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Genes to Cells (2006) 11, 623-631. doi:10.1111/j.1365-2443.2006.00964.x
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Decreased basal mucus secretion by Slp2-a-deficient gastric surface mucous cells

Chika Saegusa1, Toru Tanaka2, Satoru Tani2, Shigeyoshi Itohara3, Katsuhiko Mikoshiba4,5 and Mitsunori Fukuda1,6,*

1 Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
2 Faculty of Pharmaceutical Sciences, Josai University, Sakado, Saitama 350-0295, Japan
3 Laboratory for Behavioral Genetics, Brain Science Institute, and
4 Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
5 Division of Molecular Neurobiology, Department of Basic Medical Science, the Institute of Medical Science, the University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
6 Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Synaptotagmin-like protein (Slp) 2-a is a putative Rab27A/B-effector protein and is implicated in intracellular membrane transport. However, the precise tissue distribution of Slp2-a protein and its functions remain largely unknown. In this study we used a specific anti-Slp2-a antibody to investigate the tissue distribution of Slp2-a in mice and found that Slp2-a is most abundantly expressed in mouse stomach. Co-immunoprecipitation experiments indicated that Slp2-a interacts with Rab27A/B in vivo. We also discovered that Slp2-a and Rab27A/B are predominantly localized at the apical region of gastric-surface mucous cells, where mucus granules are accumulated. Analysis of Slp2-a mutant mice generated by homologous recombination showed a reduced number of mucus granules, a deficiency of granule docking with the apical plasma membrane in the gastric-surface mucous cells and reduction of mucus secretion by Slp2-a-deficient gastric primary cells. Based on these results, we propose that Slp2-a is part of the mucin secretory machinery in surface mucous cells of mouse stomach.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Rab GTPases are thought to be involved in the regulation of various types of membrane transport in various cell types by mediating the interaction between vesicular carriers and their specific acceptor compartments (Zerial & McBride 2001). Rab27A is a member of the Rab family that is expressed in a cell-type specific manner (Barral et al. 2002; Tolmachova et al. 2004) and it is involved in the intracellular transport of melanosomes in melanocytes (Hume et al. 2001; Wu et al. 2001), lytic granules in cytotoxic T lymphocytes (Haddad et al. 2001; Stinchcombe et al. 2001), dense granules in platelets (Wilson et al. 2000) and secretory granules in pancreatic ß-cells (Kasai et al. 2005). Mutations in the rab27A gene cause human type II Griscelli syndrome and the corresponding model mouse, ashen, both of which exhibit pigment dilution and immunodeficiency (Ménaschéet al. 2000; Wilson et al. 2000). Rab27B is a closely related isoform of Rab27A that is also involved in different types of vesicular transport in different cell types, including pituitary cells (Zhao et al. 2002), amylase-secreting cells (Chen et al. 2004; Imai et al. 2004) and urothelial umbrella cells (Chen et al. 2003). It has been suggested that Rab27A/B interacts with organelle-specific effectors, e.g. synaptotagmin-like proteins (Slps), Slp homologs lacking C2 domains (Slac2s), rabphilin, Noc2, and Munc13-4 (Izumi et al. 2003; Fukuda 2005), to properly regulate multiple membrane transport events.

The Slp family consists of five members (Slp1-5) in humans and mice (Fukuda & Mikoshiba 2001; Fukuda et al. 2001; Kuroda et al. 2002a) and all members share an N-terminal Slp homology domain (SHD) and C-terminal tandem C2 domains. The SHD specifically interacts with Rab27 in a GTP-dependent manner in vitro (Kuroda et al. 2002b; Strom et al. 2002), while the C2 domains of Slps interact with phospholipids in the plasma membrane, such as phosphatidylinositol 3,4,5-trisphosphate (Catz et al. 2002) and phosphatidylserine (PS) (Fukuda 2002; Kuroda & Fukuda 2004), in vitro. Although the function of Slp2-a in the peripheral melanosome distribution of melan-a cells has been reported in vitro at the cell culture level, the physiological function of Slp2-a in mice has never been elucidated.

