Identification of glycosylated marker proteins of epithelial polarity in MDCK cells by homology driven proteomics

BMC Biochemistry
Joachim FüllekrugKai Simons

Abstract

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. 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. 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.

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Citations

Jan 27, 2009·Journal of Proteomics·Andrej ShevchenkoMagno Junqueira
Feb 1, 2011·The Journal of Biological Chemistry·Jacqueline TorresMartin B A ter Beest
Nov 14, 2008·Molecular Biology of the Cell·Christian E OvergaardCharles Yeaman
Aug 17, 2012·American Journal of Physiology. Cell Physiology·Vinita TakiarMichael J Caplan
Oct 31, 2008·American Journal of Physiology. Renal Physiology·Olga VaginGeorge Sachs
May 12, 2012·Stem Cell Research & Therapy·Harold S Bernstein, William C Hyun
Nov 23, 2006·Proceedings of the National Academy of Sciences of the United States of America·Otilia V VieiraKai Simons
Jan 25, 2007·Proceedings of the National Academy of Sciences of the United States of America·Diego GravottaEnrique Rodriguez-Boulan
Mar 15, 2013·American Journal of Physiology. Cell Physiology·Erika K WellsMichael J Caplan
Jul 22, 2009·The Journal of Cell Biology·Glen A FarrMichael J Caplan
Jul 5, 2012·Integrative Biology : Quantitative Biosciences From Nano to Macro·Mirren CharnleyMarcus Textor
May 16, 2019·Molecular Biology of the Cell·Diego GravottaEnrique Rodriguez-Boulan
Jul 24, 2020·International Journal of Molecular Sciences·Wolfgang H ZieglerDieter Haffner
Feb 20, 2021·Journal of Cell Science·Eric CortadaMarcel Verges
Mar 20, 2008·Journal of Cell Science·Juha M TorkkoKai Simons
Oct 26, 2006·Journal of Cell Science·Katrin MilgerJoachim Füllekrug
Apr 25, 2021·Science Advances·Sicong WangKenichiro Taniguchi

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Methods Mentioned

BETA
glycosylation
electrophoresis
immunoprecipitation

Software Mentioned

BLAST
Mascot
BLAST BLAST

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