PIP by sublingual gland is addressed below using immunocytochemistry.
PIP Protein in Salivary Secretions
PIP protein was visualized in salivary secretions by Western blot using a rabbit antiserum raised against a glutathione-S-transferase-PIP fusion protein (Figure 3A). The middle lanes (ASMG, 5d SMG, and APRG) contained 1.5 µg of salivary secretory protein. In each case, the anti-PIP antiserum reacted primarily with an Mr 17,000 protein, which was at highest relative levels in adult submandibular gland. The Mr of this protein was slightly larger than the 14 kD predicted from the deduced amino acid sequence of PIP. Adult submandibular gland also contained an approximately Mr 22,000 protein(s) reactive with the anti-PIP antiserum (Figure 3A). A similar minor higher Mr band(s) was observed in parotid and neonatal submandibular gland secretion (APRG-9 and 5d SMG-9) when a sixfold greater amount of secretory protein was examined. No reactive bands were observed in sublingual secretion at either protein concentration (data not shown). The appearance and relative proportion of the PIP protein in salivary secretions is shown in the silver-stained gel in Figure 3B.
The PIP sequence has a single N-glycosylation consensus site (NET, amino acids 2931; Figure 1A). Lanes 1 and 2 of Figure 3A contain adult submandibular secretion product incubated overnight in digestion buffer with or without addition of PNGase F to remove N-linked oligosaccharides. The relative proportion of Mr 17,000 to Mr 22,000 protein in the -PNGase F sample was similar to that in adult secretion (ASMG). In the +PNGase F sample, however, the Mr 22,000 protein was undetectable, demonstrating that the higher molecular mass band represents N-glycosylated PIP. Incubation of adult submandibular secretion overnight under the conditions of PNGase F digestion with or without enzyme resulted in the appearance of additional immunoreactive species at approximate Mr 33,000 and 60,000, as well as at the stacking/resolving gel interface. These may represent aggregation of PIP monomers and/or complexing with other salivary molecules.
Immunolocalization of PIP in Neonatal and Adult Salivary Glands
Immunogold silver staining of 1-µm plastic sections showed that PIP was present in the fetal submandibular gland as early as 20 days of gestation (Figure 4A). Reactivity was restricted to the Type III (proacinar) cells of the perinatal acini; no labeling of the Type I (terminal tubule) cells was observed. At 5 days after birth, the secretory granules present in the apical cytoplasm of Type III cells (
In the sublingual gland, differentiation of the mucous acinar cells and serous demilune cells occurs before birth (
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Discussion |
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In this report we have described the sequence and cell specificity of PIP in rat salivary glands. As visualized by SDS-PAGE, rat PIP is an Mr 17,000 protein which is a relatively minor component of submandibular and parotid salivas. The presence of PIP in adult rat submandibular gland acinar cells was observed by
The presence of PIP in both neonatal and adult submandibular gland acinar cells contrasts with the distribution of the previously described major secretory products (PSP, CSP-1, SMGB1, and SMGB2) of neonatal submandibular gland acinar cell progenitors. None of these proteins is present in adult submandibular acinar cells. In parotid gland, PSP, like PIP, is consistently an acinar cell product and is at sustained peak levels after weaning (
Human PIP is present only at exceedingly low levels in parotid gland and saliva (
The conservation of PIP sequence and abundance between rodent and human salivas implies that the function of these proteins is conserved. Rat PIP is predominantly not glycosylated, although Mr 22,000 N-glycosylated forms are secreted by the parotid and submandibular glands. In human and T-47D cells, the majority of PIP is N-glycosylated, whereas mouse PIP does not contain an N-glycosylation consensus sequence (
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Acknowledgments |
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Supported by National Institutes of Health grant DE09428.
We thank Abigail J. Miranda and Mary W. Goss for expert technical assistance, and Don Rio for providing protocols and advice on affinity chromatography of the anti-PIP antibodies.
Received for publication December 3, 1997; accepted May 19, 1998.
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