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From the Department of Internal Medicine,*
Division of
Respiratory Disease, Kanazawa Medical University, Ishikawa, Japan; the
Department of Medicine and Biochemistry,
National Jewish Research and Medical Center, Denver, Colorado; and the
Department of Medicine,
Division of Pulmonary
Medicine and Critical Care, University of Colorado Health Science
Center, Denver, Colorado
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The rab family has more than 30 members, the largest number among small G proteins. The Rab proteins are known to localize to specific cell organelles, and are found in both membrane-bound and cytosolic forms.2,3 Thus, they are believed to mediate intracellular vesicle transport among restricted intracellular compartments. Although current information in the sequence database indicates more than 30 members, there are few Rab proteins for which intracellular localization and function have been clarified.
A novel cDNA has been cloned from the rat lung cDNA library encoding a rab-related small G protein (GenBank accession no. M94043). Analysis of the cDNA analysis revealed that this protein shares large similarity with other Rab proteins and the predicted molecular weight from the deduced amino acid sequence is 24 kd. Recently, a cDNA encoding the same protein was cloned from a human melanoma cDNA library and was numbered as Rab38.4 Although the cDNA sequence of the protein has been elucidated, the cells expressing this protein and its intracellular distribution and function remain primarily unknown. In this study, we sought to clarify the expressions of the native protein and mRNA of rab38 in the lung, and examine the localization of the protein in specific lung cells and subcellular organelles.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Common chemicals and reagents were purchased from Sigma (St. Louis, MO) or Wako Chemicals (Osaka, Japan). Cell culture plasticware was from Falcon (Becton Dickinson, Tokyo, Japan). Metrizamide was from Sigma. Fetal calf serum and culture media were from Life Technologies, Inc. (Rockville, MD). Porcine pancreatic elastase was from Worthington (Freehold, NJ). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were from Novex (San Diego, CA). Nitrocellulose membranes were from Bio-Rad (Hercules, CA). ABC method-based histochemical staining kit [Histofine SAB-PO (R)] was from Nichirei (Tokyo, Japan). Restriction enzymes, BamHI, HindIII, and DNA molecular weight markers were from WAKO Chemicals. Designated DNA primers were from Funakoshi Life Science (Tokyo, Japan).
Cells
Alveolar type II cells were isolated from specific pathogen-free adult male Sprague-Dawley rats (Sankyo Labo Service, Tokyo, Japan) by pancreatic elastase digestion and metrizamide density-gradient centrifugation, according to the method described by Dobbs and colleagues.5 Alveolar macrophages were isolated by bronchoalveolar lavage. In SPF rats, >98% of the lavaged cells were macrophages and these were used without further purification. Spodoptera frugiperda cells (Sf9 cells) (Invitrogen, Carlsbad, CA) were cultured in TNM-FH medium in 25-cm plastic culture dishes.
Expression Strategy of rab38
The original cDNA clone was constructed in a pET-3 vector. The
rab38 cDNA (Figure 1)
was
amplified by polymerase chain reaction (PCR) using specific primers.
The 5' primer was 5'-TCCCGGATCCATGCAGACACCGCACAAG-3'. The primer was
designed to have the BamHI restriction sequence at its 5'
site upstream of the initiation codon in the cDNA sequence. The 3'
primer was 5'-TTAAAAGCTTGTAAACACTGTGCTGAC-3'. The primer
originally contained the HindIII restriction sequence
present in downstream site of the termination codon. The PCR product
was inserted into the pBlueBacHis 2A vector. The DNA sequencing of the
recombinant vector was performed with the ABI BigDye terminator cycle
sequencing ready reaction kit (PE Applied Biosystems, Foster City, CA).
Co-transfection of the recombinant plasmid and lethal-depleted
baculovirus (Bac-N-Blue DNA, Invitrogen) was performed with Sf9 cells
using cationic liposome (InsectinPlus, Invitrogen) according to the
instructions in the manufacturer’s manual. The resultant culture
supernatant was used for plaque purification of recombinant virus. The
presence of rab38 containing recombinant virus was verified
by PCR. The PCR-positive virus was amplified for large-scale culture.
