Murine mCLCA6 Is an Integral Apical Membrane Protein of Non-goblet Cell Enterocytes and Co ...

15 Pages·2010·0.38 MB·English

Volume56(5):495–509,2008
JournalofHistochemistry&Cytochemistry
http://www.jhc.org
ARTICLE
Murine mCLCA6 Is an Integral Apical Membrane Protein of
Non-goblet Cell Enterocytes and Co-localizes With the Cystic
Fibrosis Transmembrane Conductance Regulator
Melanie K. Bothe, Josephine Braun, Lars Mundhenk, and Achim D. Gruber
DepartmentofVeterinaryPathology,FacultyofVeterinaryMedicine,FreieUniversita¨tBerlin,Berlin,Germany
y
r
t
s
i
m SUMMARY The CLCAfamilyofproteins consistsofa growing numberofstructurallyand
e functionally diverse members with distinct expression patterns in different tissues. Several
h CLCAhomologshavebeenimplicatedindiseaseswithsecretorydysfunctionsintherespiratory
c
andintestinaltracts.Herewepresentbiochemicalproteincharacterizationanddetailsonthe
o
t cellular and subcellular expression pattern of the murine mCLCA6 using specific antibodies
y
C directed against the amino- and carboxy-terminal cleavage products of mCLCA6. Computa-
tionalandbiochemicalcharacterizationsrevealedproteinprocessingandstructuralelements
&
shared with hCLCA2 including anchorage in the apical cell membrane by a transmembrane KEY WORDS
y domain in the carboxy-terminal subunit. A systematic light- and electron-microscopic calcium-activatedchloride
r immunolocalization found mCLCA6 to be associated with the microvilli of non-goblet cell channel
t
s enterocytes in the murine small and large intestine but in no other tissues. The expression mCLCA6
i
m patternwasconfirmedbyquantitativeRT-PCRfollowinglaser-capturemicrodissectionofrel- non-gobletcellenterocytes
e evanttissues.ConfocallaserscanningmicroscopycolocalizedthemCLCA6proteinwiththe mouse
h cystic fibrosis transmembrane conductance regulator CFTR at the apical surface of colonic intestine
c
o cryptcells.Togetherwithpreviouslypublishedfunctionaldata,theresultssupportadirect cysticfibrosis
t orindirectroleofmCLCA6intransepithelialanionconductanceinthemouseintestine. CFTR
s
i (JHistochemCytochem56:495–509,2008) membraneprotein
H
f
o
l THE CLCA GENE FAMILY is a heterogeneous multigene etal.2006).Todate,theCLCAgenefamilycomprises
a family with cell-type-specific expression patterns and 18 members known in eight mammalian species with
n
diverse protein structures and processing of different six murine and four human homologs reported
r
u
familymembers(LoewenandForsyth2005).Theterm (Cunningham et al. 1995; Gandhi et al. 1998; Gruber
o
CLCAhadinitiallybeencoinedduetoanovelcalcium- et al. 1998a; Gruber and Pauli 1999; Komiya et al.
J
e activated chloride current observed following heterol- 1999; Lee et al. 1999; Romio et al. 1999; Elble et al.
h ogous expression of several CLCA proteins in various 2002;LeverkoehneandGruber2002;Evansetal.2004;
T
celllines,implyingafunctionsuchascalcium-activated Antonetal.2005;Al-Jumailyetal.2007).Thehuman
chloridechannels.However,severalotherfunctionshave andmurineCLCAproteinsareofconsiderableinterest
been proposed and include adhesion molecule function based on their role in diseases with secretory dysfunc-
(Abdel-Ghanyetal.2003),bicarbonatechannelactivity tionincludingasthma(Nakanishietal.2001;Hoshino
(Thevenod et al. 2003), modulation of other channel et al. 2002; Range et al. 2007). Human hCLCA1 and
proteins (Greenwood et al. 2002; Loewen et al. 2002), hCLCA4 and murine mCLCA2 and mCLCA3 have
inhibition of tumor growth by promoting apoptosis also been proposed to modulate the basic chloride se-
(ElbleandPauli2001),andproteaseactivity(Pawlowski cretorydefectandthediseasephenotypeincysticfibro-
sis(CF)patientsandmousemodelsofCF(Ritzkaetal.
2004; Leverkoehneetal. 2006; Younget al. 2007).
Correspondenceto:AchimD.Gruber,DepartmentofVeterinary Murine mCLCA6 has recently been discovered as
Pathology,FreieUniversita¨tBerlin,Robert-von-Ostertag-Strasse15, the murine ortholog of human hCLCA4 (Evans et al.
14163Berlin,Germany.E-mail:[email protected]
2004).Bothproteinsarethoughttobeexpressedinthe
Received for publication December 16, 2007; accepted January
30,2008[DOI:10.1369/jhc.2008.950592]. intestine(Agneletal.1999;Evansetal.2004),andthe
CTheHistochemicalSociety,Inc. 0022-1554/08/$3.30 495
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496 Bothe, Braun, Mundhenk, Gruber
hCLCA1/hCLCA4 gene locus has been shown to play tivities, the anti-N-terminal antibody am6-N-1a and
a role as modulator of the basic intestinal chloride se- the anti-carboxy-terminal antibody am6-C-1b were af-
cretorydefectinCF(Ritzkaetal.2004).However,the finityimmunopurifiedusingthecorrespondingpeptides
cell type expressing these proteins and their subcellu- coupledtoanEAH–Sepharosecolumn.Immunopurified
larlocationareasyetunknown.Also,thefunctionand antisera were designated am6-N-1ap and am6-C-1bp
biomedical significance of the mCLCA6 and hCLCA4 (Figure1A).
proteins have not yet been established in detail. Of
particularinterestfordeterminingtheirfunctionwould TransientTransfectionofHEK293Cells
be the establishment of their protein structures and
HEK293cellsweregrownonsix-wellplatesinDMEM
association with the plasma membrane. Specifically,
containing 10% heat-inactivated fetal calf serum, 2%
it seems critical to establish whether mCLCA6 is an
glutamate, and 1% penicillin/streptomycin (culture
entirely secreted protein like its homolog mCLCA3
medium) at 37C in the presence of 5% CO . If not
(Mundhenketal.2006)orwhetheritisanchoredtothe 2
mentioned otherwise, cells were washed between me-
plasma membrane, similar to hCLCA2 (Elble et al.
