title: Asn54-linked glycan is critical for functional folding of intercellular adhesion molecule-5.
abstract: Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a dendritically polarized type I membrane glycoprotein, and promotes dendritic filopodia formation. Although we have determined the N-glycan structures of ICAM-5 in a previous report, their function is unknown. Here, we produced fifteen ICAM-5 gene constructs, in which each potential N-glycosylation site was mutated, to elucidate the function of the N-glycans of ICAM-5, and observed the effects of transfection of them on a neuronal cell line, Neuro-2a (N2a). Only the N54Q mutant, which is the mutant for the most N-terminal glycosylation site, failed to induce filopodia-like protrusions in N2a cells. Immunofluorescence staining and cell surface biotinylation revealed that N54Q ICAM-5 was confined to the ER and also could not be expressed on the cell surface. This is further supported by the biochemical evidence that almost all N-glycans of N54Q ICAM-5 were digested by Endo glycosidase H and peptide:N-glycanase, indicating that almost all of them retain high-mannose-type structures in ER. In additon, it also failed to form disulfide bonds or functional protein complexes. The stable transformants of N54Q ICAM-5 showed retarded cell growth, but it was interesting that there was no apparent ER stress, because the mutant was sequentially degraded via ER associated degradation pathway by comparing the susceptibilities of the responses to various inhibitors of this pathway in wild-type and N54Q ICAM-5 transfectants. Taken together, the Asn(54)-linked glycan is necessary for normal trafficking and function of ICAM-5, but is unassociated with ER-associated degradation of it.
authors.affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Yamazaki, Noda, Chiba, Japan. ohgomori@med.nagoya-u.ac.jp
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Ohgomori
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Nanao
authors.forename: Akinori
authors.initials: A
authors.lastname: Morita
authors.forename: Masahiko
authors.initials: M
authors.lastname: Ikekita
mesh_terms: D000818:Animals; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry; D008358:Mannose / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011554:Pseudopodia / Q000737:chemistry; D051381:Rats; D014162:Transfection / Q000379:methods
chemical_list: D015815:Cell Adhesion Molecules; C547413:ICAM5 protein, rat; D009419:Nerve Tissue Proteins; D011134:Polysaccharides; D001216:Asparagine; D006026:Glycoside Hydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D008358:Mannose