Virus titers were determined by plaque assay.
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Protein production was performed using Sf9 cells cultured in 25-cm plastic dishes. Cells at 80 to 90% confluency were infected with the recombinant virus at a multiplicity of 10. Four days after infection, the cells were harvested, washed twice with cold phosphate-buffered saline (PBS), rapidly frozen with dry-ice in ethanol, and stored at -80°C until use. The frozen cells were rapidly thawed and suspended in lysis buffer (1% Triton X-114/50 mmol/L Hepes at pH 7.4, 150 mmol/L NaCl, 1.5 mmol/L EGTA, 10% glycerol) containing protease inhibitors (1 mmol/L phenylmethyl sulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin) on ice for 30 minutes. The cell suspension was centrifuged at low speed (360 x g, 10 minutes), and the supernatant was collected. The supernatant was extracted using Triton X-114 partitioning.6,7 Briefly, the supernatant with 1% Triton X-114 was layered on 6% sucrose/50 mmol/L Hepes at pH 7.4/150 mmol/L NaCl/0.06% Triton X-114. It was then incubated at 30°C in a water bath for 30 minutes and centrifuged at a low speed for 10 minutes. The upper layer was recovered, adjusted to 0.5% Triton X-114, incubated at 30°C for 30 minutes, again layered on the previous sucrose cushion, and centrifuged at a low speed. The detergent pellet in the bottom of the tube was redissolved in PBS and used for further processing. The Triton X-114-extracted cell lysate was loaded on a Ni++-charged affinity column (Probond, Invitrogen) under native conditions, according to the manufacturer’s manual. Elution was performed with imidazole gradients. The purified fraction was monitored with SDS-PAGE using 8 to 16% gradient gels under reducing conditions and subsequent Coomassie Blue staining.
Polyclonal Antibody Production
Based on the amino acid sequence predicted for rab38, a
polypeptide consisting of 20 amino acid residues of the C-terminal
domain was synthesized. Keyhole limpet hemocyanin (Calbiochem, San
Diego, CA) was coupled with
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
(Calbiochem) and fractionated with Sephadex G-25 column chromatography
(Pharmacia, Tokyo, Japan). The keyhole limpet
hemocyanin-m-maleimidobenzoyl-N-hydroxysuccinimide ester
conjugate was allowed to couple with the synthesized polypeptide and
was stored at -20°C until use. An emulsion of the
polypeptide-m-maleimidobenzoyl-N-hydroxysuccinimide-keyhole
limpet hemocyanin preparation and a complete Freund’s adjuvant was
prepared and injected into the subcutaneum of two female New Zealand
White rabbits (body weight, 
2.5 kg). A booster injection with an
incomplete Freund’s adjuvant was added 3 weeks after the first
injection, and the third injection was given 2 weeks later. The titer
of rabbit blood was monitored by the dot blot method using an
immobilized Sf9-cell lysate infected with the recombinant virus. Serum
was collected and an IgG fraction was purified with protein
A-conjugated Sepharose CL4B beads (Sigma). The IgG fraction was further
purified with the synthesized polypeptide-coupled
2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP) activated
Cellulofine (Seikagakukogyo, Tokyo, Japan).
Immunohistochemistry
Excised rat lungs were slowly infused endotracheally with an OCT-compound (Sakura Finetechnical, Tokyo, Japan) and were frozen rapidly with ethanol/dry ice. Lung slices were prepared and fixed with 4% paraformaldehyde for 20 minutes, followed by acetone for 30 seconds. Immunostaining was performed based on the ABC method using a commercial kit [Histofine SAB-PO(R) Kit, Nichirei, Tokyo]. The polyclonal antibody for Rab38 was layered onto the fixed lung slices using a concentration of 8 µg/ml. Control staining was performed in two ways by using the same concentration of nonimmune rabbit IgG, and by using anti-Rab38 plus the synthesized polypeptide in a 100-fold molar excess. A biotin-labeled goat anti-rabbit IgG antibody was added as the second antibody followed by horseradish peroxidase-conjugated streptavidin. Color development was performed for 3 minutes in the presence of diaminobenzidine and H2O2.