diumchangeswithprewarmedPBS.HEK293cellswere
y 2006).Moreover,basedontheintestinalexpressionof
r transfected with pcDNA3.1 plasmid containing the
t both mCLCA3 and mCLCA6, it is tempting to spec-
s mCLCA6openreadingframe(Evansetal.2004)using
i ulate that mCLCA6 may be able to compensate for a
m Lipofectamine 2000 (Invitrogen; Karlsruhe, Germany)
loss of mCLCA3 function in mCLCA3 knockout mice
e according to the manufacturer’s protocol. Cells trans-
h as these mice do not display any obvious spontaneous fected with pcDNA3.1-vector alone served as negative
c pathology(Robichaud et al. 2005).
controls (mock transfected).
o
Herewepresentdetailedstudiesonthecellularpro-
t
y cessing, membrane association, and cellular and ultra-
C ImmunoblotAnalyses
structurallocalizationofthemCLCA6protein.Results
& suggest that mCLCA6 occupies an epithelial niche dis- Forty eight hr after transfection, HEK293 cells were
washedthreetimeswithPBSfollowedbyincubationin
tinct from and complementary to that of mCLCA3 in
y
DMEM supplemented with 2% glutamate for 6 hr.
r the murine intestine.
st Medium was removed, and cells were lysed on ice in
mi 100mlstandardlysisbuffer(25mMTris–HCl,pH8.0,
Materials and Methods
50 mM NaCl, 0.5% DOC, 0.5% Triton X-100) and
e
h BioinformaticsandAntibodyGeneration supplemented with a mixture of protease inhibitors
c (1 mM PMSF, 1 mg/ml pepstatin, 1 mg/ml aprotinin,
o Computational analysis of the mCLCA6 amino acid
t (aa) sequence (GenBank accession #AAS86332) was 5 mg/ml antipain, 5 mg/ml leupeptin, 100 mg/ml
s
Hi performed using the SignalP 3.0 (Nielson et al. 1997), tmryepdsiuinm–cshaymmpoltersywpseirneisnphuibnitaotr)1.0T0o0r3emgofvoera5nymcinellast,
Kyte–Doolittle (Kyte and Doolittle 1982), SOSUI
of (Hirokawa et al. 1998), HMMtop (Tusnady and 4C, the pellet was discarded, and the remaining
supernatant was spun at 10,000 3 g for 15 min at
Simon2001),TMPRED(Hofmann1993),DAS(Cserzo
l
a et al. 1997), and PSORT II (Nakai and Horton 1999) 4C. This second supernatant was ethanol precipitated
n overnight at 220C, and pellets were resuspended in
algorithms. Peptide sequences of high predicted immu-
ur 100 ml of standard lysis buffer. Tissue samples were
nogenicity were selected from the mCLCA6 open
o lysed in standard lysis buffer using a Precellys 24 ho-
reading frame using computer-aided antigenicity anal-
J
mogenizer (Peqlab Biotechnologie GmbH; Erlangen,
e yses. Four oligopeptides were synthesized, two located
h in the predicted N-terminal cleavage product of Germany). Medium and lysate samples were boiled in
T mCLCA6 (am6-N-1 corresponding to aa 85–98, 3-foldSDS-PAGELaemmlibuffercontaining150mM
WKDSPQYRRPKQES, and am6-N-2 corresponding DTT, each split into two, and separated by 10%
SDS-PAGE. Immunoblot analysis was performed as
to aa 106–119, VAPPTVEGRDEPYT) and two in the
predictedcarboxy-terminalcleavageproduct(am6-C-1 described (Leverkoehne and Gruber 2002) with am6-
N-1a andam6-C-1b as primaryantibodies (1:500).
correspondingtoaa803–818,DNFNNSPRVDTTNLT,
andam6-C-2correspondingtoaa870–882,QAEPDP-
DESPSSS). The four oligopeptides were coupled to AcidRelease
keyhole limpet hemocyanin (KLH) and used for stan- Forty eight hr after transfection, HEK293 cells trans-
dard immunization of two rabbits each. Preimmune fected with mCLCA6 grown on a 60-mm culture dish
sera were collected before immunization and used as werewashedthreetimeswithprewarmedPBSandsplit
controlsinallimmunodetectionexperiments.Theeight into two equal volumes. Release of non-covalently
antisera were designated am6-N-1a/am6-N-1b, am6- boundproteinbytreatmentwithpH2.5wasperformed
N-2a/am6-N-2b,am6-C-1a/am6-C-1b,andam6-C-2a/ as described earlier (Gibson et al. 2005). In brief, cells
am6-C-2b. Following initial testings of antiserum reac- werepelletedbybriefcentrifugationandresuspendedin
Downloaded from jhc.sagepub.com by guest on May 2, 2016
Processing and Expression Pattern of mCLCA6 497
either PBS, pH 7.5, or 0.9% NaCl equilibrated to pH
2.5 with acetic acid. Cells were rotated end-over-end
for 20 min at 4C and then collected by brief cen-
trifugation. Supernatants were spun at 40,000 3 g
for 30 min at 4C and then subjected overnight to eth-
anol precipitation. Precipitates were resuspended in
20 ml standard lysis buffer and boiled in 3-fold SDS-
PAGE Laemmli buffer containing 150 mM DTT. Im-
munoblotanalysiswasperformedasdescribedabove.