To protect the gastric surface from chemical, enzymatic, mechanical and microbial damage, the surface mucous cells and neck cells of the mammalian stomach secrete a defensive factor, mucin, that forms a mucous gel layer overlying the luminal surface of stomach (Forstner & Forstner 1994). Despite the physiological importance of mucus secretion, the biochemical and genetic characterization of mucin has been limited by its large size and abundant glycosylation (Perez-Vilar & Hill 1999), and the molecular mechanism of its secretion has not been fully clarified. Identification of the key molecules involved in mucus secretion would be an important step toward clarifying the molecular mechanism of mucus secretion.

In this study we investigated the pattern of expression of Slp2-a protein in the mouse in vivo and found the highest levels of expression in the surface mucous cells of the stomach. To investigate the physiological functions of Slp2-a, we generated and analyzed mutant mice with a functional disruption in the slp2-a gene. The results revealed that Slp2-a, a putative Rab27A/B effector, is a positive mediator of mucus secretion by the surface mucous cells of mouse stomach.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Endogenous expression of Slp2-a

We first investigated the tissue distribution of Slp2-a protein in mice by immunoblot analysis with the anti-Slp2-a-N antibody. As shown in Fig. 1A, Slp2-a was much more strongly expressed in stomach than in any of the other organs tested and low levels of expression of Slp2-a were found in the lung and thymus. The abundant expression of Slp2-a in the stomach was also confirmed by RT-PCR analysis (Fig. 1B). Since both Rab27A and Rab27B, putative ligands of Slp2-a, were also abundantly expressed in mouse stomach (Fig. 1A,C) (Barral et al. 2002; Tolmachova et al. 2004), we next investigated whether Slp2-a interacts with these Rab proteins in vivo. Consistent with the results of our previous in vitro binding assays in COS-7 cells (Kuroda et al. 2002b; Fukuda 2003), both Rab27A and Rab27B were efficiently co-immunoprecipitated with Slp2-a from the total cell lysates of mouse stomach, whereas two other Rabs (Rab8 and Rab11) endogenously expressed in stomach (Fig. 1A), neither of which interacts with Slp2-a in vitro (Kuroda et al. 2002b), were not detected in the anti-Slp2-a-immunoprecipitates (Fig. 1C).


Figure 1
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  		Figure 1  Slp2-a is a Rab27A/B-binding protein in mouse stomach. (A) Tissue distribution of mouse Slp2-a, Rab27A/B, Rab8 and Rab11 proteins as determined by immunoblot analysis. Equal amounts of total protein (100 µg) from the mouse tissue homogenates indicated were analyzed by 7.5% or 12.5% SDS-PAGE and immunoblotted with antibodies shown on the right. Note the presence of strong Slp2-a and Rab27A/B signals in the mouse stomach. The positions of the molecular mass markers ( x 10–3) are shown on the left. The blots stained with Amido black are shown at the bottom. (B) Tissue distribution of Slp2-a as determined by RT-PCR analysis. G3PDH-specific primers were used as a control. Abundant Slp2-a transcripts were detected in the wild-type stomach, but not in the Slp2-a–/– stomach, and the signal was much stronger in the stomach than in the brain, consistent with the results shown in Fig. 1A. (C) Interactions between Slp2-a and Rab8, Rab11, Rab27A, and Rab27B were analyzed by immunoprecipitation. Lysate from mouse stomach was immunoprecipitated with anti-Slp2-C2B antibody, and the immunoprecipitates were immunoblotted with anti-Rab8, -Rab11, -Rab27A or -Rab27B antibody (top panels). The same blots were then immunoblotted with anti-Slp2-a-SHD antibody (bottom panels). Slp2-a interacts with Rab27A and Rab27B in the stomach, but not with Rab8 or Rab11.