Reverse Transcriptase (RT)-PCR
Total RNA was extracted from perfused rat lung, isolated alveolar type II cells, and isolated alveolar macrophages using an RNA isolation kit (Trizol, Life Technologies, Inc.). DNase I (a RQ1 RNase-free DNase, Promega) was added to the total RNA sample and incubated at 37°C for 15 minutes. The reaction mixture was extracted with phenol:chloroform, and RNA was precipitated with methanol. First-strand DNA was synthesized with random hexamer primers using a reverse transcriptase (Superscript RT, Life Technologies, Inc.). RNase H (Life Technologies, Inc.) was next added. Designated primers, Taq DNA polymerase (Takara Ex Taq; Takara, Shiga, Japan), and other reaction components for PCR were subsequently added. PCR was started at 94°C for 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 90 seconds using a Thermal Cycler (Gene Amp PCR System 2400, Perkin Elmer). Aliquots of the PCR product were electrophoresed on a 2% agarose gel and stained with ethidium bromide. The primers for rab38 were 5'-ATGCAGACACCGCACAAG-3' and 5'-AGGGAGAGTTAACTTTGAGTC-3'. The primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'.
Western Blot
Total protein was extracted from perfused rat lungs, isolated
alveolar type II cells, alveolar macrophages, and Sf9 cells using a
lysis buffer containing protease inhibitors. The protein concentration
was determined by a deoxycholate-trichloroacetic acid precipitation and
a BCA microprotein assay kit (Pierce, Rockford, IL). SDS-PAGE was
performed under reducing conditions with 1-mm-thick, 8 to 16%-gradient
precast minigels (IWAKI, Tokyo, Japan). The nitrocellulose membranes
were blocked with 3% skim milk/1% Triton X-100/PBS. The membranes
were reacted with a rabbit anti-rat Rab38 polyclonal antibody at 8
µg/ml/3% skim milk/1% Triton X-100/PBS overnight at 4°C.
Subsequently the membranes were reacted with a horseradish
peroxidase-conjugated anti-rabbit IgG antibody (Bio-Rad) at 1:1000
dilution. Color development was performed in the presence of
H2O2 and diaminobenzidine
as the chromogen for 3 minutes.
In Situ Hybridization
Perfused rat lungs were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced into 5- to 7-µm-thick sections. PCR amplification of rab38 using recombinant pBlueBacHis2A as the template was performed using the same primers used in RT-PCR. The PCR products were isolated from the gels with Geneclean (BIO101, Vista, CA) and directly inserted into the pGEM-T Easy vector (Promega, Madison, WI). Competent E. coli cells (One Shot Cells, Invitrogen) were transformed with the recombinant pGEM-T Easy plasmid. E. coli was grown and plasmid DNA was isolated with a plasmid purification kit (Plasmid Mini; Qiagen, Valencia, CA). Digoxigenin-labeled RNA riboprobes were synthesized using a DIG RNA labeling kit (SP6/T7) (Boehringer Mannheim, Indianapolis, IN). The rab38 cDNA-pGEM-T EASY plasmid was linearized with BamHI or HindIII. The linearized plasmids were used as DNA templates to synthesize RNA riboprobes (antisense and sense riboprobes) with SP6 or T7 RNA polymerase in the presence of digoxygenin-labeled UTP. An antisense or sense digoxygenin-labeled RNA probe was warmed to 85°C for 3 minutes, quickly put on ice, and hybridized with the lung-tissue section at 42°C for 16 hours. Concentrations of the RNA probes in the hybridization buffer were 0.1 to 0.5 µg/ml. The sections were treated with RNase, blocked with 10% normal sheep serum, and hybridized for 30 minutes at room temperature with sheep polyclonal anti-digoxygenin Fab fragments conjugated with alkaline phosphatase (750 U/ml), that was diluted to 1:500 before use. Color development was performed in the nitroblue tetrazolium salt / 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) solution overnight in the dark.
