\set{final}

\def\Author{Boehlke}
\def\author{boehlke}
\def\vol{10}
\def\year{2004}
\def\anum{104}
\def\pages{867-873}
\def\txt_title{Cytokeratin 12 in human ocular surface epithelia is the antigen reactive with a commercial anti-G[alpha] q antibody}
\def\txt_authors{Christopher S. Boehlke, Ching Yuan, Winston W.-Y. Kao, Andrew J. W. Huang}

\def\rcvd{21 June 2004}
\def\accept{15 November 2004}
\def\publ{16 November 2004}
\def\pdfsize{}
\def\PMID{}


\include{mvstyle.hsm}

\| External links

\def\mascot{http://www.matrixscience.com/}
\def\mowsescore{http://www.matrixscience.com/help/scoring_help.html}

\| Internal defs


\article{

\title{Cytokeratin 12 in human ocular surface epithelia is the antigen
reactive with a commercial anti-G\alpha q antibody}

\authors{Christopher S. Boehlke,\sup{1} \mailto{yuanx019@umn.edu}{Ching
Yuan},\sup{1} Winston W.-Y. Kao,\sup{2} \mailto{huang088@umn.edu}{Andrew
J. W. Huang}\sup{1}}

\institutions{\sup{1}Department of Ophthalmology, University of
Minnesota, Minneapolis, MN; \sup{2}Department of Ophthalmology,
University of Cincinnati, Cincinnati, OH}

\correspondence{Andrew J. W. Huang, MD, MPH, Department of
Ophthalmology, University of Minnesota, 420 Delaware Street SE, MMC493,
Minneapolis, MN, 55455; Phone: (612) 625-4400; FAX: (612) 626-4455;
email: huang088@umn.edu}

\abstract

\abs_purpose{In our initial attempt to identify differentiation markers
for ocular surface epithelia, we observed a unique staining pattern by a
commercial anti-G\alpha q antibody. We further isolate and characterize
the protein reactive with this anti-G\alpha q antibody in human ocular
surface epithelia.}

\abs_methods{Human donor corneoscleral buttons were sectioned and
stained with a battery of commercial antibodies against G\alpha\
proteins. Western blot analysis of cell lysates of corneal epithelial
cells and HEK 293 cells transfected with G\alpha q cDNA was used to
determine the identity of the protein reactive with the anti-G\alpha q
antibody (E-17). Comparisons were made with another anti-G\alpha q
antibody (G4415) and an anti-cytokeratin 12C (J7) antibody. The isolated
proteins reactive with E17 and J7 were then analyzed with two
dimensional isoelectric focusing. Polypeptide sequences were identified
using matrix assisted laser desorption/ionization time of flight mass
spectrometry (MALDI-TOF MS) after in-gel protein digestion.}

\abs_results{The E-17 anti-G\alpha q antibody preferentially stained the
entire corneal epithelia and the suprabasal layers of the limbus with
complete absence of staining in the basal limbus and conjunctiva.
Western blot analysis of corneal epithelial cells showed that E-17
antibody identified a protein with a molecular weight of 55 kDa.
However, the antibody did not react with the purported antigen, G\alpha
q protein (42 kDa) produced by G\alpha q cDNA. Another anti-G\alpha q
antibody (G4415) did not react with the 55 kDa protein but did react
with the 42 kDa G\alpha q protein. Further comparison of the E-17
antibody with the J7 antibody revealed that both recognized the 55 kDa
band in one and two dimensional analysis. MALDI-TOF MS analysis
confirmed that the 55 kDa protein of interest was actually cytokeratin
12 (CK12), rather than G\alpha q protein.}

\abs_conclusions{The commercial E-17 anti-G\alpha q antibody did not
react with G\alpha q protein, its purported antigen. Instead, it
recognized a 55 kDa protein, which was characterized to be cytokeratin
12 by isoelectric focusing and peptide fingerprinting with mass
spectrometry. Based on its reactivity with CK12, this commercial E-17
can be used as a differentiation marker to study ocular surface
epithelia.}

\introduction

\p{Antibodies are required for research techniques such as
immunohistochemistry, western blot analysis, and immunoprecipitation.
Commercially available antibodies help to facilitate laboratory
investigations by eliminating the need for antibody production. However,
there are a limited number of antibodies currently available that are
specific for epithelial proteins in the human ocular surface.}

