1 Experimental Animal Models, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, Københavns Universitet2 Carroll University3 Porse Group, BRIC Research Groups, BRIC, Københavns Universitet4 Section of Biomedicine, Department of Veterinary Disease Biology, Faculty of Life Sciences, Københavns Universitet5 Experimental Animal Models, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, Københavns Universitet6 Porse Group, BRIC Research Groups, BRIC, Københavns Universitet7 Section of Biomedicine, Department of Veterinary Disease Biology, Faculty of Life Sciences, Københavns Universitet
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, in order to avoid immune recognition. Virus infection and inflammation alter protein N-glycosylation and we have previously shown that changes in cellular N-glycosylation, is 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 (N8) 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 (T24) in the extracellular domain of MICA018 was essential for the N-glycosylation dependency, while 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 dependency. In addition we show that this regulatory mechanism of MICA surface expression is likely targeted during different pathological conditions.
Journal of Biological Chemistry, 2014, Vol 289, Issue 29, p. 20078-20091