Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 283, Issue 24, Pages 16469-16476Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M709175200
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- Associazione Italiana per la Ricerca sul Cancro Funding Source: Custom
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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.
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