Golgi phosphoprotein 3 determines cell binding properties under dynamic flow by controlling golgi localization of core 2 N-acetylglucosaminyltransferase 1

Mohamed F. Ali, Vishwanath B. Chachadi, Armen Petrosyan, Pi Wan Chengs

Research output: Contribution to journalArticle

41 Scopus citations

Abstract

Core 2 N-acetylglucosaminyltransferase 1 (C2GnT1) is a key enzyme participating in the synthesis of core 2-associated sialyl Lewis x (C2-O-sLex), a ligand involved in selectin-mediated leukocyte trafficking and cancer metastasis. To accomplish that, C2GnT1 needs to be localized to the Golgi and this step requires interaction of its cytoplasmic tail (CT) with a protein that has not been identified. Employing C2GnT1 CT as the bait to perform a yeast two-hybrid screen, we have identified Golgi phosphoprotein 3 (GOLPH3) as a principal candidate protein that interacts with C2GnT1 and demonstrated that C2GnT1 binds to GOLPH3 via the LLRRR9 sequence in the CT. Confocal fluorescence microscopic analysis shows substantial Golgi co-localization of C2GnT1 and GOLPH3. Upon GOLPH3 knockdown, C2GnT1 is found mainly in the endoplasmic reticulum and decorated with complex-type N-glycans, indicating that the enzyme has been transported to the Golgi but is not retained. Also, we have found that a recombinant protein consisting of C2GnT1 CT1-16-Leu17-32-Gly33-42-GFP is localized to the Golgi although the same construct with mutated CT (AAAAA 9) is not. The data demonstrate that the C2GnT1 CT is necessary and sufficient for Golgi localization of C2GnT1. Furthermore, GOLPH3 knockdown results in reduced synthesis of C2-O-sLex associated with P-selectin glycoprotein ligand-1, reduced cell tethering to and rolling on immobilized P- or E-selectin, and compromised E-selectin-induced activation of spleen tyrosine kinase and cell adhesion to intercellular adhesion molecule-1 under dynamic flow. Our results reveal that GOLPH3 can regulate cell-cell interaction by controlling Golgi retention of C2GnT1.

Original languageEnglish (US)
Pages (from-to)39564-39577
Number of pages14
JournalJournal of Biological Chemistry
Volume287
Issue number47
DOIs
StatePublished - Nov 16 2012

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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