Identification of glycosylated marker proteins of epithelial polarity in MDCK cells by homology driven proteomics.
BACKGROUND
MDCK cells derived from canine kidney are an important experimental model system for investigating epithelial polarity in mammalian cells. Monoclonal antibodies against apical gp114 and basolateral p58 have served as important tools in these studies. However, the molecular identity of these membrane glycoproteins has not been known.


RESULTS
We have identified the sialoglycoprotein gp114 as a dog homologue of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. Gp114 was enriched from tissue culture cells by subcellular fractionation and immunoaffinity chromatography. The identification was based on tandem mass spectrometry and homology based proteomics. In addition, the p58 basolateral marker glycoprotein was found to be the beta subunit of (Na+)(K+)-ATPase.


CONCLUSIONS
Gp114 has been characterized previously regarding glycosylation dependent trafficking and lipid raft association. The identification as a member of the canine CEACAM family will enable synergy between the fields of epithelial cell biology and other research areas. Our approach exemplifies how membrane proteins can be identified from species with unsequenced genomes by homology based proteomics. This approach is applicable to any model system.
Max-Planck-Institute of Molecular Cell Biology and Genetics, Dresden, Germany. Joachim.Fuellekrug@med.uni-heidelberg.de
Joachim
J
Füllekrug
Anna
A
Shevchenko
Andrej
A
Shevchenko
Kai
K
Simons
D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D016764:Cell Polarity; D002846:Chromatography, Affinity; D019295:Computational Biology; D004285:Dogs; D004847:Epithelial Cells / Q000737:chemistry* / Q000378:metabolism; D023281:Genomics; D006031:Glycosylation; D007668:Kidney / Q000166:cytology; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification
D015415:Biomarkers; D015815:Cell Adhesion Molecules; D008562:Membrane Glycoproteins; D000254:Sodium-Potassium-Exchanging ATPase