EndoglycosidaseTreatment
Incubation of intestinal lysate, cell lysate, or superna-
tantwithendoHorPNGaseF(NewEnglandBiolabs;
Frankfurt, Germany) prior to SDS-PAGE analysis was
y
r performed according to the manufacturer’s protocol.
t
s Briefly, an aliquot of each sample was incubated with
mi 50U/mlendoHor25U/mlPNGaseF,respectively,for
e 1 hr.Onesamplewas left untreatedas anegativecon-
h trol ineach experiment.
c
o
t RT-PCRAssayforDetectionofthemCLCA6
y
C SpliceVariant
& Total RNA was extracted from HEK293 cells trans-
fectedwithmCLCA6asdescribedabove.Primersused
y
for RT-PCR were designed using the Beacon Designer
r
st 3.0 software (Premier Biosoft International; Palo Alto,
mi CA). Primer sequences for mCLCA6 were 5¶-AAT-
CAAGGCTGCAATTCAGGTAT-3¶ (forward) and 5¶-
e
h TGTCTTTCACCTCATCAATACAGT-¶3 (reverse)
c with a predicted amplicon size of 122 bp. To dis-
o Figure 1 Generation of antibodies against mCLCA6. (A) Antisera criminate between mCLCA6 and its splice variant
st wereraisedagainstsyntheticpeptidescorrespondingtoaminoacid
i (aa) sequences within the amino-(N)-terminal cleavage product mCLCA6sv (Evans et al. 2004), an additional primer
H (am6-N-1a)andthecarboxy-(C)-terminalcleavageproduct(am6-C- pairwasdesignedasfollows:5¶-TACAGATGAAGCC-
f 1b). Hydrophobicity analyses predicted a signal sequence (ss) at CAGAACAAT-3¶(forward)and5¶-TCCAGGTGACAA-
o theamino-terminusandatransmembranedomain(TM)attheend GAAAGAATGT-3¶ (reverse), spanning the portion of
ofthecarboxy-terminalcleavageproduct.Dotsindicateconsensus
al sitesforN-linkedglycosylation.Arrowshowsputativecleavagesite. exon10absentinmCLCA6withapredictedamplicon
n (B) When lysates of heterologously mCLCA6-transfected HEK293 size of 191 bp when amplifying mCLCA6 and 126 bp
ur cells were immunoblotted with am6-N-1a, two specific proteins when amplifying mCLCA6sv. The optimized 25-ml
o were detected of 125 and 110 kDa in size, respectively (Lane 1). reaction mix contained 2.5 ml 10X buffer, 300 nM of
J Lysates of intestinal tissues contained only the 110-kDa protein
e (Lane3).Preimmuneserumfromthesamerabbitfailedtodetectany each primer, 100 mM probe, 5.5 mM MgCl2, and
h proteininthesamelysates(Lanes7and8).Inadditiontospecific RNasefreewater(Roth;Karlsruhe,Germany)toafinal
T detection of mCLCA6, the unpurified immune serum am6-N-1a volume of 25 ml. Thermal profile used was 2 min at
reacted unspecifically with an additional protein of z115 kDa in
transfected HEK293 cells (Lane 2) and two proteins of z70 and 95C followed by 34 cycles of 50 sec at 95C, 40 sec at
60 kDa (crosses) in the intestinal lysate (Lane 4). However, these 57C,and20secat72Cwithatimeincrementof2sec
proteinswerenotdetectedbytheimmunopurifiedserumam6-N- andfinally10 min at 72C.
1ap. (C) Anti-carboxy-terminal antibody am6-C-1b also detected
the 125-kDa primary translation product only in overexpressing
HEK293cells(Lane1),notinintestinaltissuelysates(Lanes4and5). AnimalsandTissueProcessing
Thevariousproteinspeciesofz35kDawerereducedbydeglyco-
sylationwithPNGaseFtoasingleproteinofz30kDainsize(Lane2, Five male and five female 12-week-old C57BL/6 mice
m6D5celllysate,deglycosylated;Lane5,int.D5intestinallysate, were sacrificed by cervical dislocation in accordance
deglycosylated). Vector alone transfected cells served as controls with national guidelines. The following tissue samples
(Lane3,mock).Allantibodiesweredilutedat1:500.Crosses5un- were collected and immersion fixed in 4% neutral-
specificbackgroundstaining.
buffered formalin: liver, gallbladder, spleen, kidneys,
urinary bladder, pancreas, oral cavity, parotid and
sublingual salivary glands, esophagus, stomach, duo-
denum, jejunum, ileum, cecum, colon, rectum, nasal
Downloaded from jhc.sagepub.com by guest on May 2, 2016
498 Bothe, Braun, Mundhenk, Gruber
cavity, trachea, lung, heart, adrenal glands, thyroid staining withhematoxylindissolvedindiethylpyrocar-
glands, ovaries, uterus, mammary glands, testes, epi- bonate (DEPC)-treated water for 2 min, rinsing in
didymides,maleaccessorysexglands(seminalvesicular DEPC-treated water for 2 min, and staining with 1%
glands, coagulating glands, ampullary glands, bulbo- eosin dissolved in DEPC-treated water for 30 sec. Sec-
urethral glands), lymph nodes, brain (cortex, cerebel- tions were dehydrated in ascending graded ethanol
lum, brainstem, medulla), eyes, Harderian gland, skin, (30%, 50%, 70%, 100%), air dried at room temper-
adiposetissue,skeletalmuscle,and bone. ature for 5 min, and stored at 280C. Total RNA was
isolated from four different locations: luminal third,
medial third, and basal third of the jejunal villi and
Immunohistochemistry
jejunal smooth muscle. A total area of 1.5 3 106 mm2
Immunohistochemical (IHC) analyses were performed
cellsfromeachregionofinterestwasseparatedbylaser
as described previously (Leverkoehne and Gruber
microdissection and laser-pressure catapulted into the
2002). Briefly, fixed tissue samples were embedded in
caps of 0.5-ml Eppendorf tubes containing 60 ml RLT
paraffin, cut at 4-mm thickness, and mounted on ad-
buffer (RNeasy Mini Kit; Qiagen). The tubes were
hesive glass slides for IHC. Consecutive tissue sections
y closed,inverted,brieflycentrifuged,andfilledto350ml
r were routinely stained with hematoxylin and eosin
t with RLT buffer. Total RNA was extracted and pu-
s (HE)forhistologicalexamination.ForIHC,theavidin–
i rifiedaccordingtothemanufacturer’sprotocolinclud-
m biotin–peroxidase complex (ABC) method (Vectastain
ing a digestion with RNase free DNase I (Qiagen).
e Elite ABC Kit; Vector Laboratories, Burlingame, CA)
h Eight ml total RNA was reverse transcribed into
was applied with antigen retrieval by 15-min micro-
c 21 ml cDNA (SuperScript III First-Strand Synthesis
o wave heating (750 W) in 10 mM citric acid, pH 6.0,
System; Invitrogen) using random hexamer primers in
t supplemented with 0.05% Triton X-100. Slides were
y the presence of RNase Out following the manufac-
C incubatedwiththepurifiedseraam6-N-1apandam6-
turer’s protocol.
