Asn 54-linked glycan is critical for functional folding of intercellular adhesion molecule-5
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Yamazaki, Noda, Chiba, Japan. Glycoconjugate Journal
(Impact Factor: 2.52).
12/2011; 29(1):47-55. DOI: 10.1007/s10719-011-9363-0
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.
Available from: Akhil Agarwal
- "Glycosylation which is one of the most common post-translational modifications of proteins is known to modulate a variety of biological activities, including protein folding, trafficking, stability and the expression of proteins on the cell surface     . The glycosylation of N-linked proteins starts in the rough endoplasmic reticulum (RER) and progresses in the Golgi. "
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ABSTRACT: Lysosome Associated Membrane protein-1 (LAMP1) which lines the lysosomes, is often found to appear on the surface of several cells involved in migratory and/or invasive functions including metastatic tumor cells. However, the mechanism of its surface translocation in these cells is still poorly understood. Glycosylation, one of the major post-translational modifications of membrane proteins, regulates a variety of functions of such glycoproteins. The levels of poly-N-acetyllactosamine (polyLacNAc) substituted β1,6 branched N-oligosaccharides on B16 melanoma variants has previously been shown to correlate with the metastatic potential of these cells. Lysosomal protein LAMP1 is one of the major carriers of these oligosaccharides and its expression on the cell surface also correlates with metastatic potential of B16 murine melanoma cells. To investigate whether these oligosaccharides have any role in increasing surface expression of LAMP1, low (B16F1) and high metastatic (B16F10) variants of B16 melanoma cells were treated with N-glycosylation inhibitor, swainsonine (SW). SW treatment resulted in significantly decreased expression of polyLacNAc substituted β1,6 branched N-oligosaccharides on these cells. This was also accompanied with significantly reduced expression of LAMP1 on the cell surface, although total levels of LAMP1 in these cells remained unaffected. This points towards a possibility that glycosylation modulates the surface expression of LAMP1 on tumor cells. The present study thus clearly underscores a novel role of N-glycosylation in regulating the surface translocation of a lysosomal membrane protein, LAMP1.
Available from: sciencedirect.com
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ABSTRACT: The endoplasmic reticulum is a major compartment of protein biogenesis in the cell, dedicated to production of secretory, membrane and organelle proteins. The secretome has distinct structural and post-translational characteristics, since folding in the ER occurs in an environment that is distinct in terms of its ionic composition, dynamics and requirements for quality control. The folding machinery in the ER therefore includes chaperones and folding enzymes that introduce, monitor and react to disulfide bonds, glycans, and fluctuations of luminal calcium. We describe the major chaperone networks in the lumen and discuss how they have distinct modes of operation that enable cells to accomplish highly efficient production of the secretome. This article is part of a Special Issue entitled:Functional and structural diversity of endoplasmic reticulum.
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ABSTRACT: The neuron-specific intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a member of the ICAM family of adhesion proteins. It has a complex structure with nine external immunoglobulin domains followed by a transmembrane and a cytoplasmic domain. The external part binds to beta1- and beta2-integrins and the matrix protein vitronectin, whereas its transmembrane domain binds to presenilins and the cytoplasmic domain to alpha-actinin and the ERM family of cytoplasmic proteins. In neurons it is confined to the soma and dendrites and it is enriched in dendritic filopodia with less expression in more mature dendritic spines. ICAM-5 strongly stimulates neurite outgrowth. ICAM-5 is cleaved by matrix metalloproteases upon activation of glutamate receptors or degraded through endocytosis resulting in increased spine maturation. Ablation of ICAM-5 expression increases functional synapse formation. The cleaved soluble fragment of ICAM-5 is immunosuppressive, which may be important in neuronal inflammatory diseases.
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