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.
- SourceAvailable from: Akhil Agarwal[Show abstract] [Hide abstract]
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, LAMP1.05/2014; 52014:556-563.