\p{G-proteins are ubiquitous heterotrimeric guanine nucleotide binding
proteins that function as signal transducers for several distinct
intracellular signaling pathways. G-proteins are composed of \alpha,
\beta, and \gamma\ subunits, of which there are several \alpha\ subunits
including G\alpha s, G\alpha i, and G\alpha q. G\alpha s stimulates
adenylyl cyclase to produce cyclic AMP (cAMP) to activate protein kinase
A (PKA), whereas G\alpha i inhibits the same cascade [1]. G\alpha q
stimulates phospholipase C (PLC) to produce the intracellular second
messengers inositol triphosphate (IP3) and diacylgylcerol (DAG) [2]. IP3
causes the release of intracellular calcium while DAG activates protein
kinase C (PKC) [2].}

\p{To the best of our knowledge, the roles of G proteins in the ocular
surface epithelia have never been explored. We surmise that G-proteins
may be involved in signal transduction in the ocular surface epithelia.
For example, in corneal epithelium, mucin-like glycoprotein secretion is
mediated by cAMP and PKC signaling pathways [3]. PKC may play a role in
corneal epithelial gene expression and wound healing, as increased PKC
activity has been observed in corneal epithelial wound healing and
inhibition of PKC results in a significant delay in corneal
re-epithelialization [4-6].}

\p{In our attempt to identify G-protein and its subunits as epithelial
differentiation markers specific to ocular surface epithelia, we
initially characterized the specific immunohistochemical staining
pattern of a battery of commercially available anti-G protein antibodies
in the human ocular surface. We noted that a commercial anti-G\alpha q
(\alpha\ subunit) antibody (E-17), differing from other anti-G\alpha q
antibody, had a unique staining pattern in the ocular surface epithelia.
Herein, we performed a detailed biochemical analysis to identify the
unique corneal epithelial protein reactive with this E-17 antibody.}

\methods

\subsection{Tissue}

\p{Twelve fresh human donor corneoscleral buttons with small
conjunctival skirts were obtained from the Minnesota Lions Eye Bank.
Tissues were stored in the standard Optisol-GS corneal storage media
(Bausch \and\ Lomb, New York, NY). An exemption was obtained from the
Human Subjects Committee of the University of Minnesota.}

\p{For protein analyses using western blot analysis and two dimensional
isoelectric focusing (IEF), the donor corneoscleral buttons were
prepared by first trimming away the conjunctival skirts. Using a cell
scraper, corneal epithelial cells were then gently scraped from the
entire corneal surface into a solution of 1X PBS and protease inhibitor
cocktail (Sigma Co., St. Louis, MO). After centrifuging for 15 min, the
supernatant was removed and the epithelial cells were preserved at -80
\deg C until further analysis.}

\subsection{Antibodies}

\p{Anti-G\alpha q (E-17; Santa Cruz Biotechnology, Inc, Santa Cruz, CA),
a rabbit polyclonal antibody was purportedly developed against a peptide
within the N-terminal domain of G\alpha q. Two distinct batches of the
G\alpha q (E-17) antibody from the same company were tested.}

\p{The following antibodies were obtained from Sigma (St. Louis, MO);
Anti-G\alpha q (G4415), developed against a synthetic peptide
corresponding to amino acids 277-294 of G\alpha q, alkaline phosphatase
conjugated goat anti-rabbit IgG, and goat anti-mouse IgG (as secondary
antibodies).}

\p{The anti-cytokeratin 12C antibody (J7) was previously developed
against the C-terminal domain of cytokeratin 12 (CK12) by one of the
co-authors (WWYK).}

\subsection{Immunohistochemistry}

\p{Corneoscleral buttons were vertically bisected, embedded in optimal
cutting temperature (OCT) compound, and snap frozen in liquid nitrogen.
Tissues were cryosectioned to 6 \mu m thickness and mounted on
colorfrost plus slides (Fisher Scientific, Pittsburgh, PA). At room
temperature, the tissues were blocked with 10% goat serum in phosphate
buffered saline (PBS, pH 7.4) for 1.5 h and then incubated with primary
antibody diluted in PBS (1:100) for 1 h. The sections were washed twice
for 15 min each with PBS/0.05% Tween 20 and then incubated for 1 h with
alkaline phosphatase conjugated secondary antibodies. The
5-bromo-4-chloro-3-indolyl-phosphate/nitrotetrazolium blue chloride
(BCIP/NBT) liquid substrate system (Sigma) was used to develop the
tissues. Control slides were prepared with primary antibody substituted
by pre-immune serum, but were otherwise subjected to the same protocol.
Photomicrographs were obtained using a Zeiss Axiovert 200 microscope
(Zeiss, Thornwood, NY). Tissues from three unrelated donors were used to
confirm the specific staining pattern for each antibody and the
immunostaining was repeated twice for each antibody to ensure the
findings were reproducible. Two different batches of E-17 from the same
manufacturer were also tested to reconfirm the staining pattern in
ocular surface epithelia.}