C-1bp or the respective preimmune sera (dilutions
&
rangingfrom1:100to1:40,000)inPBScontaining1%
QuantitativeReal-timePolymeraseChainReaction
y BSA in coverslides at 4C overnight followed by bio-
r Primers and probes used for quantitative real-time
t tinylatedsecondarygoatanti-rabbitantibodies(1:200)
s polymerasechainreaction(qPCR)weredesignedusing
i andhorseradishperoxidase-coupledstreptavidin.DAB
m the Beacon Designer 3.0 software (Premier Biosoft
was used as substrate for color development. Slides
e International; Palo Alto, CA). Primer sequences for
h were counterstained with hematoxylin, dehydrated
mCLCA6 were described above. The corresponding
c through ascending graded ethanol, cleared in xylene,
o Taqman probe sequence was 5¶-FAM-TGGCTG-
and coverslipped.
t TGCTGTCCTCACCATCAC-TAMRA-3¶. Primer
s
Hi pairs and probes were designed to specifically dis-
Laser-captureMicrodissection,RNAIsolation,and criminate among all known mCLCA homologs and
of ReverseTranscription to encompass an intron to exclude amplification of
For laser-capture microdissection, isolation of total contaminating genomic DNA. No cross-reactions
l
a RNA and quantitative real-time polymerase chain re- could be detected when linearized cloned cDNA sam-
n
action (qRT-PCR) tissue samples were shock frozen in ples of mCLCA1, mCLCA2, mCLCA3, and mCLCA5
r
u liquid nitrogen and kept at 285C until further use. or PCR-derived fragments of mCLCA4 were used as
o
Total RNA was extracted from whole tissue samples templates containing 107 to 109 copies. To correct for
J
e usingtheTrizolmethod(Invitrogen)andpurifiedusing variations in RNA amounts and cDNA synthesis ef-
h the RNeasy Mini Kit (Qiagen; Hilden, Germany) ficacy, a segment of the cDNA-encoding murine
T according to the manufacturer’s protocol including a elongation factor 1a (EF1a) was amplified in parallel
digestionwithRNase-freeDNaseI(Qiagen).Fivehun- in all experiments as internal housekeeping gene stan-
dred ng total RNA was reverse transcribed into 20 ml dard (Gruber and Levine 1997). Primer sequences for
cDNA (Omniscript; Qiagen) using random hexamer EF1a were 5¶-CAAAAACGACCCACCAATGG-3¶
primers (Promega; Mannheim, Germany) in the pres- (forward) and 5¶-GGCCTGGATGGTTCAGGATA-3¶
ence of RNase Out (Invitrogen) according to the man- (reverse) with a predicted amplicon size of 68 bp. The
ufacturer’s protocols. corresponding Taqman probe sequence was 5¶-FAM-
Frozen jejunum samples from three mice were cut AGCAGCTGGCTTCACTGCTCAGGTG-TAMRA-
into multiple consecutivecross-sectionsof6-mmthick- 3¶. Gen-Bank accession numbers for mCLCA6 and
nessandmountedontoglassslidescoatedwithapoly- EF1a were AY560902 and BC003969, respectively.
ethylene naphthalate membrane (PALM membrane QRT-PCRanddataanalyseswereconductedusingthe
slides; PALM Microlaser Technologies, Martinsried, MX 3000P Quantitative PCR System and MX Pro
Germany). Sections were fixed for 2 min in 95% Software (Stratagene; La Jolla, CA). PCR reactions
ethanol at 220C and air dried for 5 min followed by were carried out in 96-well polypropylene plates
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Processing and Expression Pattern of mCLCA6 499
covered with optical caps (83 strips; Stratagene). The taining 0.5% BSA and 0.1% liquid gelatin (washing
plates contained triplicates of each cDNA sample, buffer) and 5% heat-inactivated normal goat serum.
duplicates of no-template controls, and duplicates of Purified sera am6-N-1ap and am6-C-1bp were incu-
positive controls. The optimized 25-ml reaction mix bated in washing buffer at 4C overnight in a humid
contained 12.5 ml qPCR MasterMix Plus Low ROX chamber (dilutions ranging from 1:600 to 1:12,000)
(Eurogentec; Seraing, Belgium), 300 nM of each followed by repeated washes in washing buffer and
primer, 100 mM probe, 1 ml cDNA, 5.5 mM MgCl by incubation for 1 hr with 10-nm gold particle-
2
(mCLCA6only),andRNasefreewater(Roth)toafinal conjugated goat anti-rabbit immunoglobulins (Sigma–
volume of 25ml.The thermalprofileused was 10min Aldrich;Munich,Germany)dilutedat1:40inwashing
at95Cfollowedby40cyclesof15secat95Cand1min buffer. Sections were postfixed, contrasted for 15 min
at 60C. cDNA samples were used with primers and in aqueous uranyl acetate (Merck; Darmstadt, Ger-
probesforbothmCLCA6andEF1aonthesameplate many), and washed in distilled water. Grids were air
to ensure equal amplification conditions. QRT-PCR dried and examined with a Zeiss EM 10 CR trans-
protocols were initially established and optimized mission electron microscope (Zeiss; Oberkochen, Ger-
y using 10-fold serial dilutions (ranging from 1022 to many).Negativecontrols wereincluded using immune
tr 1026ng/m l or109 to103copies of mRNA)ofpurified sera from rabbitagainstirrelevantantigensasprimary
s
i mCLCA6-pcDNA3.1 (Evans et al. 2004) or purified antibodiesthatfailedtoyieldanysignalsintherelevant
m
(QIAquickPCRPurificationKit;Qiagen)PCR-derived cell typesat dilutionsof 1:2000 or higher.
