ARTICLE |
Correspondence to: Osamu Shimada, Dept. of Anatomy, Yamanashi U. School of Medicine, 1110 Tamaho-cho, Yamanashi 409-3898, Japan. E-mail: oshimada@swallow.res.yamanashi-med.ac.jp
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Summary |
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VIP36 (36-kD vesicular integral membrane protein), originally purified from MadinDarby canine kidney (MDCK) epithelial cells, belongs to a family of animal lectins and may act as a cargo receptor. To understand its role in secretory processes, we performed morphological analysis of the rat parotid gland. Immunoelectron microscopy provided evidence that endogenous VIP36 is localized in the trans-Golgi network, on immature granules, and on mature secretory granules in acinar cells. Double-staining immunofluorescence experiments confirmed that VIP36 and amylase co-localized in the apical regions of the acinar cells. This is the first study to demonstrate that endogenous VIP36 is involved in the post-Golgi secretory pathway, suggesting that VIP36 plays a role in trafficking and sorting of secretory and/or membrane proteins during granule formation.
(J Histochem Cytochem 51:10571063, 2003)
Key Words: VIP36, immunoelectron microscopy, parotid acinar cells, post-Golgi secretory pathway, animal lectin
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Introduction |
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Newly synthesized secretory and membrane proteins exit the ER in vesicles targeted to the plasma membrane through the Golgi apparatus (
VIP36 (36-kD vesicular integral membrane protein) was originally purified from detergent-insoluble complexes of MadinDarby canine kidney (MDCK) epithelial cells (
Endoplasmic reticulumGolgi intermediate compartment (ERGIC)-53 is also a member of the lectin family and proved to be a mannose-specific lectin localized to the ERGIC (
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Materials and Methods |
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Reagents and Antibodies
All the chemical reagents used, most of which were purchased from Sigma (St Louis, MO), were of analytical grade. The anti-VIP36 polyclonal antibody immunoglobulin against the luminal tail of VIP36 was raised in rabbits in our laboratory. The specificity of the antibody used was described in detail in a previous paper, in which no other band was detectable by immunoblotting and overexpression of VIP36 caused a specific increase of a single band corresponding to a 36-kD protein in MDCK cells (
Immunoelectron Microscopy
Young male Wistar rats weighing 80100 g were used. They were anesthetized with pentobarbital and fixed by perfusing 5% acrolein in 1/15 M phosphate buffer (PB), pH 7.4, through the aorta at 37C. Then the parotid glands were removed, cut into small pieces, which were washed well with PB, and immersed in a fixative composed of 2% paraformaldehyde, 0.125% glutaraldehyde, and 0.1% picric acid in PB overnight at 4C (
Confocal Laser Scanning Microscopy
For comparison with the electron microscopic data, we used the same fixing procedure for the tissues subjected to immunofluorescence analysis. As described above, the tissues were fixed with acrolein, washed well with PBS, then fixed with paraformaldehyde, a very small amount of glutaraldehyde and picric acid, washed well with PBS, and treated with hydrogen peroxide and ammonium chloride to quench any aldehyde remaining in the specimens. The tissues were immersed in 30% sucrose in PBS overnight at 4C, frozen on dry ice, and 6-µm-thick frozen sections were cut with a cryostat at -20C and mounted on gelatin-coated cover glass slides. The sections were permeated with PBS containing 1% Triton X-100 and 1% BSA for 1 hr, treated with 10% normal goat serum and/or donkey serum for 1 hr, and then incubated with the required appropriately diluted antibody or cocktail of antibodies [anti-VIP36 (1:200) and/or anti-amylase (1:100)] in PBS containing 1% BSA for 24 hr at 4C. After washing with PBS, the cells were incubated with the required fluorescence-labeled secondary antibody (1:100) in PBS containing 1% BSA for 1 hr at 20C. Then all the specimens were washed well and mounted in 50% glycerol in Tris-HCl buffer (pH 8.6) containing 50 mg/ml 1,4-diazabicyclo-octane (Aldrich Chemical; Milwaukee, WI), which was used as an anti-bleaching agent. The present fixation procedure completely inhibited nonspecific fluorescence in tissues. The results of the confocal laser scanning microscopy (Olympus, LSM300) and double-labeling experiments were obtained simultaneously to exclude any artifacts resulting from sequential acquisition. Both channels were adjusted to ensure that the maximal fluorescence intensity remained within the recording range.
Cell Culture
As endocrine cells, GH3 cells, which are prolactin (PRL)- and growth hormone (GH)-producing rat pituitary adenoma cells, were provided by Dr. R. Horiuchi (Gunma University School of Medicine). As epithelial cells, Vero cells (African green monkey kidney epithelial cell line) were also provided. They were grown in DMEM supplemented with sodium pyruvate (1 mM), glutamine (2 mM), non-essential amino acids (200 µM), epidermal growth factor (20 nM), 10% horse serum, 2.5% fetal calf serum, 48 mg l-1 penicillin and 100 mg l-1 streptomycin on a glass slide in 100-mm plastic culture dishes at 37C in 5% CO2.
