Time course of intracellular associations, processing, and cleavages of Ii forms and class II major histocompatibility complex molecules.
To determine how changing forms of class II major histocompatibility complex proteins and associated Ii molecules in intracellular compartments of human B lymphocytes might regulate or catalyze antigen processing or presentation, we analyzed immunoprecipitates of such molecules from subcellular fractions of [35S]methionine pulse-chase-labeled, 3-day-activated B lymphocytes after homogenization and distribution in Percoll density gradients. Two-dimensional gel electrophoresis of immunoprecipitates of subcellular fractions demonstrated: 1) progressive sialic acid addition to class II major histocompatibility complex beta chains and Ii but not to gamma 2, gamma 2', gamma 3, gamma 3' (p35), or p41 and its satellites; 2) association of p35 and p41 with class II complexes at 30-60 min after pulse labeling; 3) cleavage of an immature form of Ii without sialic acid at 15-30 min after pulse labeling to a COOH-terminal, 25,000-dalton fragment, p25, with a 60-90-min half-life; 4) the presence of Ii-related p29 at only 30-min chase times; 5) an effect of chloroquine or monensin, at maximal nontoxic doses, to increase (a) the time for associations of p35 and p41 with class II complexes and (b) the half-life of p25, which was then formed from Ii at reduced levels. In addition, while the half-lives of class II alpha and beta chains and Ii were comparable within intracellular fractions of any one density, in intracellular fractions of intermediate densities the complexes appeared to be longer lived (much greater than 6 h) than in lighter fractions (2-3-h half-lives).
Department of Pediatrics, University of Massachusetts Medical School, Worcester 01655.
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Nguyen
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Humphreys
D001402:B-Lymphocytes / Q000378:metabolism*; D002451:Cell Compartmentation; D002499:Centrifugation, Density Gradient; D000090266:Chromobox Protein Homolog 5; D006207:Half-Life; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D006801:Humans; D008715:Methionine / Q000493:pharmacokinetics; D008985:Monensin / Q000494:pharmacology; D019158:N-Acetylneuraminic Acid; D011499:Protein Processing, Post-Translational; D012794:Sialic Acids / Q000378:metabolism; D013997:Time Factors
C000715594:CBX5 protein, human; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D012794:Sialic Acids; D000090266:Chromobox Protein Homolog 5; D008985:Monensin; D008715:Methionine; D019158:N-Acetylneuraminic Acid