e
h fragments (1114 bp) generated from cDNA templates
c from mouse colon for EF1a. Positive controls had Co-localizationWithCFTR
o highly reproducibleamplificationefficaciesofbetween For confocal laser scanning microscopy, 10-mm tissue
t
y 95% and 105%. sectionswereprocessedasdescribedabovewithminor
C
modifications. For double staining, fresh murine
& QuantificationofTargetGeneExpression intestinal tissue samples were fixed in 3% (w/v) para-
formaldehyde for 16 hr, prior to standard paraffin
y For each sample analyzed, the mean cycle threshold
tr (Ct) value and its corresponding standard deviation embedding. Paraffin sections were cut at 5 mm and
s mounted on adhesive glass slides. Deparaffinization
i (SD) were given by the MX Pro Stratagene analysis
m and rehydration were followed by antigen retrieval by
software.Ctvaluewascalculatedbasedonthenormal-
e ized baseline corrected fluorescence (DRN). Relative boilingthesectionsin0.01Msodiumcitratebufferina
h
c geneexpressionswerecalculatedwiththeDCt-method microwaveovenat750Wforintervalsof7min,3min,
o (ratio52CtEF1a2CtGOI52DCt)(vonSmolinskietal. and 3 min. Sections were cooled for 45 min and
t endogenous peroxidase was blocked using 0.6% w/v
s 2005).Ratiosrepresentthecopynumbersofthegeneof
Hi interest (GOI) relative to the internal reference (EF1a) H2O2 in PBS containing 0.1% w/v sodium azide for
30minfollowedbyrepeatedwashesinPBSandwashes
and were thus normalized to the housekeeping gene
of EF1aasinternal standard. in PBS containing 0.5% BSA. Sections were incubated
at room temperature with the primary anti-CFTR-
l
a antibody R3195 diluted 1:50 in PBS containing 0.5%
n StatisticalAnalyses
BSAfor90min.AntibodyR3195(Doucetetal.2003)
r
u For each cDNA sample analyzed the SD of the trip-
was a kind gift of Dr. Hugo DeJonge (University of
o licates was calculated. Differences of expression levels
Rotterdam, The Netherlands). After washing in PBS
J were considered statistically significant for p values
e containing 0.5% BSA, sections were incubated with
h #0.05 ina two-group t-test. secondary FITC-labeled antibody (Sigma–Aldrich) di-
T
luted 1:80 in PBS containing 0.5% BSA for 60 min
ImmuneElectronMicroscopy and washed in PBS containing 0.5% BSA and then in
Immune electron microscopy was performed as de- PBS only. Slides were blocked in PBS containing 2%
scribed earlier (Leverkoehne and Gruber 2002). In BSA and 20% heat-inactivated normal goat serum
brief, samples of the small intestine were embedded in followed by incubation with the purified antibody
gelatin capsules filled with LR White resin containing am6-N-1apinPBS(1:100)containing1%BSAincover
LR White accelerator (Plano; Wetzlar, Germany), and slides at 4C overnight. Sections were washed in PBS/
resin was allowed to polymerize at room temperature Triton X-100 and incubated at room temperature
overnight. Ultrathin sections cut at 60–90 nm were for 1 hr with lissamine rhodamine sulfonyl chloride
collected on uncoated 180-mesh nickel grids (Pelco (LRSC)-labeled fluorescent antibody (Jackson Immu-
International; Clovis, CA). For immunolabeling, grids noresearch Europe Ltd; Newmarket, UK) diluted 1:50
were immersed upside down in drops of the respective in PBS followed by repeated washes in PBS/Triton X-
solution.AfterblockingofaldehydeswithPBScontain- 100. Sections were counterstained with To-Pro-3-
ing 50 mM glycine, sections were blocked in PBS con- iodide (Invitrogen; Breda, The Netherlands) and
Downloaded from jhc.sagepub.com by guest on May 2, 2016
500 Bothe, Braun, Mundhenk, Gruber
analyzedusingaconfocalmicroscope(TCSSP2;Leica, 125-kDaprecursormoleculeinheterologouslytransfec-
Wetzlar, Germany). FITC was excited at 495 nm, and ted cells and several poorly defined protein species of
emissionofgreenfluorescencewasdetectedat517nm. z35 kDa, most likely representing different glycovar-
LRSC was excited at 570 nm and emission of red iants of the carboxy-terminal cleavage product (Fig-
fluorescence was detected at 590 nm. Single optical ure 1C). Again, only the cleavage product, but not the
scans were repeated several times and recorded sepa- full-lengthpolypeptide,wasdetectedinthetissuelysate.
rately to be subsequently combined as an average of Thepresenceofdifferentglycovariantswasconfirmedby
three images. reduction of the various low molecular mass bands to
a single band of z30 kDa in size (Figure 1C, Lane 2).
Immunopurification of the am6-C-1b antiserum re-
Results
sulted in a complete loss of its reactivity on Western
AntibodyGeneration
blots, probably because of strong immobilization of
AhallmarkofvirtuallyallCLCAproteinsdiscoveredso high affinity antibodies on the purification columnthat
far is a posttranslational cleavage of the precursor wereinsolubilizablebytheelutionbufferused.Boththe
y molecule,resultinginalargerN-terminalandasmaller additionalantibodiesam6-N-2aandam6-C-2adirected
tr carboxy-terminal cleavage product of z90–110 and against the amino- and the carboxy-terminal cleavage
s
mi 35kDa,respectively.Previousstudiesonthebiochem- products,respectively,showedareactionpatternsimilar
istry and expression patterns of other CLCA proteins toam6-N-1aandam6-C-1b(datanotshown).AnRT-
e
h have either used antibodies directed only against the PCRassaydesignedtodetecttheshortersplicevariantof
c N-terminal subunit (Leverkoehne and Gruber 2002; mCLCA6 (Evans et al. 2004) detected the full-length
o
Connonetal.2005;Elbleetal.2006)orepitope-tagged openreadingframeonlybutnotthesplicevariantbothin
t
y constructs (Gruber et al. 1998b; Gruber and Pauli transfectedHEK293cellsandtheintestinallysates(data
C
1999;Evansetal.2004;Gibsonetal.2005;Elbleetal. notshown).