Cell Fractionation
Freshly isolated alveolar type II cells (60 x 106 cells) were disintegrated with a Dounce glass homogenizer. The homogenate was divided into two samples. One homogenate was centrifuged at 360 x g for 10 minutes. The resulting pellet was referred to as the nucleus fraction. The supernatant was centrifuged at 15,000 x g for 10 minutes, and this pellet was referred to as the heavy vesicle fraction. The supernatant was centrifuged at 100,000 x g for 1 hour, and this pellet was referred to as the light vesicle fraction. The supernatant was referred to as the cytosol fraction. Another homogenate was quickly adjusted to 0.9 mol/L sucrose concentration and 3 ml of the homogenate in a 0.9 mol/L sucrose was layered above 3 ml of 1.48 mol/L sucrose in a centrifuge tube for an SW 28 rotor (Beckman). Next, 1.5-ml steps of sucrose (in 0.1 mol/L increments) from 0.8 mol/L to 0.2 mol/L were layered above the 0.9 mol/L sucrose, and the gradient was centrifuged at 100,000 x g for 3 hours. Under these conditions, low-density lamellar bodies, characteristic secretory granules of lung surfactant in alveolar type II cells, migrate against the direction of the gravitational field from the 0.9 mol/L sucrose zone to the 0.4 to 0.5 mol/L sucrose zone.8,9 The sucrose gradient was fractionated from low- to high-density sucrose gradients by controlled vacuum aspiration of fractions.
Confocal Laser Microscopy
Freshy isolated rat alveolar type II cells were seeded in Chamber Slide (Lab-Tek 177437; Nalgen Nunc International Corp., Naperville, IL) and cultured for 24 hours. The adherent cells were washed with cold PBS and fixed with 4% paraformaldehyde/PBS for 10 minutes, and acetone for 30 seconds. The slides were sealed and stored at -80°C until use. The slides were blocked with 10% normal goat serum/5% BSA/PBS and were reacted with a rabbit anti-rat Rab38 polyclonal antibody (8 µg/ml), and one of the following mouse monoclonal antibodies: BiP/GRP78 at 1:100, GM130 at 1:100, TGN38 at 1:100, EEA1 at 1:100 (Transduction Laboratories, Lexington, KY), or an affinity-purified goat polyclonal antibody: Lamp-1 at 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA). The slides were reacted for 30 minutes with second antibodies: Alexa 488-labeled goat anti-rabbit IgG antibody at 1:400 dilution and Alexa 594-labeled goat anti-mouse IgG antibody at 1:400 dilution (Molecular Probes, Eugene, OR). For the Lamp-1, fluorescein isothiocyanate-conjugated affinity-purified donkey anti-rabbit IgG antibody (Jackson ImmunoResearch, West Grove, PA) at 1:100 dilution followed by Alexa 488-labeled rabbit anti-fluorescein IgG (Molecular Probes) at 1:200 and Cy3.5-conjugated affinity-purified donkey anti-goat IgG antibody at 1:200 dilution (Rockland, Gilbertsville, PA). Confocal microscopic images were obtained using a computer-interfaced, laser-scanning microscope (Leica TCS-4D). Immunolabeled slides (n = 3 to 4 representative fields per slide), were sectioned optically through the cell monolayer to obtain the appropriate focal depth. The representative image chosen contained the nucleus and relevant organelle of interest (ER, Golgi, trans-Golgi network, endosomes, and lamellar bodies). Simultaneous wavelength scanning allowed superimposition of fluorescent labeling with Alexa 488 and Alexa 594 (or Cy 3.5) fluorophores. Confocal images were obtained using the following parameters for Alexa 488-labeled secondary antibody: voltage 700 to 800, offset -1, and for Alexa 594-labeled secondary antibody: voltage 600 to 700, offset -1. Laser power was adjusted between 75 to 90% to obtain the best images. The pinhole setting, which was identical for both Alexa 488 and Alexa 594 images because of simultaneous scanning, was fixed to 100. Image sizes (Zoom) were X: 100.00 and Y: 100.00. Image output was at 512 x 512 pixels.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The affinity-purified rabbit anti-rat rab38 polyclonal
antibody reacted with a protein of 26-kd in both total lung
homogenate and the alveolar type II cell lysate, by Western blot
(Figure 2)
. The reaction seemed to be
specific and appeared as one band. The 26-kd band still was found in
the detergent phase after Triton X-114 extraction, suggesting that the
26-kd protein had significant hydrophobic character. Only a faint
signal of the 26-kd protein was recovered in the water-soluble fraction
after Triton X-114 partitioning of the alveolar type II cell lysate.
