N-Glycosylation of Asparagine 8 Regulates Surface Expression of Major Histocompatibility Complex Class I Chain-related Protein A (MICA) Alleles Dependent on Threonine 24

• Published in: The Journal of biological chemistry 2014-07, Vol.289 (29), p.20078-20091
• Main Authors: Mellergaard, Maiken, Skovbakke, Sarah Line, Schneider, Christine L., Lauridsen, Felicia, Andresen, Lars, Jensen, Helle, Skov, Søren
• Format: Article
• Language: English
• Subjects: Alleles; Amino Acid Sequence; Amino Acid Substitution; Asparagine - chemistry; Binding Sites - genetics; Carrier Proteins - immunology; Carrier Proteins - metabolism; Cell Line; Cell Membrane - immunology; Cell Membrane - metabolism; Genetic Polymorphism; Glycosylation; Herpesvirus; Herpesvirus 7, Human - immunology; Histocompatibility Antigens Class I - chemistry; Histocompatibility Antigens Class I - genetics; Histocompatibility Antigens Class I - metabolism; Humans; Immunology; Innate Immunity; Ligands; Melanoma; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Natural Killer Cells (NK Cells); NK Cell Lectin-Like Receptor Subfamily K - metabolism; Protein Processing, Post-Translational; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Threonine - chemistry; Viral Proteins - immunology; Viral Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.573238

NKG2D is an activating receptor expressed on several types of human lymphocytes. NKG2D ligands can be induced upon cell stress and are frequently targeted post-translationally in infected or transformed cells to avoid immune recognition. Virus infection and inflammation alter protein N-glycosylation, and we have previously shown that changes in cellular N-glycosylation are involved in regulation of NKG2D ligand surface expression. The specific mode of regulation through N-glycosylation is, however, unknown. Here we investigated whether direct N-glycosylation of the NKG2D ligand MICA itself is critical for cell surface expression and sought to identify the essential residues. We found that a single N-glycosylation site (Asn8) was important for MICA018 surface expression. The frequently expressed MICA allele 008, with an altered transmembrane and intracellular domain, was not affected by mutation of this N-glycosylation site. Mutational analysis revealed that a single amino acid (Thr24) in the extracellular domain of MICA018 was essential for the N-glycosylation dependence, whereas the intracellular domain was not involved. The HHV7 immunoevasin, U21, was found to inhibit MICA018 surface expression by affecting N-glycosylation, and the retention was rescued by T24A substitution. Our study reveals N-glycosylation as an allele-specific regulatory mechanism important for regulation of surface expression of MICA018, and we pinpoint the residues essential for this N-glycosylation dependence. In addition, we show that this regulatory mechanism of MICA surface expression is likely targeted during different pathological conditions. Background: Immune activation through surface expression of the human NKG2D ligand MICA is important in clearance of virus-infected or cancerous cells. Results: Molecular characterization of N-glycosylation in regulation of MICA cell surface expression. Conclusion: Surface expression of MICA alleles vary in dependence for N-glycosylation. Significance: We identify N-glycosylation as an allele-specific regulatory mechanism of MICA and pinpoint the essential residues.