Cells cultured on glass slides were fixed for 5 min with 5% acrolein in 1/15 M phosphate buffer (PB), pH 7.4, at 37C, washed three times with PB, then fixed with PB containing 1% paraformaldehyde, 0.125% glutaraldehyde, and 0.1% picric acid for 1 hr at 37C followed by 3 hr at 4C (
Immunoblotting Analysis
Using the method of
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Results |
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The serous acinar cells of the rat parotid gland contained secretory granules that had accumulated in the subcellular portion between the nucleus and the apical cell membrane surface. The Golgi apparatus consisting of three to six cisternae, arranged in typical stacks of a cis-face to a trans-face according to polarity, was usually found in an apical, paranuclear position in the cells. Gold particles were observed on almost all the secretory and immature granules and in the neighboring trans-Golgi cisternum (Fig 1). We have shown a control section treated by preadsorption with VIP36 (Fig 2). On the control section, we observed only a few gold particles in various areas, including the nucleus, although their number was not significant. Another control section stained with preimmune serum instead of antibody immunoglobulin showed similar results. Significant numbers of gold particles were observed only on sections stained with the specific antibody immunoglobulin against VIP36. Compared to a control section (Fig 2), slight but significant numbers of gold particles were also distributed in the area of the endoplasmic reticulum.
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Immunofluorescence microscopy showed that VIP36 was widely distributed in the apical regions of the terminal serous cells. In an attempt to investigate co-localization of secretory proteins, we compared the distributions of VIP36 and amylase. Marked but not complete overlap of VIP36 and amylase in acinar cells was observed (Fig 3a and Fig 3b). Co-localization of VIP36 and amylase was obvious in the apical paranuclear regions of the acinar cells of the gland.
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An entire gel showing immunoblot analysis of VIP36 in the parotid gland tissue yielded a strong immunopositive single band with a molecular mass of about 36 kD (Fig 4). In the parotid gland, no other band could be detected except for a single band corresponding to a 36-kD protein (Fig 4).
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Immunoelectron microscopy of endocrine GH3 cells and epithelial Vero cells provided evidence that endogenous VIP36 is localized mainly in 70100-nm-diameter vesicles around the endoplasmic reticulumGolgi intermediate compartment (ERGIC) between the endoplasmic reticulum and the neighboring cis-Golgi cisternum (Fig 5a and Fig 5b).
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Although we prepared two VIP36 antibody immunogobulins from two different rabbits, we obtained almost all the same results from the two antibodies. Two different antibodies against amylase also showed the same results.
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Discussion |
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Recently, intracellular lectins were demonstrated to play important roles in vesicular transport (
Inconsistent with parotid acinar cells,
In a preliminary experiment, we examined the localization of VIP36 in various cells. In some endocrine cells and many exocrine cells, including submandibular glandular and pancreatic amylase-secreting acinar cells, large and small intestinal goblet cells and lacrimal and thyroid glandular secretory cells, VIP36 immunoreactivity was detected along the post-Golgi secretory pathways also. However, in many endocrine and some epithelial cells, such as GH3 cells, Vero cells, and MDCK cells, the distribution of VIP36 was prominent in the pre-Golgi early secretory pathway. These data suggest that VIP36 is involved in cellular vesicular transport not only from the endoplasmic reticulum to the Golgi apparatus but also from the TGN to the plasma membrane. To elucidate the function(s) of VIP36, further investigations on a wide variety of cells are needed.
Consistent with the view that the lectin activity of VIP36 is involved in the transport and sorting of glycoproteins, several studies have indicated that N-linked glycan is one of the sorting signals for apical transport (
In parotid acinar cells, localization of VIP36 was not detected on the plasma membrane in the present experiment. The distribution appeared to contradict the previous data for MDCK cells but could not rule out the possible distribution of VIP36 on the plasma membrane. The failure to find any distribution might have been due to the present fixation method, in which animals were perfused with saline and then subjected to chemical fixation. Unlike the case for separate cells, long-term fixation is needed for rats, and we considered that the changes in distribution might have occurred particularly in the surface plasma membrane. Our present chemical fixation method could not resolve the question of whether the VIP36 was recycled or exocytosed. Rapid freeze-fixation might resolve this problem in the future.
Comparing to endocrine GH3 cells and epithelial Vero cells, the parotid acinar cells showed a remarkable distribution following three portions: (a) the Golgi apparatus, (b) immature granules, and (c) mature secretory granules. This raises another question: what cargo protein(s) does the lectin VIP36 carry in the parotid acinar cells? Zymogen granules contain amylase. These is a possibility that membrane fractions containing VIP36 were dispersed and deposited in the secretory granules with cargo protein(s). Alpha-amylase contains glycosylation sequences and glycosylation modification might occur during the processing of a multitude of precursor proteins (
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Acknowledgments |
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We thank Drs S. Oono and K. Hori (Yamanashi University School of Medicine), respectively, for valuable suggestions and letting us carry out confocal laser scanning microscopy freely.
Received for publication September 20, 2002; accepted February 19, 2003.
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