\subsection{HEK 293 cell transfection}

\p{Full length human G\alpha q subunit cDNA in pCDNA 3.1 was obtained
from Guthrie cDNA resource center (Sayre, PA). Plasmid DNA (5 \mu g) was
mixed with 50 \mu l of lipofectamine (Invitrogen, Carlsbad, CA) in 0. 5
ml of Opti-Mem and incubated at room temperature for 30 min. The
liposome-DNA complex was then added to a 10 cm plate of HEK 293 cells
(ATCC, Manassas, VA) at approximately 80% confluency and incubated at 37
\deg C for 4 h. Cells were harvested 48 h later, gently scraped,
centrifuged, and re-suspended in sample buffer for collection of G\alpha
q protein.}

\subsection{Gel electrophoresis and immunoblotting}

\p{Lysates from corneal epithelial cells and HEK 293 cells transfected
with G\alpha q cDNA were used for comparison. Cells were homogenized in
an SDS containing buffer (1X PBS, 2% SDS, and \beta-mercaptoethanol) and
the protein concentration was measured using the bicinchoninic acid
(BCA) protein assay kit (Pierce, Rockford, IL) according to the
manufacturer's instructions. One dimensional SDS polyacrylamide gel
electrophoresis (SDS-PAGE) was used to characterize the molecular weight
of the protein recognized by the E-17 antibody. Equal amounts of protein
(about 20 \mu g/lane) were electrophoresed on 10% SDS-PAGE gels.
Proteins were then transferred to nitrocellulose membranes (Immobilon;
Millipore, Bedford, MA) and the membranes were blocked with a 1X Tris
buffered saline (TBS, 10 mM Tris, pH 8.0 and 150 mM NaCl)/5% dry milk
solution. Membranes were incubated with primary antibody for 1 h at room
temperature or overnight at 4 \deg C and then washed twice with a
solution of 1X TBS and 0.05% Tween 20. The blots were then incubated
with secondary antibodies for 1 h at room temperature, washed, and
developed with the BCIP/NBT liquid substrate system.}

\subsection{Preparative 2D electrophoresis}

\p{Definitive analysis of the corneal epithelial protein of interest was
performed with two dimensional isoelectric focusing, silver staining,
and in-gel digestion by trypsin for mass spectrometry. Soluble proteins
were removed by combining corneal epithelial cells with a
radioimmunoprecipitation assay solution (RIPA, 50 mM Tris-HCl, pH 7.5,
150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS),
protease inhibitor, and phenylmethylsulfonyl fluoride (PMSF) for 1 h at
4 \deg C. After centrifugation and removal of supernatant, the pellet
was then resuspended in an SDS containing sample buffer and the protein
concentration was measured using bovine serum albumin (BSA) as a
standard.}

\p{Two dimensional electrophoresis was performed using precast
immobilized pH gradient (IPG) strips (pH 3-10, 7 cm; Bio-Rad, Hercules,
CA) in the first dimension. Samples were applied via active rehydration
of IPG strips using a PROTEAN IEF cell (Bio-Rad) according to the
manufacturer's recommendations. Typically 500-600 \mu g of proteins were
loaded on each IPG strip and focusing was carried out for 20,000
volt-hour. After IEF separation, the strips were immediately
equilibrated twice for 15 min in an SDS containing buffer (6 M urea,
0.375 M Tris, pH 8.8, 2% SDS, and 20% glycerol). In the first
equilibration solution DTT (2%) was included and 2.5% w/v iodoacetamide
was added in the second equilibration step to alkylate thiols. SDS-PAGE
was performed using 1.0 mm thick, 10% SDS/polyacrylamide gels and
electrophoresis was carried out at constant voltage (100 V). Prior to
silver staining, gels were fixed in 50% methanol/10% acetic acid,
incubated in 20% ethanol, and then washed with water one time each.
Following incubation with sodium thiocyanate (0.1 g/500 ml) for one min
and then silver nitrate (0.25 g/125 ml) for 30 min, the gels were
developed in buffer containing sodium carbonate, formaldehyde, and
sodium thiocyanate. The reaction was stopped with 1% acetic acid and the
proteins of interest were excised from the gel.}