Thus, the majority of native Rab38 exists as a hydrophobic protein in
the cell. The recombinant baculovirus-infected Sf9 cell lysate showed
abundant immunoreactive protein with a molecular weight of 27 kd.
However, Sf9 cells contained approximately equal amounts of soluble
Rab38 and Triton X 114-partitioning Rab38 (data not shown). The
difference in the molecular weights of native Rab38 and the recombinant
Rab38 seemed to be because of an N-terminal fusion peptide (37 amino
acids) that had been constructed in the pBlueBacHis2A, including the
six histidine-tag and the enterokinase restriction site.
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The RT-PCR for rab38 showed one distinct band
corresponding to the molecular size predicted from the chosen primers.
The PCR product was specifically observed in a total RNA sample derived
from whole lungs or freshly isolated alveolar type II cells, but it was
not detected in a total RNA sample from alveolar macrophages (Figure 3B)
. In contrast, the PCR products for
GAPDH were observed in similar amounts, both in alveolar type II cells
and in alveolar macrophages. Western blotting of the Rab38 using the
rabbit anti-rat Rab38 antibody clearly showed the existence of the
Rab38 protein in isolated alveolar type II cells, but not in alveolar
macrophages (Figure 3A)
. Thus, rab38 is selectively
expressed in alveolar type II cells but not in alveolar macrophages.
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Native Rab38 Protein
Immunostaining of rapidly frozen and paraformaldehyde-fixed lung
tissue showed specific immunoreactivity of the anti-rat
rab38 antibody with a number of alveolar corner cells
(Figure 4A)
and some bronchial epithelial
cells (Figure 4B)
. This immunoreactivity was abolished when the
antibody was added together with the synthesized polypeptide for Rab38.
Moreover, when the same amount of nonimmune rabbit IgG was used as the
primary antibody, these cells showed no positive signal (data not
shown). The alveolar corner cells seemed to be exclusively alveolar
type II cells. Some, but not all, of the airway epithelial cells showed
an immunoreactive signal, and most of the terminal airway epithelial
cells were immunoreactive. Alveolar epithelial cells not existing in
alveolar corners, alveolar macrophages, pulmonary vessels, airway
smooth muscle cells, connective tissue cells, and other lung cells were
not immunoreactive.
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In situ hybridization of rab38 using a
digoxigenin-labeled antisense RNA probe clearly demonstrated
localization of rab38 mRNA in airway epithelial cells and
alveolar corner cells (Figure 5)
; this
was consistent with the results of immunostaining. Most of the terminal
airway epithelial cells showed positive signals (Figure 5
, inset). No
other lung cells showed positive signals. The sense RNA probe did not
produce a significant signal. Because the in situ
hybridization used a paraffin-embedded lung section, preservation of
the structure of the lung tissue was better than that of the rapidly
frozen immunostained sections.
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Freshly isolated alveolar type II cells were homogenized with a
Dounce homogenizer. The cell homogenate was fractionated by
differential centrifugation and sucrose density gradient
ultracentrifugation. Fifty µg of total protein from each cell
fraction was subjected to SDS-PAGE and transferred to a nitrocellulose
membrane. An affinity-purified anti-rat Rab38 antibody (8 µg/ml) was
used as the primary antibody for Western blotting. The total alveolar
type II cell lysate, heavy vesicles (15,000 x g, for
10 minutes), light vesicles (100,000 x g, for 1 hour),
and cytosol showed immunoreactivity, whereas the nuclear fraction was
negative (Figure 6A)
. The heavy vesicles
showed the strongest signal. There was a prominent positive signal in
the 0.9 mol/L sucrose fraction where the homogenized sample was loaded
(Figure 6B)
. The 0.4 to 0.6 mol/L sucrose fraction containing the lung
surfactant-rich lamellar bodies did not give specific immunoreactive
signal.
