STRUCTURE OF THE N-LINKED OLIGOSACCHARIDES OF MHC CLASS-I MOLECULES FROM CELLS DEFICIENT IN THE ANTIGENIC PEPTIDE TRANSPORTER - IMPLICATIONS FOR THE SITE OF PEPTIDE ASSOCIATION

Class I molecules are N-linked glycoproteins encoded by the MHC, They carry cytosolic protein-derived peptides to the cell surface, displayi ng them to enable immune surveillance of cellular processes, Peptides are delivered to class I molecules by the transporter associated with Ag processing (TAP), Peptide association is known to occur before expo sure of class I molecules to the medial Golgi-processing enzyme alpha- mannosidase II, but there is limited information regarding the locatio n or timing of peptide binding within the earlier regions of the endop lasmic reticulum (ER)-Golgi pathway, A reported association of newly s ynthesized class I molecules with the ER chaperonin calnexin raises th e possibility of persistence of the monoglycosylated N-linked oligosac charide (NLO) Glc(1)Man(8)GlcNAc(2), known to be recognized by this le ctin, To explore these matters, we determined the structure of the NLO s on the subset of newly synthesized class I molecules awaiting the lo ading of peptide, We pulse-labeled murine MHC H-2D(b) class I molecule s in RMA/S cells, which lack one of the TAP subunits, causing the grea t majority of the molecules to be retained for prolonged periods in an early secretory compartment, awaiting peptide binding, MHC molecules pulse-labeled with [H-3]glucosamine were isolated, the NLOs specifical ly released and structurally analyzed by a variety of techniques. With in the chosen window of biosynthetic time, most D-b molecules from par ental RMA cells carried mature NLOs of the biantennary complex-type, w ith one to two sialic acid residues, In RMA/S cells, such chains were in the minority, the majority consisting of the precursor forms Man(8) GlcNAc(2) and Man(9)GlcNAc(2). No glucosylated forms were detected, no r were the later processing intermediates Man(5-7)GlcNAc(2) or GlcNAc( 1)Man(4-5)GlcNAc(2). Thus, most D-b molecules in TAP-deficient cells a re retained in an early compartment of the secretory pathway, before t he point of first access to the Golgi alpha-mannosidase I, which trims alpha 1-2 linked mannose residues, but beyond the point where the alp ha 1-3-linked glucose residue is finally removed by the ER glucosidase II, Thus, structural analysis of NLOs on class I molecules within a d efined biosynthetic window has established a biochemical measure of th e timing of peptide association.