Phenotype-dependent differences in proteasome subunit composition and cleavage specificity in B cell lines.
We have compared the subunit composition and enzymatic activity of purified 26S proteasomes from Burkitt's lymphoma (BL) cells and in vitro EBV-transformed lymphoblastoid cell lines (LCLs) of normal B cell origin. Low expression of the IFN-gamma-regulated beta low molecular mass polypeptide (Lmp)2, Lmp7, and MECL-1 was demonstrated in a panel of seven BL lines that express the germinal center cell phenotype of the original tumor. Coexpression of Lmp2 and Lmp7 with the constitutively expressed subunits delta and MB1 was demonstrated in the BL lines by immunoprecipitation and two-dimensional gel fractionation of the 20S proteasomes. Coexpression of these subunits correlated with reduced levels of chymotrypsin- and trypsin-like activities detected by the cleavage of fluorogenic substrates. Down-regulation of Lmp2 and Lmp7 and decreased chymotrypsin- and trypsin-like activities were also observed in purified proteasomes from a c-myc-transfected subline of the ER/EB2-5 LCL that has adopted a BL-like phenotype. A synthetic peptide analogue of the immunodominant epitope from the EBV nuclear Ag 4 (E4416-424Y) was cleaved by proteasomes from BLs and A1, while proteasomes from LCLs were inactive. Cleavage of the E4416-424Y peptide was not affected by treatment of the BL cells with IFN-gamma despite both significant up-regulation of Lmp2 and Lmp7 and reconstitution of chymotrypsin and trypsin-like activities against fluorogenic substrates to LCL-like levels. The results demonstrate that B cell lines representing different stages of B cell activation and differentiation express proteasomes with different subunit compositions and enzymatic activity. This may result in the generation of a distinct set of endogenous peptides and influence the immunogenicity of these cells.
Frisan
T
T
Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden. teresa.frisan@mtc.ki.se
Levitsky
V
V
Polack
A
A
Masucci
M G
MG
D016175:B-Lymphocyte Subsets / Q000145:classification* / Q000201:enzymology* / Q000276:immunology; D002051:Burkitt Lymphoma; D002461:Cell Line, Transformed; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000276:immunology; D000939:Epitopes / Q000378:metabolism*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism; D004854:Herpesvirus 4, Human; D006801:Humans; D006868:Hydrolysis; D007371:Interferon-gamma / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000032:analysis / Q000276:immunology* / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis; D014407:Tumor Cells, Cultured; D014763:Viral Matrix Proteins / Q000096:biosynthesis
C037662:EBV-associated membrane antigen, Epstein-Barr virus; D000939:Epitopes; D019309:Epstein-Barr Virus Nuclear Antigens; D009097:Multienzyme Complexes; D014763:Viral Matrix Proteins; D007371:Interferon-gamma; D003546:Cysteine Endopeptidases; C077436:LMP7 protein; C098948:PSMB10 protein, human; D046988:Proteasome Endopeptidase Complex