\subsection{In-gel digestion}

\p{Digestion of protein in excised gel pieces was performed after
destaining with a 1:1 solution of 30 mM potassium ferricyanide/100 mM
sodium thiosulphate. Gel pieces were washed with 100% acetonitrile and
dried by vacuum centrifugation. Modified porcine trypsin (12.5 ng/\mu l,
sequence grade; Promega, Madison, WI) in digestion buffer (50 mM
ammonium bicarbonate/5 mM calcium chloride) was added to the dry gel
pieces and incubated on ice for 1 h for rehydration. After removing the
supernatant, 30 \mu l of digestion buffer was added and digestion was
continued at 37 \deg C overnight.}

\p{The resulting peptide mixture was extracted with 20 mM ammonium
bicarbonate once, followed by 50% acetonitrile/5% formic acid three
times. The supernatants were pooled and the peptides were dried by
vacuum centrifugation.}

\subsection{Mass spectrometry}

\p{Polypeptide sequences were identified using matrix assisted laser
desorption/ionization time of flight mass spectrometry (MALDI-TOF MS)
after in-gel protein digestion. Immediately prior to mass spectrometry,
the protein samples were reconstituted with a solution of
acetonitrile/water (5:95, vol:vol) and trifluoroacetic acid. A ziptip
was hydrated, the sample loaded, and water used to wash the sample. An
elution solution of acetonitrile/water (60:40), and 0.1% trifluoroacetic
acid was then loaded three times, \alpha-cyano-4-hydroxycinnamic acid
(CCA) matrix was added, and 1 \mu l of the sample mixture was spotted
directly on a MALDI target for analysis. Peptide mass mapping of tryptic
digests was carried out with a Brooker Biflex III MALDI-TOF mass
spectrometer (Billarica, MA) in reflectron mode. Protein identification
based on peptide mass fingerprint was performed with
\hot{\mascot}{Mascot} search software (Matrix Science Inc., Boston, MA;
version 1.8).}

\results

\p{With the E-17 anti-G\alpha q antibody, a unique staining pattern was
observed in ocular surface epithelia (\figref{1}). The antibody
preferentially stained the suprabasal epithelial layers of the limbus
(\figref{1}{A}) and entire corneal epithelium (\figref{1}{B}). Complete
absence of staining in the conjunctival epithelium (\figref{1}{C}) was
noted.}

\p{In order to confirm the identity of the protein stained by the E-17
antibody, we first transfected HEK 293 cells with G\alpha q cDNA to
generate genuine G\alpha q protein (42 kDa). With western blot analysis,
the E-17 antibody recognized a unique protein of 55 kDa from corneal
epithelial lysates (\figref{2}{A}, lane 1), but it did not recognize any
proteins from the HEK 293 cells transfected with G\alpha q cDNA
(\figref{2}{A}, lane 2). Two different batches of E-17 from the same
manufacturer showed similar results in immunohistochemistry and western
blot analysis. For comparison, further analysis was also performed with
an anti-G\alpha q antibody (G4415) from another manufacturer (Sigma).
The G4415 anti-G\alpha q antibody did not recognize any protein from the
corneal epithelial lysates (\figref{2}{B}, lane 1). Instead, the G4415
antibody did react to the 42 kDa protein (G\alpha q) in the HEK 293
cells transfected with G\alpha q cDNA (\figref{2}{B}, lane 2). The data
indicated the two commercial antibodies, both presumably reactive with
the G\alpha q, instead recognized two distinct proteins.}

\p{Since our immunohistochemical data with E-17 antibody showed an
epithelial staining pattern that is in striking resemblance to the well
known distribution of cytokeratin pair CK3/CK12 in ocular surface
epithelia, we further analyzed the identity of the unknown protein
recognized by E-17. Western blots using the E-17 antibody
(\figref{2}{C}) and a J7 (anti-cytokeratin 12C) antibody (\figref{2}{D})
revealed that both antibodies recognized an equivalent 55 kDa band in
the corneal epithelial cells.}

\p{To confirm that the identity of the epithelial protein recognized by
E-17 was indeed CK12, 2D IEF and subsequent western blots were
performed. Both E-17 and J7 antibodies stained equivalent bands of 55
kDa at a low pH (\figref{3}{A,B}). This is consistent with CK12 being a
member of the acidic cytokeratins.}

