title: Ninjurin1 Assembles Into a Homomeric Protein Complex Maintained by N-linked Glycosylation.
abstract: Ninjurin1 (Ninj1) is a cell surface protein known as a homophilic adhesion molecule. Previous studies have shown a trans-interaction of Ninj1 between immune cells and endothelial cells; however, little is known about Ninj1 modification and structure in the cis-interaction. We showed that Ninj1 assembles into a homomeric complex via a cis-interaction mediated by the intracellular region and N-glycosylation at Asn60. We identified cis-interaction between Ninj1 proteins using CFP- and YFP-tagged Ninj1 by Förster resonance energy transfer using a confocal microscope and fluorescence-activated cell sorter. We further observed the Ninj1 homomeric complexes composed of two to six monomeric Ninj1 molecules by a formaldehyde cross-linking assay. Co-immunoprecipitation assays with epitope-tagged truncated Ninj1 suggested that the intracellular region encompassing Leu101-Ala110participates in Ninj1 homomer assembly. Ninj1 N-glycosylation was characterized by treatment of tunicamycin and substitution of Asn to Gln or Ala. Fluorescence-activated cell sorting-based Förster resonance energy transfer assays further demonstrated that N-glycosylation is indispensable for the Ninj1 cis-interaction, and a formaldehyde cross-linking assay confirmed that interruption of N-glycosylation by Asn substitution disrupted Ninj1 homomeric complex formation. In silico analysis revealed that Ninj1 is highly conserved in vertebrates and that the conserved sequence contains an N-glycosylation motif and cis-interacting intracellular region, which participate in Ninj1 homomer assembly. Taken together, these data show that Ninj1 assembles into a homomeric protein complex and that N-glycosylation is a prerequisite for Ninj1 homomer assembly. J. Cell. Biochem. 118: 2219-2230, 2017. © 2017 Wiley Periodicals, Inc.
authors.affiliation: SNU-Harvard NeuroVascular Protection Research Center, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, 08826, Korea.
authors.forename: Sung-Jin
authors.initials: SJ
authors.lastname: Bae
authors.affiliation: SNU-Harvard NeuroVascular Protection Research Center, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, 08826, Korea.
authors.forename: Min Wook
authors.initials: MW
authors.lastname: Shin
authors.affiliation: SNU-Harvard NeuroVascular Protection Research Center, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, 08826, Korea.
authors.forename: Ran Hee
authors.initials: RH
authors.lastname: Kim
authors.affiliation: SNU-Harvard NeuroVascular Protection Research Center, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, 08826, Korea.
authors.forename: Dongyoon
authors.initials: D
authors.lastname: Shin
authors.affiliation: SNU-Harvard NeuroVascular Protection Research Center, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, 08826, Korea.
authors.forename: Taekwon
authors.initials: T
authors.lastname: Son
authors.affiliation: SNU-Harvard NeuroVascular Protection Research Center, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, 08826, Korea.
authors.forename: Hee-Jun
authors.initials: HJ
authors.lastname: Wee
authors.affiliation: SNU-Harvard NeuroVascular Protection Research Center, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, 08826, Korea.|Crop Biotechnology Institute, GreenBio Science and Technology, Seoul National University, Pyeongchang, 25354, Korea.
authors.forename: Kyu-Won
authors.initials: KW
authors.lastname: Kim
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D006031:Glycosylation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization / Q000187:drug effects / Q000235:genetics; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D015816:Cell Adhesion Molecules, Neuronal; C484438:NINJ1 protein, human; D009414:Nerve Growth Factors; C484439:Ninj1 protein, mouse; D014415:Tunicamycin
keywords: FRET; HOMOOLIGOMER; N-GLYCOLISATION; NINJURIN1; PROTEIN ASSEMBLY; cis-INTERACTION