Interaction of mitogenic bacterial lipoprotein and a synthetic analogue with mouse lymphocytes. Isolation and characterization of binding proteins.
Lipoprotein from the outer membrane of Escherichia coli constitutes a potent mitogen and polyclonal activator for B lymphocytes of different species. The binding of lipoprotein to murine spleen cells was investigated using water-soluble 125I-labelled citraconylated lipoprotein from E. coli B/r. Our results indicate that the binding of this B-cell mitogen to splenocytes is a saturable, time- and dose-dependent, reversible process; about 9.7 X 10(8) lipoprotein molecules were bound to each cell. The mechanism of the binding of lipoprotein to lymphocytes was investigated by using the synthetic analogue of its N-terminal part, S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteinyl-( S)-seryl- (S)-seryl-(S)-asparaginyl-(S)-alanine (tripalmitoyl pentapeptide). This compound had been shown by us previously to be the molecular part of lipoprotein responsible for mitogenicity and exhibited, in all experiments performed, a stimulatory activity towards B lymphocytes comparable, or even superior, to native lipoprotein. Binding proteins for the synthetic N-terminus were enriched by affinity chromatography, using an affinity column prepared by coupling the mitogenic compound to CPG-aminopropyl controlled-pore glass beads by the carbodiimide method. [3H]Leucine-labelled murine spleen cells were solubilized by the nonionic detergent NP40 and applied to the affinity adsorbent. Proteins bound to the column were selectively eluted by a solution of tripalmitoyl pentapeptide, and the fractions were analyzed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and autoradiography. Our results indicate the presence of a major binding protein of Mr 35000 on mouse primary lymphocytes for the biologically active N-terminal structure of lipoprotein, which might play a role as membrane receptor in mitogenic B lymphocyte activation.
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D055666:Lipopeptides; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008934:Mitogens / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D010779:Photofluorography; D013154:Spleen / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D055666:Lipopeptides; D008934:Mitogens; D009842:Oligopeptides; C051659:mitogenic bacterial lipoprotein, E coli; C038293:2,3-bis(palmitoyloxy)-2-propyl-N-palmitoyl-cysteinyl-seryl-seryl-asparaginyl-alanine