 
Gene targeting of Slp2-a

We produced Slp2-a mutant mice to investigate the physiological functions of Slp2-a. The targeting vector, in which the exon 1 of Slp2-a (which encodes amino acids 1-34, which are critical for Rab27-binding activity (Fukuda 2005)) is replaced by a pgk-neo cassette, was electroporated into E14 ES cells (Fig. 2A). Homologous recombination was assessed by Southern blot analysis (Fig. 2B) and immunoblot analysis of homogenates of stomach (Fig. 2C). Slp2-a mutant mice were born in accordance with the Mendelian ratio (+/+:+/–:–/– = 51 : 112 : 61). Related proteins, such as Slp1, Slp2-b, Slp3-a or Slp4-a (Fukuda et al. 2001), were unlikely to compensate for the functional disruption of Slp2-a, since our immunoblot analysis did not show any up-regulation in the Slp2-a–/– mouse (Fig. 2C).


Figure 2
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  		Figure 2  Generation of Slp2-a mutant mice. (A) Schematic representation of the mouse slp2-a gene (top), targeting vector (middle), and targeted allele (bottom). The protein-coding exon is represented by closed box. Restriction enzyme sites shown are: B, BamHI; Bg, BglII; H, HindIII; and Xb, XbaI. (B) Southern blot analysis of genomic DNA. Blotted membranes were hybridized with either 5'-outside or 3'-outside probe. The sizes for the wild-type and recombinant genotypes are shown on the right. (C) Immunoblot analysis. Homogenates extracted from the stomachs of Slp2-a+/+ or Slp2-a–/– mice were subjected to 7.5% or 10% SDS-PAGE and were immunoblotted with anti-Slp2-a-N, -Slp1-SHD, -Slp2-b, -Slp3-a-SHD, -Slp4-a-SHD, -Rab27A, -Rab27B or -syntaxin 3 antibodies. No immunoreactive Slp2-a signal was detected in the Slp2-a–/–, whereas signals for other proteins were detected in the Slp2-a–/–, the same as in the Slp2-a+/+.

 
An immunohistochemical analysis with specific antibodies was performed to identify the types of cells in the stomach that express Slp2-a. As shown in Fig. 3, Slp2-a signals (red in Fig. 3A,C,E,G,M) were detected mainly in the surface mucous cells of the mouse stomach, identified by UEA-1 (Ulex europaeus type 1) staining (green in Fig. 3B,C,M) and PAS (periodic acid-Schiff) staining (Fig. 3P), but not detected in the neck cells, identified by GS II (Grifforia simplifolica II) staining (green in Fig. 3F,G). Abundant Rab27A and Rab27B expression was also detected in the surface mucous cells (Fig. 3I,K), and the Slp2-a, Rab27A, and Rab27B in the surface mucous cells were predominantly localized at the apical region (Fig. 3M–O). A higher magnification view of gastric surface mucous cells stained for Slp2-a and UEA-1 is also shown in Fig. S1. Since the distribution of Slp2-a and Rab27A/B in the apical region was quite similar to the localization of PAS-stained signals (Fig. 3P), they were suspected of being present on mucus granules. Based on the subcellular localizations of Slp2-a and Rab27A/B, we hypothesized that the Slp2-a·Rab27A/B complex in gastric surface mucous cells is involved in granule biogenesis, polarized transport (from the perinuclear region to the apical plasma membrane) and/or the exocytosis of mucus granules (e.g. docking, priming, and/or fusion of granules). Staining with mucus granule markers, lectins (UEA-1 and GSII), anti-pepsinogen or anti-H/K ATPase antibody was performed to investigate the morphology of the epithelia of stomach of the Slp2-a–/– mice, but no clear abnormalities were observed in thickness of epithelia or numbers of other cells including parietal, neck, or chief cells (Fig. 3Q–X), indicating that the growth and differentiation of gastric epithelial cells were not significantly impaired by the Slp2-a deficiency.