\p{The band corresponding to the 55 kDa protein of the epithelial
lysates was excised from the 2D gel (\figref{4}{A}) and subjected to
in-gel digestion, followed by MALDI-TOF MS analysis. MALDI-TOF MS
revealed a monoisotopic peptide mass fingerprint spectrum
(\figref{4}{B}). When the spectrum was analyzed with Mascot search
software, it matched 25 peptides (51% sequence coverage) of human
cytokeratin-12 (\figref{4}{C}). A probability based Mowse score was 147
(\figref{4}{D}), where the score is derived from (-10)log\sub{10}(P) in
which P is the probability that the observed match is a random event.
Mowse scores greater than 63 (or p\lt 0.05) indicate a significant match
of the protein sequences (\hot{\mowsescore}{Mowse scoring} is described
in detail on the Matrix Science web site) [7]. These results confirmed
that the isolated peptides matched the CK12 sequence with a high degree
of certainty and the protein recognized by E-17 was indeed CK12 with
very high probability.}

\discussion

\p{Our results demonstrated that E-17 stained the entire corneal
epithelium and suprabasal epithelia of the limbus with complete absence
of staining in the conjunctiva and basal limbus. It reacted with a
unique 55 kDa protein in corneal epithelial cells. Surprisingly, this
presumed anti-G\alpha q antibody did not react with the bona fide 42 kDa
G\alpha q from transfected HEK293 cells. Taken together, the data thus
suggest that E-17 antibody preferentially stains the more differentiated
cells of corneal epithelial lineage, sparing the limbal stem cell zones
and conjunctiva. Such a unique pattern is in striking resemblance to the
well known distribution of cytokeratin pair CK3/CK12 in ocular surface
epithelia [8,9].}

\p{Cytokeratins are a family of water insoluble polypeptides that form
10 nm intermediate filaments, major components of the epithelial cell
cytoskeleton. In vivo, an acidic keratin is usually paired with a basic
keratin to form a heterodimer [10]. Both cytokeratins, acidic CK12 (55
kDa) and basic CK3 (64 kDa), are known to be expressed in the epithelia
of the suprabasal limbus and entire cornea, but not in the basal limbal
epithelium where corneal stem cells are thought to reside [8,9]. This
CK3/CK12 pair is expressed in the corneal epithelia of humans, cows,
guinea pigs, rabbits, and chickens and they have been regarded as
specific markers of terminal differentiation in the corneal epithelium
[11-19]. Our extensive biochemical analyses indicate that the 55 kDa
protein was indeed CK12, which reacted equally with the E-17 and J7
antibodies. Consequently, we believe that E-17 represents the first
commercially available rabbit derived antibody to recognize CK12 (55
kDa). Monoclonal antibodies to CK12 such as J7 [17,19] have been
developed in the past, but are not commercially available. In addition
to J7 and E-17, there are other anti-CK12 antibodies available such as
goat derived sc-17098, sc-17099 and sc-17101 from Santa Cruz
Biotechnology, Inc and a less selective monoclonal antibody (reacting to
CK9, CK10, CK11, and CK12) from Biomeda Corporation (Foster City, CA).}

\p{Similar to other self renewing epithelial tissues such as skin or
oral mucosa, the ocular surface epithelia are in a state of constant
renewal, requiring stem cell participation. In contrast to human skin in
which epidermal stem cells are uniformly distributed along the basal
layer of the epidermis, the progenitors of the cornea reside in the
narrow transition zone of limbal cells between the cornea and the
conjunctiva [20-23]. The theory that corneal stem cells reside in the
basal layers of the limbus was first proposed based, in part, on
labeling patterns of CK3 by the monoclonal antibody AE5 [8,20]. It was
observed that CK3 exists in the epithelia of the suprabasal limbus and
entire cornea, but not in the epithelia of the basal limbus or
conjunctiva. CK3 was also expressed in post-confluent stratified cell
cultures but was not present in undifferentiated cells in culture. These
data suggest that the basal limbal epithelium is less differentiated
than the suprabasal limbal epithelium and entire corneal epithelium, and
that the putative corneal stem cells reside in the basal limbus. The
commercial AE5 monoclonal antibody has been used widely to study
epithelial differentiation. The availability of commercial E-17 antibody
which reacts to CK12 (the counterpart of CK3) should further augment the
armamentarium for research on ocular surface epithelial
differentiation.}

\p{The study of CK12 is important because the keratin 12 gene is the
second most abundant gene isolated from a full length cDNA library of
human corneal epithelium [24]. Furthermore, CK12 knockout mice have
fragile corneal epithelia that are easily abraded [19], and mutations of
human CK3 and CK12 genes are known to result in autosomal dominant
Meesmann's corneal epithelial dystrophy [25,26]. Thus, the
identification of a commercial antibody such as E-17 as a specific
marker of corneal epithelial differentiation will help to facilitate
further study of ocular surface epithelial differentiation and stem cell
development.}