& 2006; Mundhenk et al. 2006). To study both cleavage
products without risk of artifacts associated with epi-
y CellularProcessingandAssociationWiththe
r tope tagging, we generated specific antibodies against
st the amino- and carboxy-terminal cleavage products of PlasmaMembrane
i
m themCLCA6protein(Figure1A).Sizesoftheproteins Distinct cellular processing and associations with the
e detected by these antibodies in lysates of mCLCA6- plasma membrane have been reported for different
h transfected HEK293 cells and in lysates of mouse in- CLCA proteins (Gibson et al. 2005; Elble et al. 2006;
c
o testineareconsistentwiththegeneralmodelpreviously Mundhenk et al. 2006). To unravel which of these
t proposed for CLCA protein processing (Gruber et al. models may be true for mCLCA6, we performed
s
Hi 2002). Antibody am6-N-1a detected a faint 125-kDa computer-aided sequence analyses followed by bio-
and a 110-kDa protein in mCLCA6 overexpressing chemical assays using heterologously transfected
of HEK293 cells, corresponding to the glycosylated pre- HEK293 cells, finally verified on lysates of murine
cursor molecule and the glycosylated N-terminal intestinaltissues.TheSignalP3.0algorithmpredicteda
l
a cleavage product, respectively (Figure 1B). When ap- signal sequence atthe amino-terminus, consistent with
n
plied on intestinal tissue lysates, the same antibody the protein entering the secretory pathway. A single
r
u detected only the 110-kDa protein, most likely repre- potential transmembrane domain was predicted by
o senting the glycosylated N-terminal cleavage product. Kyte–Doolittle, SOSUI, and DAS algorithms at zaa
J
e Consistently, only the cleavage products but not the 885–911(Kyte–Doolittlezaa900,SOSUIaa885–908,
h precursor protein have previously been detected in and DAS aa 885–908), implying a cytoplasmic tail of
T
tissue lysates of other CLCA proteins (Leverkoehne nolessthan13hydrophilicaa.Incontrast,theTMPred
and Gruber 2002; Mundhenk et al. 2006). Preim- and PSORTII algorithms predicted two potential
mune serum failed to detect any proteins of relevant transmembrane regions, one at aa 436–456 and one
sizes in the lysates of transfected cells or intestinal tis- close to the carboxy-terminus between aa 889 and
sue lysates, and unspecific reactions with a few other 911 (TMPred: aa 889–911 and PSORT/PSORT II: aa
proteins were eliminated when using the immuno- 889–905). Thus, computer-aided predictions of po-
purified antiserum am6-N-1ap (Figure 1B, compare tential transmembrane domains for mCLCA6 resulted
Lanes2and3withLanes5and6).Unfortunately,the inaconsistentlypredictedtransmembraneregionclose
faint 125-kDa band of the precursor was poorly de- to the carboxy terminus and a possible additional
tected by the immunopurified serum; therefore, the transmembrane region in the predicted N-terminal
following immunoblot experiments were performed cleavage product, predicted by two of the five algo-
withthenon-immunopurifiedantibodyam6-N-1a.An- rithms used.
tibody am6-C-1b that was raised against the carboxy- To corroborate these predictions biochemically, the
terminal cleavage product also faintly detected the cell lysate and conditioned supernatant of transiently
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Processing and Expression Pattern of mCLCA6 501
mCLCA6 transfected HEK293 cells were immuno- tectedwhenusingtherespectiveantibodies(Figures1B
blotted with antibodies am6-N-1a and am6-C-1b. A and1C).Anacid-releaseexperimentwasperformedas
precursor molecule of z125 kDa was detected in the described (Gibson et al. 2005) to establish whether
cell lysate by antibody am6-N-1a (Figure 2A). This mCLCA6,likehCLCA1andhCLCA2,canbereleased
precursor molecule was never detected in the superna- from the plasma membrane by mild acidic hydrolysis.
tant. In contrast, the N-terminal cleavage product of Similar to hCLCA1 and hCLCA2 (Gibson et al. 2005;
z110kDawaspresentbothinthecelllysateandinthe Elble et al. 2006), the N-terminal cleavage product of
supernatant 6 hr after replacement of the medium mCLCA6 was released into the medium following
(Figure 2A). However, the carboxy-terminal cleavage treatmentwithmildacid,whereasthecarboxy-terminal
product of z35 kDa in size was detected in the cell cleavage product was not (Figure 2B). We then ex-
lysate only but not in the supernatant (Figure 2A). In amined the glycosylation patterns of the mCLCA6
the intestinal lysate, both cleavage products were de- proteins before and after cleavage of the precursor
moleculetofurthercharacterizethecellularprocessing
andtolocalizethecellularcompartmentwhereprotein
y cleavage occurs. Cell lysates and supernatants were
r
t treated with endoglycosidases endo H or PNGase F
s
mi to identify the extent and kind of glycosylation. The
125-kDa precursor molecule of mCLCA6 was sensi-
e
h tive to both Endo H and PNGase F (Figure 3A), in-
c dicatedbyitsdisappearanceinLanes2and3,wherethe
o
deglycosylatedproductislikelyobscuredbylowermo-
t
y
lecular mass species. Thus, the precursor molecule
C
shows an immature glycoprotein pattern of the high-
&
mannose type. In contrast, the N-terminal subunit
y found in the cell lysate and in the supernatant and the
r carboxy-terminal cleavage product found in the cell
t
s
i lysate only were fully resistant to endo H but sensitive
m
to PNGase F (Figure 3). The N-terminal cleavage
e product was reduced in size to a protein of z90 kDa,
h
c whereas the carboxy-terminal cleavage product was
o reduced to an z30-kDa product. Glycosylation pat-
t
s terns of both cleavage products detected here are
i
H consistent with the six consensus glycosylation sites
predicted in the N-terminal cleavage product (Fig-
f
o ure 1A) and two glycosylation sites predicted in the
l carboxy-terminalcleavageproductearlier(Evansetal.