Figure 3
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  		Figure 3  Histological studies of Slp2-a+/+ and Slp2-a–/– mouse stomach. Sections were prepared from Slp2-a+/+ mouse (A–P, U, and W) or Slp2-a–/– mouse (Q–T, V and X) stomach. In A, C, E, G, M, Q and S, immunostaining was performed with antibodies against Slp2-a (red). Surface mucous cells and neck cells were identified by Ulex europaeus type 1 (UEA-1)-FITC (green in B, C, M, and Q) and Grifforia simplifolica II (GS II)-Alexa 488 (green in F, G, and S), respectively. Note that the Slp2-a in mouse stomach was predominantly detected in the surface mucous cells. In I, K, N and O, sections were immunostained with anti-Rab27A (green in I and N) and anti-Rab27B (green in K and O) antibody, respectively. Rab27A and Rab27B were also strongly expressed in the surface mucous cells of the mouse stomach. Observation of the surface mucous cells at higher magnification indicated that Slp2-a, Rab27A and Rab27B were predominantly localized at the apical region (M, N, O), and their distribution profiles in the apical region was quite similar to the localization of PAS-stained signals (P). In (U–X), immunostaining was performed with anti-pepsinogen (green in U and V) and anti-H/K ATPase (green in W and X) antibody, respectively. Specific loss of Slp2-a expression was observed in the Slp2-a–/– mice (in Q and S). Lectin staining and immunostaining on Slp2-a–/– stomach shows that the growth and differentiation of gastric epithelial cells were not significantly impaired by the Slp2-a deficiency in comparison with the wild-type controls. Nuclear staining was performed with DAPI (blue). The corresponding DIC images are shown in the right column (D, H, J, L, R, T). Scale bars: A–L, Q–X, 50 µm; M–O, 10 µm; and P, 10 µm.

 
Defects in granule biogenesis and docking in Slp2-a mutant mice

Although no clear morphological alterations were detected in the mutant stomach by examination with a light microscope, electron microscopy revealed definite differences between the number and the distribution of mucus granules in the Slp2-a+/+ and Slp2-a–/– stomach (Fig. 4). When mucus granules in 20 randomly selected cell sections were counted, an average of 125.9 ± 19.7 granules were found in a typical section of Slp2-a+/+ surface mucous cells (Fig. 4A,C). This number decreased to 71.1 ± 7.69 for Slp2-a–/– cells (Fig. 4B,C) (+/+, n = 20 cells from 3 mice; –/–, n = 26 cells from 3 mice; P < 0.01). The number of mucus granules beneath the apical plasma membrane (within ~0.5 µm below the apical plasma membrane) (Fig. 4E,F) in one cell was also reduced in the Slp2-a–/– (+/+, 20.83 ± 3.20, n = 18 cells from 3 mice; –/–, 10.58 ± 4.16, n = 19 cells from 4 mice; P < 0.01) (open bars, Fig. 4G). Approximately 28% of the granules localized beneath the apical plasma membrane in the Slp2-a+/+ (n = 386) were docked with the apical plasma membrane (arrows in Fig. 4E) and 72% were undocked (arrowheads in Fig. 4E), whereas the number of mucus granules docked with the apical plasma membrane was only 10% in the absence of Slp2-a, thereby revealing a ~2.8-fold reduction in the Slp2-a–/– (n = 224, P < 0.05) (gray bars, Fig. 4G). The mucus granules in the Slp2-a–/– were also larger than in the Slp2-a+/+. Granules having a long axis > 500 nm were more frequently observed in the Slp2-a–/– than in the Slp2-a+/+ (+/+, n = 579; –/–, n = 335) (Fig. 4D), but it is unknown whether the biochemical properties of granule contents were affected. Moreover, the absence of Slp2-a affected the morphology of the apical plasma membrane, because irregular membranes were frequently observed in the Slp2-a–/–, as opposed to flat in the Slp2-a+/+ (compare Fig. 4E and F). Slp2-a may be directly involved in maintenance of the integrity of the structure of the apical plasma membrane, or the altered features of the apical plasma membrane may be a secondary defect caused by the impaired association between the mucus granules and the apical plasma membrane.


