2 Department of Biochemistry, Sasaki Institute, 2-2 KandaSurugadai, Chiyoda-ku, Tokyo 101-0062, Japan; and 3 Department of Anatomy, Yamanashi University School of Medicine, Yamanashi, Japan
Received on January 27, 2004; revised on March 14, 2004; accepted on March 17, 2004
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Abstract |
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Key words:
-amylase
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high mannose
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salivary glands
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secretion
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VIP36
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Introduction |
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VIP36 was originally isolated from MDCK cells as a component of detergent-insoluble, glycolipid-enriched complexes containing apical marker proteins and was shown to have a carbohydrate-recognition domain similar to that of ERGIC-53 (Fiedler et al., 1994). In our previous studies, we demonstrated that VIP36 is an intracellular lectin recognizing high-mannose type glycans (Hara-Kuge et al., 1999
). Furthermore, we reported that VIP36 is involved in intracellular transport and the secretion of glycoproteins in polarized MDCK cells (Hara-Kuge et al., 2002
). However, currently the function(s) of VIP36 in mammalian organs remains unknown. In this study, we demonstrate that VIP36 and
-amylase are colocalized and that they coprecipitate in secretory vesicles of the rat parotid gland, suggesting that VIP36 is involved in
-amylase secretion in this gland.
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Results |
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To exclude the possibility that the absence of high- mannose type glycan(s) in fraction 7 reflected a specific localization of endo-ß-N-acetylglucosaminidase, we determined the activity of this enzyme in fractions 1 and 7. Although endo-ß-N-acetylglucosaminidase activity was detected in both fractions, there was no significant difference between fractions 1 and 7 with respect to this activity (Table I).
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As expected, VIP36 was recovered in the detergent phase (Figure 5). In fraction 1, -amylase was recovered in both the detergent and aqueous phases (Figure 5). In contrast,
-amylase in fraction 7, which was the fraction lacking VIP36, was recovered only in the aqueous phase (Figure 5). These results implied that
-amylase in the detergent phase of fraction 1 was associated with VIP36. To further examine this association,
-amylase in parotid gland homogenates was immunoprecipitated with anti-
-amylase, and the precipitated proteins were analyzed by immunoblotting with anti-VIP36 and by lectin staining with Con A (Figure 6). The precipitated
-amylase reacted with Con A, and this staining intensity was greatly reduced by endo-ß-N-acetylglucosamidase H (endo H) treatment of the homogenate prior to the immunoprecipitation step. Immunoblotting of the precipitated proteins with anti-VIP36 revealed that VIP36 coprecipitated with
-amylase in an endo H treatmentsensitive manner. These results indicated that within fraction 1 a substantial portion of
-amylase was associated with VIP36 via high-mannose type glycan(s). Further studies will be required to determine whether the N-glycan(s) of
-amylase in the aqueous phase of fraction 1 undergo deglycosylation or further processing to complex-type glycan(s).
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Discussion |
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Human parotid -amylase can be divided into two groups: non-glycosylated and glycosylated, both of which are secreted into the mouth cavity. In this study, we showed the presence of two types of
-amylase; one (fraction 1 in Figure 2) contains high-mannose type glycans, and the other (fraction 7 in Figure 2) contains no or very few glycans. We previously showed the involvement of VIP36 in the secretion of clasterin, which has at least one high-mannose type glycan (Hara-Kuge et al., 2002
). These facts indicate that
-amylase in fraction 1 binds to VIP36 and is targeted toward the secretion on the apical surface of parotid cells. On the other hand, how is
-amylase in the fraction 7 secreted? Rat nonglycosylated
-amylase should be secreted into the mouth cavity like as that of human. One speculation is that
-amylase in fraction 1 may be deglycosylated by intrinsic endo-ß-N-acetylglucosaminidase and be detached from VIP36, and then be accumulated in other type of vesicles; those in fraction 7 are destined to secrete like as those in fraction 1. It is necessary to assess whether nonglycosylated
-amylase contains a GlcNAc residue, which is a reaction product of the action of endo-ß-N-acetylglucosaminidase.
Rat parotid endo-ß-N-acetylglucosaminidase acts on both high-mannose type glycans and complex-type glycans (as summarized in Table I), whereas human parotid endo-ß-N-acetylglucosaminidase has specific activity for complex-type glycans (Ito et al., 1993). Glycan structures of human glycosylated
-amylase are complex type (Yamashita et al., 1980
, 1981
), and are recognized by human endo-ß-N- acetylglucosaminidase. On the contrary, in this study, it is clear that rat parotid
-amylase contains high-mannose type glycans. At this time, it is unlikely that VIP36 is involved in the intracellular transport and secretion of human glycosylated-
-amylase. Thus the mechanism of parotid
-amylase secretion could differ substantially between human and rat.
Recently, a VIP36-like protein, VIPL, that displays molecular similarities toVIP36, was identified (Neve et al., 2003; Nufer et al., 2003
). The polyclonal anti-VIP36 antibody, which we raised against the luminal domain of VIP36 (Hara-Kuge et al., 1999
) and used in this study, also might recognize VIPL, reflecting their very close amino acid homology. Because it has been reported that VIPL localizes to the ER and may affect the recycling of ERGIC-53 (Neve et al., 2003
; Nufer et al., 2003
), the protein we identified in secretory vesicles using this anti-VIP36 antibody should be VIP36.
Furthermore, there appeared to be two types of secretory vesicle that contained -amylase, one residing in the lightest Percoll fraction and one in the densest fraction. Both types of vesicle might function in secreting glycoproteins, because
-amylase in each vesicle was enzymatically active. Although the
-amylase of fraction 1 seems to be regulated by VIP36, other functional differences between fraction 1 and fraction 7 secretory vesicles may emerge in the near future.
