N-linked glycosylation selectively regulates the generic folding of HLA-Cw1.
To resolve primary (glycosylation-assisted) from secondary (glycosylation-independent) quality control steps in the biosynthesis of HLA (human leukocyte antigen) class I glycoproteins, the unique N-linked glycosylation site of the HLA-Cw1 heavy chain was deleted by site-directed mutagenesis. The non-glycosylated Cw1S88G mutant was characterized by flow cytometry, pulse-chase, co-immunoprecipitation, and in vitro assembly assays with synthetic peptide ligands upon transfection in 721.221 and 721.220 cells. The former provide a full set of primary as well as secondary chaperoning interactions, whereas the latter are unable to perform secondary quality control (e.g. proper class I assembly with peptide antigens) as a result of a functional defect of the HLA-dedicated chaperone tapasin. In both transfectants, Cw1S88G displayed a loss/weakening in its generic chaperoning interaction with calreticulin and/or ERp57 and became redistributed toward calnexin, known to bind the most unfolded class I conformers. Despite this, and quite unexpectedly, a weak interaction with the HLA-dedicated chaperone TAP was selectively retained in 721.221. In addition, the ordered, stepwise acquisition of thermal stability/peptide binding was disrupted, resulting in a heterogeneous ensemble of Cw1S88G conformers with unorthodox and unprecedented peptide assembly features. Because a lack of glycosylation and a lack of tapasin-assisted peptide loading have distinct, complementary, and additive effects, the former is separable from (and upstream of) the latter, e.g. primary quality control is suggested to supervise a crucial, generic folding step preliminary to the acquisition of peptide receptivity.
Martayan
Aline
A
Laboratory of Immunology, Regina Elena National Cancer Research Institute, Centro della Ricerca Sperimentale, Via delle Messi d'Oro 156, 00158 Rome, Italy.
Sibilio
Leonardo
L
Setini
Andrea
A
Lo Monaco
Elisa
E
Tremante
Elisa
E
Fruci
Doriana
D
Colonna
Marco
M
Giacomini
Patrizio
P
D001671:Biochemistry / Q000379:methods; D006031:Glycosylation; D015236:HLA-C Antigens / Q000737:chemistry*; D006801:Humans; D047468:Immunoprecipitation; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000737:chemistry; D017510:Protein Folding; D014162:Transfection
D015236:HLA-C Antigens; C047431:HLA-Cw1 antigen; D008024:Ligands; D026901:Membrane Transport Proteins; D010455:Peptides; C107762:tapasin; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human