Figure 4
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  		Figure 4  Fewer mucus granules were observed in the surface mucous cells of the Slp2-a–/– stomach. Electron-microscopic appearance of surface mucous cells in Slp2-a+/+ (A) and Slp2-a–/– stomach (B). N denotes nucleus. Scale bars: (A, B), 1 µm; (E, F), 500 nm. (C) Quantification of mucus granules in a cell. The number of granules in surface mucous cells was calculated (+/+, n = 20 cells from 3 mice; –/–, n = 26 cells from 3 mice). Error bars indicate the means +/– SE. Slp2-a–/– values are significantly different from those of Slp2-a+/+ (**P < 0.01). (D) Profiles of the size of the mucus granules. The length of the long axis of the mucus granules was measured (+/+, 579 granules from 14 cells; –/–, 335 granules from 14 cells). Mucus granules of the surface mucous cells of the stomach are likely to be enlarged in the Slp2-a–/–. White and black bars represent the Slp2-a+/+ and Slp2-a–/– granules, respectively. Mucus granules can be seen in closer apposition to the apical plasma membrane in Slp2-a+/+ cells (E) than in the Slp2-a–/– cells (F). Granules close to, but not associated with the apical plasma membrane, are observed in Slp2-a–/– cells (F). Arrows in E point to granules docked with the apical plasma membrane, and arrowheads in E and F identify undocked granules. (G) Quantification of mucus granules beneath the apical plasma membrane. The ratios of granules docked with the apical plasma membrane were calculated and shown as gray bars (+/+, 386 granules in 18 cells from 3 mice; –/–, 224 granules in 19 cells from 4 mice). Error bars indicate the mean ± SD. Slp2-a–/– values are significantly different from those of Slp2-a+/+ (*P < 0.05; **P < 0.01).

 
Decreased mucus secretion in Slp2-a mutant mice

Finally, we investigated the mucus secretion activity of the mutant mouse stomach to determine the function of Slp2-a in mucus secretion. Since it is technically difficult to measure the mucin secreted into the gastric cavity of mice in vivo, we measured the mucin secreted by isolated gastric cells by an enzyme-linked lectin assay (ELLA) with SBA (soybean agglutinin) and WGA (wheat germ agglutinin) as described in Experimental procedures. Basal mucin secretion by gastric epithelial cells was significantly reduced in Slp2-a–/– cells than in Slp2-a+/+ cells (Fig. 5). In contrast, we did not observe any significant differences in mucus secretion between the Slp2-a+/+ and Slp2-a–/– cells when they were stimulated with A23187 [GenBank] (data not shown). Consistent with this finding, it has recently been reported that strong stimulation obscures the subtle functional differences in insulin secretion between rab27A-deficient and wild-type pancreatic ß-cells (Kasai et al. 2005), which suggests involvement of the Rab27A·Slp2-a complex in the similar docking process during exocytosis. Alternatively, A23187 [GenBank] may stimulate mucus secretion from the granules undocked to the apical plasma membrane independently of the function of Slp2-a (Gomi et al. 2005).