\p{In summary, we set out to investigate the unique immunohistochemical
staining pattern of a commercial anti-G\alpha q antibody (E-17) in human
ocular surface epithelia, and found that CK12, instead of the G\alpha q
protein, is the protein in ocular surface epithelia responsible for the
selective staining by E-17. Based on a protein sequence search and
further comparison with other antibodies such as J7 and G4415, we did
not find any antigenic similarity or crossreactivity between G\alpha q
and CK12. Thus, we conclude that E-17 antibody represents another
commercially available differentiation marker of corneal epithelial
phenotype.}

\acknowledgements

\p{Supported in part by the Minnesota Medical Foundation (Research Grant
3180-9227-02 [AJWH]), Student Research Grant 3171-9295-02 (CSB), and by
an unrestricted grant from Research to Prevent Blindness (AJWH, CY).}

\references

\p{1. Sunahara RK, Dessauer CW, Gilman AG. Complexity and diversity of
mammalian adenylyl cyclases. Annu Rev Pharmacol Toxicol 1996; 36:461-80.
\pubmed{8725398}}

\p{2. Wu DQ, Lee CH, Rhee SG, Simon MI. Activation of phospholipase C by
the alpha subunits of the Gq and G11 proteins in transfected Cos-7
cells. J Biol Chem 1992; 267:1811-7. \pubmed{1309799}}

\p{3. Nakamura M, Endo K, Nakata K. Mucin-like glycoprotein secretion is
mediated by cyclic-AMP and protein kinase C signal transduction pathways
in rat corneal epithelium. Exp Eye Res 1998; 66:513-9. \pubmed{9628798}}

\p{4. Chandrasekher G, Bazan NG, Bazan HE. Selective changes in protein
kinase C (PKC) isoform expression in rabbit corneal epithelium during
wound healing. Inhibition of corneal epithelial repair by PKCalpha
antisense. Exp Eye Res 1998; 67:603-10. \pubmed{9878223}}

\p{5. Lin N, Bazan HE. Protein kinase C subspecies in rabbit corneal
epithelium: increased activity of alpha subspecies during wound healing.
Curr Eye Res 1992; 11:899-907. \pubmed{1330442}}

\p{6. Hirakata A, Gupta AG, Proia AD. Effect of protein kinase C
inhibitors and activators on corneal re-epithelialization in the rat.
Invest Ophthalmol Vis Sci 1993; 34:216-21. \pubmed{8425827}}

\p{7. Pappin DJ, Hojrup P, Bleasby AJ. Rapid identification of proteins
by peptide-mass fingerprinting. Curr Biol 1993; 3:327-32.
\pubmed{15335725}}

\p{8. Schermer A, Galvin S, Sun TT. Differentiation-related expression
of a major 64K corneal keratin in vivo and in culture suggests limbal
location of corneal epithelial stem cells. J Cell Biol 1986; 103:49-62.
\pubmed{2424919}}

\p{9. Kurpakus MA, Stock EL, Jones JC. Expression of the 55-kD/64-kD
corneal keratins in ocular surface epithelium. Invest Ophthalmol Vis Sci
1990; 31:448-56. \pubmed{1690687}}

\p{10. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. The catalog
of human cytokeratins: patterns of expression in normal epithelia,
tumors and cultured cells. Cell 1982; 31:11-24. \pubmed{6186379}}

\p{11. Tseng SC, Jarvinen MJ, Nelson WG, Huang JW, Woodcock-Mitchell J,
Sun TT. Correlation of specific keratins with different types of
epithelial differentiation: monoclonal antibody studies. Cell 1982;
30:361-72. \pubmed{6183000}}

\p{12. Tseng SC, Hatchell D, Tierney N, Huang AJ, Sun TT. Expression of
specific keratin markers by rabbit corneal, conjunctival, and esophageal
epithelia during vitamin A deficiency. J Cell Biol 1984; 99:2279-86.
\pubmed{6209290}}

\p{13. Cooper D, Sun TT. Monoclonal antibody analysis of bovine
epithelial keratins. Specific pairs as defined by coexpression. J Biol
Chem 1986; 261:4646-54. \pubmed{2420789}}

\p{14. Rodrigues M, Ben-Zvi A, Krachmer J, Schermer A, Sun TT.
Suprabasal expression of a 64-kilodalton keratin (no. 3) in developing
human corneal epithelium. Differentiation 1987; 34:60-7.
\pubmed{2440750}}

\p{15. Chaloin-Dufau C, Sun TT, Dhouailly D. Appearance of the keratin
pair K3/K12 during embryonic and adult corneal epithelial
differentiation in the chick and in the rabbit. Cell Differ Dev 1990;
32:97-108. \pubmed{1707331}}