a
n 2004). In conclusion, results of the deglycosylation
r experiments indicated that cleavage of the precursor
u
o molecule most likely occurs after its exit from the en-
J doplasmic reticulum and that only the two cleavage
e products havepassedthe Golgiapparatus.
h
Figure 2 Cellular processing of mCLCA6. (A) Immunoblotting
T
analyses identified both the amino- and the carboxy-terminal
cleavage productsof mCLCA6 inthecell lysate (L, Lanes 1and 5) TissueExpressionPatternofthemCLCA6Proteinand
but only the N-terminal subunit in the conditioned medium (M,
QuantitationofmRNAExpressionLevels
Lane 2). HEK293 cells transiently transfected with mCLCA6 were
analyzedbyimmunoblottingusingantibodiesam6-N-1a(1:500)or When a systematic IHC analysis was performed in-
am6-C-1b (1:500), respectively. For analysis of the conditioned cludingvirtuallyallorgansandtissuesofatotaloffive
medium, a medium change was performed 42 hr after transfec-
C57BL/6 mice, the mCLCA6 protein was exclusively
tion followed by harvest and analysis of the medium 6 hr later.
Vectoralonetransfectedcellsservedascontrols(mock,Lanes7and detected in the intestine (Figure 4) but in no other or-
8). Crosses 5 unspecific background staining. (B)When cells were gan or tissue. Only weak staining was observed in the
incubated at pH 2.5, only the N-terminal cleavage product of
duodenum, with an increasing intensity along the
mCLCA6waspartiallyreleasedintothesupernatant.Fortyeighthr
aftertransfection,HEK293cellstransientlytransfectedwithmCLCA6 proximal-to-distal axis of the intestinal tract. Similar
wereincubatedwithNaCl,pH2.5(pHadjustedwithaceticacid)to staining patterns were obtained when the purified an-
release non-covalently associated proteins from the plasma mem- tibodies against the amino- or the carboxy-terminal
brane.TreatmentwithPBSservedascontrol.Antibodiesam6-N-1a
and am6-C-1b were diluted at 1:500. Crosses 5 unspecific back- cleavage products of mCLCA6 were used, whereas no
groundstaining. staining was observed with the respective preimmune
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502 Bothe, Braun, Mundhenk, Gruber
y
r
t
s
i
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c
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C
&
y
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t
s
i Figure4 mCLCA6islocatedattheapicalsurfaceofnon-gobletcell
H Figure3 Glycosylationpatternsoftheamino-andcarboxy-terminal enterocytes. Immunohistochemical analyses localized mCLCA6 at
cleavage products. (A) Deglycosylation experiments identified the theapicalsurfaceofnon-gobletcellenterocytesatthevilloustips
of N-terminalcleavageproductofmCLCA6asacomplexglycosylated of the jejunum (A,B) and in the crypts of the colon (D,E). Goblet
matureproteinformthathaspassedtheGolgiapparatus.Aliquots cellsstainednegative(arrowsinB).Tissuesectionswereincubated
al of cell lysate (L) and supernatant (M) of HEK293 cells transiently with am6-N-1ap (diluted 1:20,000 in A,B,D,E) or the respective
n transfectedwithmCLCA6weretreatedwithendoH(H),PNGaseF preimmune serum (diluted 1:20,000 in C,F). Color was developed
r (F),ornottreated(2).TheN-terminalcleavageproductofmCLCA6 using DAB as substrate (brown) with hematoxylin as counterstain
u wasresistanttoendoH(Lane2)butwasreducedinsizetoz90kDa (blue).Bar520mm.
o when treated with PNGase F (Lane 3). Crosses 5 unspecific back-
J groundstaining.(B)Thecarboxy-terminalcleavageproductinthe
e celllysate(L)wasalsoresistanttoendoH(Lane2)andwasreduced
h in size to appear 30 kDa when treated with PNGase F (Lane 3). Higher magnifications revealed that the mCLCA6
T Crosses5unspecificbackgroundstaining. protein was present only at the apical membrane of
enterocytes. In the small intestine, it was expressed
onlybynon-gobletcellenterocytesintheluminalthird
sera. Both the additional antibodies am6-N-2a and of the villi, whereas it was present along the entire
am6-C-2adirectedagainsttheamino-andthecarboxy- crypt–lumen axis in the large intestine, with no stain-
terminal cleavage product showed a detection pattern ing of goblet cells or mucus along the entire intestinal
similartoam6-N-1aandam6-C-1b(datanotshown). tract. Nostaining was observed in the stomach.
Interestingly, antisera raised against the amino- and The mCLCA6 expression pattern was verified on
carboxy-terminal cleavage products labeled the same the mRNA level. Real-time quantitative reverse tran-
structures in all tissues examined with similar signal scription–polymerase chain reaction (qRT-PCR) using
strength. Affinity-immunopurified antibodies am6- whole-tissue mRNA extracts confirmed increasing
N-1ap and am6-C-1bp were used in all subsequent mCLCA6 mRNA expression from the duodenum to
IHC experiments because of their reduced back- theileumanddecreasinglevelstowardthecolon.Max-
ground staining. imal average expression levels peaked at 0.2 copies of
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Processing and Expression Pattern of mCLCA6 503
mCLCA6 mRNA per mRNA copy of the housekeep- be associated with the apical microvilli of enterocytes
ing gene EF1a(Figure 5A). (Figure 6). In addition, small clusters of gold particles
Increase of mCLCA6 protein staining toward the were located underneath the terminal actin web con-
luminal third of the crypt–villus axis was verified on sistent with secretory vesicles (Figure 6B) and occa-
the mRNA level using laser-capture microdissection of sionally associated with the endoplasmic reticulum
selectedtissueareasfollowedbyqRT-PCR.Resultscon- (Figure 6C). Only for the antibody against the N-
firmed increasing relative copy numbers of mCLCA6 terminal cleavage product, considerably fewer signals
mRNAfromthe basalthird of the villus tothe luminal wereobservedintheentericlumenabovethemicrovilli
third (Figure 5B). During initial establishments of the (Figure 6A). No gold particles were detected in the
real-timePCRprotocols,nocross-reactivitywasdetected nucleus,mitochondria,orothercellularstructures,and
with the murine homologs mCLCA1, mCLCA2, no othercell typeswere labeled.
mCLCA3,mCLCA4,andmCLCA5,respectively.