Figure 5
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  		Figure 5  Basal mucus secretion by gastric primary cells. Gastric primary cells were cultured under 5% CO2 at 37 °C for 2 days. An aliquot of culture supernatant and an aliquot of cultured cells were subjected to ELLA to determine the amount of released mucin in 30 or 60 min and total cellular mucin, respectively. Mucus secretion was determined by the amount released into medium relative to total cellular content (expressed as percent mucus release). Basal mucus secretion by gastric primary cells from Slp2-a–/– animals (n = 6 animals) was significantly different from secretion by Slp2-a+/+ cells (n = 6 animals). Error bars indicate the mean ± SE. Significant differences are indicated (*P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The surface mucous gel layer plays an important role in protecting the stomach from endogenous agents, such as hydrochloride and pepsin, and from exogenous substances that have been swallowed. Despite the physiological importance of mucus secretion, the biochemical and genetic characterization of mucin has been limited by its large size and abundant glycosylation (Perez-Vilar & Hill 1999) and none of the molecules that regulate the docking and fusion of mucus granules in the surface epithelial cells of the mammalian stomach have been identified. In the present study in the mouse we demonstrated for the first time that Slp2-a and its binding partner Rab27A/B (Fig. 1) are predominantly localized at the apical region of the surface mucous cells of the stomach (Fig. 3). The fact that the number of mucus granules is reduced and mucus granule size is larger in the absence of Slp2-a (Fig. 4C,D) suggests a role of Slp2-a in granule formation and/or maturation. Because another striking feature of the Slp2-a mutant mice is the defect in docking of mucus granules with the apical plasma membrane of the surface mucous cells of the mouse stomach, one of the major roles of Slp2-a is suggested to be a linker of Rab27A/B-bound mucus granules to the apical plasma membrane to facilitate mucus secretion. While this manuscript was being prepared, similar defects in granule docking in mice lacking Slp4-a/granuphilin-a, another Rab27 effector predominantly expressed in pancreatic ß-cells was reported (Gomi et al. 2005), suggesting involvement of the Rab27A·Slp2-a complex in the similar docking process during exocytosis. Targeted disruption of Slp2-a also results in a reduction of the basal mucus secretion in mouse stomach (Fig. 5). At present, however, it remains unknown which of the affected parameters (granule formation, docking of the granule with the apical plasma membrane, morphology of the apical plasma membrane or reduction in the basal mucus secretion) are primarily caused by the lack of Slp2-a and which of them are secondary defects.

It is very interesting that defects in granule docking and/or granule size have also been observed in the cytotoxic T lymphocytes (Haddad et al. 2001; Stinchcombe et al. 2001), pancreatic ß-cells (Kasai et al. 2005), and platelets (Wilson et al. 2000) of ashen (i.e. rab27a-defective) mice. No abnormalities have been reported in the stomach of ashen mice, but Rab27B presumably compensates for the functions of Rab27A in gastric surface mucous cells (Barral et al. 2002). Rab27A/B may utilize different Slp members to regulate granule docking in different tissues or cell types, because Slp2-a is not expressed in pancreatic ß-cells (Waselle et al. 2003). Consistent with this notion, it has been suggested that Slp4-a is involved in the docking step of insulin granules (Gomi et al. 2005) or amylase-containing granules in parotid acinar cells (Fukuda et al. 2005) and that rabphilin is involved in the docking step of dense-core vesicles in PC12 cells through interaction with SNAP-25 (Tsuboi & Fukuda 2005). Our preliminary immunohistochemical findings have shown that other Slps are also expressed in mouse stomach, but they are expressed in different cell types: Slp1 is dominantly expressed in chief cells, which secrete pepsinogen, and Slp3 and Slp4 are expressed in parietal cells, which secrete hydrochloride (C. Saegusa and M. Fukuda, unpublished observations), suggesting that Slps are involved in cell-type-specific exocrine secretion in the mouse stomach.

As Slp2-a is also expressed in the lung (Fig. 1A), Slp2-a may be involved in the control of mucus secretion to protect tissues along the airway from bacterial infection, although, because of its low level of expression in the lung, we were unable to identify the types of cells that express Slp2-a in the lung under our immunohistochemical conditions. The discovery of Slp2-a expression in the thymus is also interesting, because lytic granules do not dock in ashen mice and another Rab27A effector, Munc13-4, is not involved in the docking process (i.e. Munc13-4 controls the priming or fusion step) (Feldmann et al. 2003; Shirakawa et al. 2004). Involvement of Slp2-a in the control of exocytosis of granules other than mucus granules is under investigation in our laboratory.