\p{16. Cooper D, Schermer A, Sun TT. Classification of human epithelia
and their neoplasms using monoclonal antibodies to keratins: strategies,
applications, and limitations. Lab Invest 1985; 52:243-56.
\pubmed{2579289}}

\p{17. Liu CY, Zhu G, Westerhausen-Larson A, Converse R, Kao CW, Sun TT,
Kao WW. Cornea-specific expression of K12 keratin during mouse
development. Curr Eye Res 1993; 12:963-74. \pubmed{7508359}}

\p{18. Liu CY, Zhu G, Converse R, Kao CW, Nakamura H, Tseng SC, Mui MM,
Seyer J, Justice MJ, Stech ME, Hansen GM, Kao WW-Y. Characterization and
chromosomal localization of the cornea-specific murine keratin gene
Krt1.12. J Biol Chem 1994; 269:24627-36. \pubmed{7523376}}

\p{19. Kao WW, Liu CY, Converse RL, Shiraishi A, Kao CW, Ishizaki M,
Doetschman T, Duffy J. Keratin 12-deficient mice have fragile corneal
epithelia. Invest Ophthalmol Vis Sci 1996; 37:2572-84. \pubmed{8977471}}

\p{20. Cotsarelis G, Cheng SZ, Dong G, Sun TT, Lavker RM. Existence of
slow-cycling limbal epithelial basal cells that can be preferentially
stimulated to proliferate: implications on epithelial stem cells. Cell
1989; 57:201-9. \pubmed{2702690}}

\p{21. Zieske JD. Perpetuation of stem cells in the eye. Eye 1994; 8 (Pt
2):163-9. \pubmed{7958017}}

\p{22. Kruse FE. Stem cells and corneal epithelial regeneration. Eye
1994; 8 (Pt 2):170-83. \pubmed{7958018}}

\p{23. Pellegrini G, Golisano O, Paterna P, Lambiase A, Bonini S, Rama
P, De Luca M. Location and clonal analysis of stem cells and their
differentiated progeny in the human ocular surface. J Cell Biol 1999;
145:769-82. \pubmed{10330405}}

\p{24. Nishida K, Adachi W, Shimizu-Matsumoto A, Kinoshita S, Mizuno K,
Matsubara K, Okubo K. A gene expression profile of human corneal
epithelium and the isolation of human keratin 12 cDNA. Invest Ophthalmol
Vis Sci 1996; 37:1800-9. \pubmed{8759347}}

\p{25. Irvine AD, Corden LD, Swensson O, Swensson B, Moore JE, Frazer
DG, Smith FJ, Knowlton RG, Christophers E, Rochels R, Uitto J, McLean
WH. Mutations in cornea-specific keratin K3 or K12 genes cause
Meesmann's corneal dystrophy. Nat Genet 1997; 16:184-7.
\pubmed{9171831}}

\p{26. Nishida K, Honma Y, Dota A, Kawasaki S, Adachi W, Nakamura T,
Quantock AJ, Hosotani H, Yamamoto S, Okada M, Shimomura Y, Kinoshita S.
Isolation and chromosomal localization of a cornea-specific human
keratin 12 gene and detection of four mutations in Meesmann corneal
epithelial dystrophy. Am J Hum Genet 1997; 61:1268-75. \pubmed{9399908}}

\endreferences

}

\beginfigures

\figfile{1}{
\figtitle{1}{Immunohistochemical analysis of human ocular surface epithelia with
anti-G\alpha q antibody (E-17)}

\p{Immunohistochemistry of human ocular surface epithelia with
anti-G\alpha q antibody (E-17) showed a preferential staining of the
suprabasal limbal epithelium (\panel{A}) and the full layer of central
corneal epithelium (\panel{B}), with complete absence of staining in the
conjunctival epithelium (\panel{C}) and basal limbal epithelium (A,
arrows). Scale bars represent 50 \mu m.}

\ctr{\jpgimage{1}{500}{417}{55}}

}

\figfile{2}{
\figtitle{2}{Western blot analyses of cell lysates by E17 antibody and
J7 (anti-cytokeratin 12) antibody}