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; a, b, and c). Major subcellular
organelles were simultaneously stained with Rab38 on the same slide,
and the same planar section of the cells was imaged with simultaneous
two wavelength scanning. The immunostained organelles included ER,
Golgi, trans-Golgi network (Figure 7a)
, and early and late
endosomes (Figure 7b)
. Surfactant apoproteins, SP-A and SP-B, were also
stained (Figure 7c)
. Among these organelles, Rab38 showed a
distribution pattern similar to resident ER protein, BiP/GRP78. Rab38
seemed to show no co-localization with the Golgi and TGN, which had a
tubular distribution pattern with juxtanuclear location. The endosomal
marker proteins, EEA1 and Lamp-1, showed a granular distribution
pattern. Although EEA1 showed minor co-localization with Rab38, Lamp-1
did not show any co-localization. Surfactant apoproteins, SP-A and
SP-B, showed granular distribution in the cytoplasm. Although SP-A did
not seem to co-localize with Rab38, SP-B showed partial
co-localization. Thus, Rab38 seemed to distribute with a pattern most
like the ER among major subcellular organelles examined in this study.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Comparison of the primary structure shows the greatest
similarity between this protein and members of the rab
family. The Rab family is the largest among the small G protein
superfamily that contains more than 50 members. Rab family members are
strictly localized to defined intracellular organelles and play
prominent roles in intracellular trafficking2,4,10-13
. No
single cell expresses all of the rab family members.
However, there are many Rab proteins whose expressions are ubiquitous
over a variety of tissues and cells, such as Rab 1, 2, 4, 5, 6–14, 18,
20, 22, 24, 28, and 30.2
Expression of some Rab proteins
are highly regulated depending on cell type (epithelial, mesenchymal,
or endothelial) and specific phenotype (differentiation, activation, or
polarization). These proteins include Rab 3, 15, 17, 19, 23, 25, 26,
and 27.2
Few reports mention expression of small numbers
of particular rab small G proteins in the lung. Although the
number of rab family members exceeds more than 30, there are
only a few members for which the molecular mechanism of vesicular
transport has been clarified. The molecular mechanism for Rab3A have
been best elucidated. Rab3A is exclusively expressed in neural tissues,
especially in presynaptic vesicles.14
There are several
regulator proteins for the rab
protein,1,2,4,15,16
including rab GDP
dissociation inhibitors (rab GDI), the rab
GDP dissociation stimulator (rab GDS), the
rab guanine nucleotide exchange protein
(rab GEP), and the rab GTPase activating
protein (rab GAP). Moreover, there are target
proteins that interfere with rab proteins. In the case of
rab 3A, Rabphillin3A is a target protein whose cDNA has been cloned and
whose recombinant protein has been made.17,18
Although many Rab proteins are expressed ubiquitously, some Rab
proteins are restricted to specific tissues and cells. Distribution of
a novel Rab protein in defined tissues and cells can provide unique
information about the role of the protein. Indeed, there are some Rab
proteins whose roles were initially identified in this way, including
Rab310,14
and Rab17.12,13
Examination of the
distribution of a novel Rab protein requires specific antibody with
relatively high affinity. In this study, we made a rabbit anti-rat
Rab38 polyclonal antibody using a C-terminal amino acid sequence. The
C-terminal sequence of small G proteins is the hypervariable region
containing the most prominent structural differences in primary
structure. It is customary to choose this region to synthesize
polypeptides for specific immunogens.19
Although there are
several bands in the Western blot, the most prominent band at 26 to 27
kd seemed to have reacted with the native and recombinant Rab38 (Figure 2)
. The native Rab38 was expressed in whole lung and alveolar type II
cells but not in alveolar macrophages by Western blot. Most Rab38 in
alveolar type II cells was present in the Triton X-114-extracted phase,
and only a trace amount was present in the water-soluble phase. This
strongly suggests that most of the native Rab38 present in alveolar
type II cells has significant hydrophobic character, most likely
attributable to prenylation.