Co-localizationofmCLCA6WiththeCFTRProtein
UltrastructuralLocalization UsingImmuneConfocalLaserScanningMicroscopy
y
r ImmuneelectronmicroscopicdetectionofthemCLCA6 Based on the well-established expression of the CFTR
st protein using 10-nm gold-particle-labeled secondary protein in the apical membrane of non-goblet cell
mi antibodies identified both the N-terminal and the enterocytesintheintestinalcrypts(Ameenetal.2000a)
e carboxy-terminal cleavage products of mCLCA6 to and a putative role of CLCA proteins as modulators
h of the CF phenotype (Ritzka et al. 2004; Leverkoehne
c
et al. 2006; Young et al. 2007), we hypothesized
o
t that the mCLCA6 and CFTR proteins may be coex-
y
C pressed at theapical plasma membrane of enterocytes.
IHC detection using antibody R3195 confirmed
&
expression of the CFTR protein in the apical mem-
y branes of large intestinal crypt enterocytes with no
r
t staining of goblet cells. When these signals were over-
s
i laid with the signals identifying the mCLCA6 pro-
m
tein, colocalization was clearly visualized in the apical
e
plasma membranes of crypt epithelial cells in the large
h
c intestine (Figure 7). However, only mCLCA6, but not
o CFTR, was strongly expressed at the apical plasma
t
s membrane of luminalenterocytes.
i
H
f Discussion
o
Sincetheirdiscoveryinthemid-1990s,thestructureof
l
a CLCA proteins has been the subject of considerable
n
debate. The first family member to be characterized
r
u
biochemicallywasthebovineendothelialLu-ECAM-1,
o
alias bCLCA1, which was considered a complex of
J
e five proteins of 130, 120, 90, 38, and 32 kDa in size,
h allbeingencodedbyasingleopenreadingframe(Zhu
T
etal.1991;Elbleetal.1997).Thesecondfamilymem-
ber to be cloned, the bovine Ca–CC, alias bCLCA2,
had initially been characterized as a homopolymeric
complex of two or four copies of a 38-kDa protein
(Ran and Benos 1992; Cunningham et al. 1995). A 2-
Figure5 QuantificationofmCLCA6mRNAexpressioninthemurine
or 3-fold transmembrane spanning heterodimer of a
intestine using quantitative RT-PCR. (A) Relative copy numbers of
mCLCA6 mRNA increased from the duodenum to the ileum and 90- and 40-kDa protein has been proposed for the
thereafterdecreasedtothecolon,confirmingthestainingintensi- human hCLCA1 and hCLCA2 proteins, respectively
tiesobservedusingimmunohistochemicalproteindetection.Bars5
(Gruber et al. 1998a; Gruber and Pauli 1999). Re-
standarddeviations.(B)Lasercapturemicrodissectionwasusedto
isolatedifferentsegmentsalongthecrypt–villusaxisofthejejunal cently,thelattermodelshavebeencorrectedbyfurther
villi prior toquantitative RT-PCR. Results confirmedincreasing ex- biochemical evidence, and two distinct models now
pressionlevelsinthevilloustips(comparewithFigure4A).Expres- seem to be accepted for different members of the gene
sionlevelsaregiveninratiosofgeneofinterest(GOI)relativetothe
copynumbersofthehousekeepinggeneEF1a(n55C57BL/6mice). family. One class of CLCA proteins consists of a het-
Noexpressionwasdetectedinjejunalsmoothmuscle(NoCt). erodimeric glycoprotein of z90–110 and 35-kDa
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504 Bothe, Braun, Mundhenk, Gruber
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s Figure 7 Colocalization of mCLCA6 and CFTR in apical plasma
i
H membranes of non-goblet cell enterocytes in crypts of the large
intestine.ThemCLCA6protein(red)wasvisualizedalongtheapical
f plasma membrane of non-goblet cell enterocytes both at the
o
intestinal lumen and in the crypts (A,C). In contrast, the CFTR
l protein was only detected at the apical plasma membrane of
a enterocytes in the deep crypt areas (green in A,B) where it was
n
clearlycolocalizedwiththemCLCA6protein(A,C).(A,D)Overlayof
r
u bothsignals.(A,B)CFTRstainingusingantibodyR3195diluted1:25
o withFITC-labeledsecondaryantibody.(A,C)mCLCA6stainingusing
J antibody am6-N-1ap diluted 1:100 with LRSC-labeled secondary
e antibody.Bar510mm.
h
T
subunitsencodedbyasingleopenreadingframe.They
enter the secretory pathway and are fully secreted by
the cell. Prototypes for this model are the human
Figure6 ImmuneelectronmicroscopiclocalizationofthemCLCA6 hCLCA1 (Gibson et al. 2005) and the murine gob-5,
protein. On the ultrastructural level, the mCLCA6 protein was alias mCLCA3 (Mundhenk et al. 2006). In contrast, a
localizedtothebrushborderofenterocytes(A,B)insmallclusters
second class with the human hCLCA2 as prototype is
underneaththeterminalactinweb,insmallclustersassociatedwith
the endoplasmic reticulum near the nucleus (C), and in small clearly anchored to the plasma membrane via a hy-
numbers in the lumen of the intestine (A). Tissue sections were drophobic transmembrane domain in the z35-kDa
incubatedwitham6-N-1ap(diluted1:12,000).Incubationwitham6- carboxy-terminal subunit but otherwise shares many
C-1bp (1:50) resulted in the same staining pattern except for the
properties of the secreted CLCA proteins (Elble et al.
lumen, which was negative for the carboxy-terminal cleavage
product. Incubation without primary antibody served as negative 2006).Computationalpredictionsandbiochemicalev-
control.ER,endoplasmicreticulum;N,nucleus.Bar5200nm. idenceobtainedformCLCA6inthisstudyclearlyshow
a structural homology with the model proposed for
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