\p{\panel{A}: Western blot analysis showed that E-17 antibody recognized
a protein of 55 kDa (arrow) in corneal epithelial lysates(lane 1), but
did not identify such a protein in lysates of HEK 293 cells transfected
with G\alpha q cDNA (lane 2). \panel{B}: Western blot analysis showed
G4415 anti-G\alpha q antibody recognized a protein of 42 kDa only in
lysates of HEK 293 transfected with G\alpha q cDNA (arrowhead, lane 2),
but not in corneal epithelial cells (lane 1). \panel{C}: Western blot
analysis of corneal epithelial lysates stained with E-17 antibody.
\panel{D}: Western blot analysis of corneal epithelial lysates stained
with J7 (anti-CK12) antibody. Both antibodies (E-17 in \panel{C} and J7
in \panel{D}) identified equivalent 55 kDa bands and reconfirmed the
finding in \panel{A} that E-17 reacted with the 55 kDa protein
(cytokeratin 12), instead of the purported G\alpha q (42 kDa).}

\ctr{\jpgimage{2}{500}{240}{30}}

}

\figfile{3}{
\figtitle{3}{Western blot analysis of corneal epithelial cell lysates
subjected to two dimensional gel electrophoresis}

\p{Isoelectric focusing by pH was run horizontally and the SDS-PAGE by
molecular weight was run vertically. Acidic proteins are on the left and
basic proteins are on the right. \panel{A}: E-17 antibody recognized a
protein band in the acidic region at about 55 kDa. \panel{B}: J7
antibody also reacted with an acidic protein with a molecular weight of
about 55 kDa.}

\ctr{\jpgimage{3}{540}{216}{25}}

}

\figfile{4}{
\figtitle{4}{Confirmation of the 55 kDa protein reactive with E17 to be
cytokeratin 12 by in-gel digestion and MALDI-TOF}

\p{\panel{A}: The 55 kDa protein band (arrow) was excised from the
silver stained, two dimensional electrophoresis gel. \panel{B}:
MALDI-TOF MS analysis showed a monoisotopic peptide mass fingerprint
spectrum of the protein extracted from the 55 kDa band. \panel{C}: A
Mascot search showed the matched peptides (in \color{\red}{red}) from
the isolated 55 kDa protein accounted for 51% of the protein sequence of
cytokeratin 12. \panel{D}: A probability based Mowse score of 147
(arrow) indicated that the identified peptides match the cytokeratin 12
protein sequence with a high degree of certainty. The shaded area
represents scores with the greatest statistical uncertainty. The Mowse
score is derived from (-10)log\sub{10}(P), where P is the probability
that the observed match is a random event. Mowse scores greater than 63
(or p\lt 0.05) are considered a significant match of the proteins. The
shaded area showed that matching of these tryptic peptides by Mascot
search with other protein sequences yielded Mowse scores lower than 63
and the tryptic peptides from 55 kDa did not match significantly (p\gt
0.05) with any protein other than cytokeratin 12
(\hot{\mowsescore}{Mowse scoring} is described in detail on the Matrix
Science web site).}

\ctr{\jpgimage{4a}{350}{273}{35}}

\p{\sp}

\ctr{\gifimage{4b}{600}{321}{54}}

\p{\sp}

\p{\panel{C}:}

\box{\pre{
  1 \color{\red}{MDLSNNTMSL SVRTPGLSR}R LSSQSVIGRP RGMSASSVGS GYGGSAFGFG
 51 ASCGGGFSAA SMFGSSSGFG GGSGSSMAGG LGAGYGR\color{\red}{ALG GGSFGGLGMG}
101 \color{\red}{FGGSPGGGSL GILSGNDGGL LSGSEK}ETMQ NLNDRLASYL DKVRALEEAN
151 TELENKIR\color{\red}{EW YETRGTGTAD ASQSDYSK}YY PLIEDLRNK\color{\red}{I ISASIGNAQL}
201 \color{\red}{LLQIDNARLA AEDFRMKYEN ELALR}QGVEA DINGLRR\color{\red}{VLD ELTLTRTDLE}
251 \color{\red}{MQIESLNEEL AYMKKNHEDE LQSFRVGGPG EVSVEMDAAP GVDLTR}LLND
301 MR\color{\red}{AQYETIAE QNRK}DAEAWF IEKSGELRKE ISTNTEQLQS SKSEVTDLRR
351 \color{\red}{AFQNLEIELQ SQLAMKK}SLE DSLAEAEGDY CAQLSQVQQL ISNLEAQLLQ
401 VRADAERQNV DHQRLLNVK\color{\red}{A RLELEIETYR RLLDGEAQGD GLEESLFVTD}
451 \color{\red}{SK}SQAQSTDS SKDPTKTRKI K\color{\red}{TVVQEMVNG EVVSSQVQEI EELM}
}}

\p{\sp}

\ctr{\gifimage{4c}{500}{254}{30}}

}