Western blotting of subcellular fractions derived from isolated
alveolar type II cells revealed that the Rab38 protein was present in
both vesicle and cytosol fractions (Figure 6)
. The Rab38 was most
prominent in the heavy vesicle fractions. This result suggests that the
Rab protein is present both in a vesicle membrane-bound form and in a
cytosol-soluble form. Rab proteins cycle between GDP- and the GTP-bound
forms. GTP-bound Rab proteins are most often membrane-associated
whereas the GDP-bound proteins are soluble.20
The Rab38
was predominantly found in the heavy vesicles rather than in the light
vesicles, and was absent from lamellar body-enriched fraction. Lamellar
bodies, characteristic structures of alveolar type II cells, are known
to store lung surfactant components, including phospholipids, neutral
lipids, and surfactant apoproteins and undergo regulated
secretion.9,21
Although this Rab38 is selectively
expressed in the surfactant-producing alveolar type II cells, it does
not seem to be involved in the exocytosis of lamellar bodies.
Confocal laser microscopic analysis of immunofluorescence cytochemistry
of isolated and cultured rat alveolar type II cells showed that the
distribution pattern of the Rab38 matched that of ER but not Golgi,
TGN, or endosomes. However, care must be taken to conclude that the Rab
protein co-localizes with ER, because the broad distribution pattern is
not specific only for the ER. As already discussed, Rab proteins
exhibit both membrane and soluble forms. This property was confirmed
for Rab38 in Figure 6
, in which the protein was found in the cytosol
fraction as well as membrane fractions. Rab38 did not seem to
co-localize with SP-A and SP-B. But, this does not necessarily exclude
the possibility that the Rab38 participates in specific steps in the
transport of surfactant components. Because SP-A and SP-B are highly
enriched in LB and also found throughout the secretory
pathway,22
the co-localization of these proteins with
Rab38 is unlikely to be extensive.
The Rab38 was exclusively expressed in alveolar corner cells and
bronchial epithelial cells in immunohistochemistry and in
situ hybridization (Figures 4 and 5)
. A number of alveolar corner
cells were positively stained for Rab38, and most of these cells were
identified as alveolar type II cells based on their characteristic
alveolar position. Among bronchial cells the Rab protein and mRNA was
predominantly expressed in terminal bronchial cells, although some
large bronchial airway epithelial cells also showed positive staining.
In rat lungs, Clara cells are bronchial epithelial cells accounting for
>80% of the cells in the terminal airway.23
Therefore,
most of the terminal airway epithelial cells staining for Rab38 are
likely to be Clara cells. It is noteworthy that Clara cells synthesize
many components of the lung surfactant, but do not contain lamellar
bodies, the characteristic lipid-containing secretory granules present
in alveolar type II cells.21,24-26
Clara cells do not
express surfactant protein C (SP-C), an extraordinarily hydrophobic
protein that is a distinct marker of alveolar type II
cells.27
Because Rab38 is not present in lamellar
body fractions and is present in Clara cells that have no lamellar
bodies, it is likely that this protein is not required for lamellar
body secretion. Many Rab proteins mediate very restricted steps in
vesicle trafficking and it is possible that Rab38 plays a role with
respect to the surfactant system. Comparison of protein and lipid
sorting in alveolar type II cells demonstrates that surfactant
components have different and independent trafficking routes in
alveolar type II cells.20
Although the precise role of Rab38 still remains to be elucidated, this study clearly demonstrates the restricted expression of a novel Rab protein (Rab38) to alveolar type II cells and terminal bronchial epithelial cells in the lung. This restricted expression of Rab38 strongly implies a unique role for the protein related to the surfactant system.
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Acknowledgements |
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Footnotes |
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Supported by a Grant-in-Aid for Scientific Research (C) (2) 08670685 and 10670566, and (A) (2) 08557042, The Ministry of Education, Science, Sports, and Culture, Japan.
Accepted for publication January 23, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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