How does Slp2-a control the docking of mucus granules to the apical plasma membrane of the surface mucous cells of mouse stomach? We speculate that Slp2-a links the Rab27A/B on mucus granules via the N-terminal SHD to the apical plasma membrane via the C-terminal tandem C2 domains. Although we previously showed that the C2A domain of Slp2-a is responsible for the plasma membrane association of melanosomes in cultured melanocytes through direct interaction with PS (Kuroda & Fukuda 2004), an additional, as yet unidentified ligand(s) of Slp2-a must be required for the docking of mucus granules to the apical plasma membrane because PS is likely to be present throughout the plasma membrane. Another possibility is that slp2-a mRNA is transported to the apical plasma membrane and the Slp2-a protein is locally synthesized in a manner similar to the apical plasma membrane localization of Drosophila Slp homolog Btsz/dm-Slp in epithelial cells (Serano & Rubin 2003), although we do not know whether mammalian slp2-a mRNA is transported to the apical plasma membrane before translation. Further study is needed to elucidate this question.

In summary, we have demonstrated that Slp2-a expression in the mouse is most abundant in the stomach and that it mediates basal mucus secretion by mammalian gastric surface cells by promoting the proper granule biogenesis and docking of mucus granules with the apical plasma membrane. As far as we have been able to determine, Slp2-a is the first Rab27 effector to have been identified that is involved in basal secretion of granules rather than stimulated secretion (e.g. hormone secretion by endocrine cells). The Slp2-a mutant mouse will continue to provide an interesting and useful model for elucidating the molecular basis of mucus secretion and features of its regulation.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Antibodies

Anti-Slp2-a-N antibody was raised against a 15-mer peptide (MIDLSFLTEEEQDAC) corresponding to amino acids 1-14 of mouse Slp2-a. Anti-Rab27A and anti-Slp2-b polyclonal antibodies were raised against GST-Rab27A and GST-Slp2-b-N (amino acids 1-240 of mouse Slp2-b), respectively, as previously described (Imai et al. 2004). Anti-Slp2-a-SHD, -Slp2-C2B, -Slp1-SHD, -Slp3-a-SHD, -Slp4-a-SHD and -Rab27B antibodies were prepared as described elsewhere (Kuroda et al. 2002b; Imai et al. 2004). Anti-Rab8, -Rab11 and -Rab27A mouse monoclonal antibodies were obtained from BD Transduction Laboratories (Lexington, KY, USA). Anti-Rab27B, -syntaxin 3, -pepsinogen and -hydrogen/potassium ATPase Beta (H/K ATPase) antibody was from Immuno-Biological Laboratories Co. (Gunma, Japan), Merck Biosciences Calbiochem (Darmstadt, Germany), Abcam (Cambridge, UK) and Affinity BioReagents Inc. (Golden, CO, USA), respectively.

Immunoblot analysis

Immunoblot analyses were performed as previously described (Saegusa et al. 2002) with modifications to the preparation of the tissue samples. Mice were perfused by cardiac injection of PBS to remove blood from the tissues. The tissues harvested were homogenized in a lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% deoxycholate, 0.1 mM phenylmethylsulfonyl fluoride, 10 µM pepstatin A, and 10 µM leupeptin), and the supernatant was obtained after centrifugation at 14 000 r.p.m. at 4 °C for 10 min.

Immunoprecipitation

The stomach was dissected from an adult mouse and homogenized in 50 mM HEPES, pH 7.2, 150 mM NaCl, 1 mM MgCl2, 1% Triton X-100, 0.5 mM GTP{gamma}S, and protease inhibitors. The homogenate was rotated at 4 °C for 1 h for solubilization, and after removing the insoluble material by centrifugation at 14 000 r.p.m. for 10 min, the supernatant was incubated at 4 °C for 1 h with 1 µg/mL anti-Slp2-C2B antibody and then with protein A-Sepharose beads (Amersham Biosciences, Amersham, UK). After washing the beads, the immunoprecipitates were subjected to 7.5% (or 12.5%) SDS-PAGE followed by immunoblotting with anti-Rab and anti-Slp2-a-SHD antibodies.

