Cytokeratin 12 in human ocular surface epithelia is the antigen reactive with a commercial anti-Galpha q antibody.
OBJECTIVE
In our initial attempt to identify differentiation markers for ocular surface epithelia, we observed a unique staining pattern by a commercial anti-Galphaq antibody. We further isolate and characterize the protein reactive with this anti-Galphaq antibody in human ocular surface epithelia.


METHODS
Human donor corneoscleral buttons were sectioned and stained with a battery of commercial antibodies against Galpha proteins. Western blot analysis of cell lysates of corneal epithelial cells and HEK 293 cells transfected with Galphaq cDNA was used to determine the identity of the protein reactive with the anti-Galphaq antibody (E-17). Comparisons were made with another anti-Galphaq 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.


RESULTS
The E-17 anti-Galphaq 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, Galphaq protein (42 kDa) produced by Galphaq cDNA. Another anti-Galphaq antibody (G4415) did not react with the 55 kDa protein but did react with the 42 kDa Galphaq 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 Galphaq protein.


CONCLUSIONS
The commercial E-17 anti-Galphaq antibody did not react with Galphaq 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.
Boehlke
Christopher S
CS
Department of Ophthalmology, University of Minnesota, Minneapolis 55455, USA.
Yuan
Ching
C
Kao
Winston W Y
WW
Huang
Andrew J W
AJ
D001324:Autoantigens / Q000276:immunology*; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western; D002454:Cell Differentiation; D003132:Commerce; D003429:Cross Reactions / Q000276:immunology; D015180:Electrophoresis, Gel, Two-Dimensional; D019573:Epithelium, Corneal / Q000166:cytology / Q000276:immunology*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000276:immunology*; D053551:Keratin-12; D007633:Keratins / Q000276:immunology*; D007668:Kidney / Q000166:cytology / Q000196:embryology; D008970:Molecular Weight; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
D001324:Autoantigens; D015415:Biomarkers; D007074:Immunoglobulin G; C507494:KRT12 protein, human; D053551:Keratin-12; D007633:Keratins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11