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Materials and methods |
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BiotinGalanthus nivalis lectin (Sigma-Aldrich, St. Louis, MO) was prepared by biotinylation using an enhanced chemiluminescence protein biotinylation module (Amersham Bioscience, Little Chalfont, U.K.), according to the manufacturer's instructions.
Anti-VIP36 antibody was prepared as described previously (Hara-Kuge et al., 2002). Anti-
-amylase goat polyclonal IgG was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-
-amylase monoclonal IgG (for immunoelectron microscopy) from QED Bioscience (San Diego, CA).
Immunoelectron microscopy
Young male Wistar rats weighing 80100 g were anesthetized with pentobarbital and fixed by perfusing 5% acroleon in 0.15 M phosphate buffer (PB), pH 7.4, though the aorta at 37°C. The parotid glands were removed and 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 4°C. The tissues were postfixed in 1% osmium tetroxide and 7% sucrose in PB for 2 h at 4°C; dehydrated with a graded ethanol series at 0°C; embedded in Lowicryl K4M; and cured under UV light for 3 days at 35°C, as described previously (Shimada et al., 2003). Ultrathin sections were treated with 3% hydrogen peroxide at room temperature for 10 min, washed three times with water, and then treated with 0.1% ammonium chloride for 10 min. After washing well with water, the ultrathin sections were incubated with 10% normal goat serum in phosphate buffered saline (PBS) for 1 h and then incubated with anti-VIP36 immunoglobulin adequately diluted with 100 mM PBS, pH 7.4, containing 1% bovine serum albumin for 24 h at 4°C. After rinsing well with PBS, the sections were incubated for 1 h with 10 nm colloidal gold-labeled anti-rabbit IgG (1:200) at room temperature, followed by rinsing with PBS and then water. For double-labeling analysis, the reverse side of the sections labeled with anti-VIP36 antibody was incubated with anti-
-amylase antibody as described. The sections were rinsed well and then incubated for 1 h with 15 nm colloidal gold-label anti-mouse IgG (1:200) at room temperature, followed by rinsing with PBS and then water. Finally, the sections were examined under an electron microscope (Hitachi H-7500).
Isolation of secretory vesicles
Secretory granules of rat parotid glands were isolated by Percoll density gradient centrifugation as described by Fujita-Yoshigaki et al. (1996, 1999
). Briefly, parotid glands were minced in ice-cold homogenizing buffer composed of 250 mM sucrose, 20 mM HEPES (pH 7.2), 1 mM ethylene glycol bis(2-aminoethyl ether)-tetra acetic acid, 0.1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride and dispersed by eight strokes in a glass homogenizer. The homogenate was centrifuged at 1,500 x g, for 10 min. The pellet was suspended in the homogenizing buffer containing 40% Percoll (Amersham Bioscience) and centrifuged at 20,000 x g for 20 min. When the sample was collected in seven fractions, secretory granules were recovered in both the lightest and the most dense fraction.
Immunoblotting and immunoprecipitation
Rat parotid gland homogenates (25 µg protein) or aliquots (50 µl) of each Percoll fraction (diluted with 10 volumes of homogenizing buffer and centrifuged at 1500 x g for 10 min) were separated on 12% or 15% polyacrylamide gels and transferred to nitrocellulose membranes. Proteins were detected by incubation with anti-VIP36 antibody (1:200), anti--amylase antibody (1:2000) or biotin-conjugated lectin (1:1000) followed by a horseradish peroxidaseconjugated secondary antibody or horseradish peroxidaseconjugated streptavidin and then visualized by enhanced chemiluminescence (Amersham Bioscience), according to the manufacturer's instructions.
For immunoprecipitation, the homogenate was incubated for 1 h at 4°C with the relevant antibody. Protein ASepharose was added to the incubation mixture, and incubation was continued for 1 h at 4°C. After washing the Sepharose beads with homogenizing buffer, the proteins bound to the beads were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis followed by immunoblotting.
Endo H assay
The homogenates and isolated secretory vesicles were suspended in 10 mM HEPES/NaOH (pH 7.2) to a concentration of 0.26 µg/µl protein and used as an enzyme source. The enzyme was assayed using 1.9 x 103 dpm/µg each of biantennary glycan (Gal2·GlcNAc2·Man3·GlcNAc2·Asn-N-[14C]Ac) or high mannose-type glycan (Man9·GlcNAc2·Asn-N-[14C]Ac) as substrate. The substrate (40,000 dpm) was mixed with enzyme solution in 50 mM HEPES/NaOH (pH 7.2), 0.1% (w/v) Triton X-100, and 10 mM CaCl2. The reaction mixture was incubated at 37°C overnight and subjected to paper chromatography (pyridine:ethyl acetate:acetic acid:H2O 5:5:1:3). The enzyme activity was measured by counting radioactivities of segregated glycans.
Phase partitioning with Triton X-114
The procedure used was as described previously with minor modifications (Brusca and Radolf, 1994). The isolated secretory vesicles were suspended in 1 ml ice-cold TNE/TX114 (25 mM Tris/HCl, pH 7.4, 150 mM NaCl, 5 mM ethylenediamine tetra-acetic acid, 1% Triton X-114, 1 mM phenylmethylsulfonyl fluoride, 100 kallikrein U/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin) and incubated on ice for 30 min. The incubation mixture was warmed to 37°C to solubilize membrane proteins. After cooling the Triton X-114 extract, residual insoluble material was removed by centrifugation. The resulting supernatants were partitioned into detergent and aqueous phases by temperature-induced phase separation. The detergent phases were reextracted with 10 volumes of TNE.
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Footnotes |
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Abbreviations |
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References |
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