Immunohistochemistry

Immunohistochemistry was performed as previously described (Saegusa et al. 2002). Stomachs were fixed in 3% paraformaldehyde (PFA), embedded in Tissue-Tek OCT compound (Sakura Finetechnical Co., Tokyo Japan) and cut into 5- or 10-µm sections. The sections were stained with specific antibodies and secondary antibodies conjugated with either Alexa 488 or Alexa 594. Gastric-surface mucous cells and neck cells were identified by PAS staining or lectin staining (Falk et al. 1994; Karam et al. 1997). FITC-conjugated UEA-1 and Alexa 488-conjugated GS II were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and Molecular Probes Inc. (Eugene, OR, USA), respectively, and used at a final concentration of 5 µg/mL.

Production of Slp2-a mutant mice

To isolate the mouse slp2-a gene, a mouse 129/Sv genomic library (Stratagene, La Jolla, CA, USA) was screened with mouse Slp2-a cDNA as a probe. In the targeting vector, the first exon of Slp2-a, which contains the Rab27-binding domain, was replaced with a neomycin-resistance gene. Linearized targeting vector was introduced into E14 embryonic stem cells (Hooper et al. 1987) by electroporation. Homologous recombinants were identified by Southern blot analyses and PCR. Germ-line transmission was obtained for two independent ES clones, and no apparent differences in phenotype between the two lines of mice were observed. As E14 ES cells contain a point mutation in the tyrosinase gene (MGI Accession ID. MGI: 98880) (Hooper et al. 1987), which is located very close to the locus of the slp2-a gene on mouse chromosome 7 (NCBI Locus ID: 83671) (within ~3 cM), we were unable to rescue the mutation in the tyrosinase gene by backcrosses to C57BL/6 J or to access the phenotypes on pigmentation of Slp2-a mutant mice. The mice used in the experiments were sex- and age-matched and were the product of crossing heterozygotes. The mice were maintained at the Research Resource Center, RIKEN Brain Science Institute. All animal experiments were carried out according to the guidelines for animal experimentation at RIKEN.

Electron microscopy

Male mice, fasted overnight with free access to water, were used for experiments. The small specimens were taken from equivalent anatomic regions of the oxyntic region (body) of the stomach of Slp2-a+/+ and Slp2-a–/– mice and fixed with 4% PFA/2.5% glutaraldehyde and, after postfixation in 1% osmium tetroxide and dehydration in a graded alcohol series, they were embedded in araldite resin. The stomachs were cut into 0.1-µm sections and examined with an electron microscope. We counted the numbers of docked granules (by direct contact between the granule and apical plasma membrane in surface mucous cells) in sections from three or four different animals of each genotype and at least two different grids per animal.

Enzyme-linked lectin assay (ELLA)

Isolation of primary cells from the epithelium of mouse stomach and ELLA were performed as previously described (Tani et al. 2002) with slight modifications. A small volume of the culture medium of the gastric primary cells was centrifuged, and the supernatant was subjected to ELLA to estimate the amount of released mucin. After removing the culture medium, the cells were frozen in medium and a small volume of thawed cells was subjected to ELLA to estimate residual mucin. "Total mucin" is determined to be "released mucin" + "residual mucin". Mucous samples were incubated overnight at 4 °C in microwell plates coated with SBA (Vector Laboratories, Burlingame, CA, USA), and then at room temperature for 2 h with biotinylated WGA (Vector Laboratories). Staining was developed with avidin-HRP (Vector Laboratories) complex, 0.03% H2O2, and 0.04% O-phenylenediamine (Sigma Chemical Co.).


   Acknowledgements
 
We thank Research Resource Center, BSI, RIKEN for generation and maintenance of the Slp2-a mutant mice, T. Yoshida for help with the electron microscopy, Dr J. Aruga for providing us the mouse genomic library, Drs T. Inoue and M. Tanaka for technical advice, and Dr K. Hironaka for valuable discussions. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (15689006, 16044248, 17024065, 17657067 to M. F.; 17790272 to C. S.).


   Footnotes
 
Communicated by: Kozo Kaibuchi

* Correspondence: E-mail: nori{at}mail.tains.tohoku.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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