Biomedical subjects
H C Wu
Publications and source records attributed to H C Wu.
Prolipoprotein signal peptidase in Escherichia coli is distinct from the M13 procoat protein signal peptidase.
We have previously reported a signal peptidase activity in Escherichia coli cell envelope which processes prolipoprotein modified with glyceride (Tokunaga, M., Tokunaga, H., and Wu, H. C. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2255-2259). To ascertain whether the processing enzyme for prolipoprotein is distinct from the signal peptidase for M13 procoat protein purified by Zwizinski and Wickner (Zwizinski, C., and Wickner, W. (1980) J. Biol. Chem. 255, 7973-7977), we have used antibody against purified procoat protein signal peptidase to study the processings of prolipoprotein and M13 procoat protein in vitro. the signal peptidase for modified prolipoprotein remained fully active in solubilized membrane preparations which had been treated with antibody against purified procoat protein signal peptidase whereas the activity towards procoat protein was completely abolished by immunoadsorption. Furthermore, both unmodified and glyceride-modified prolipoprotein were not cleaved by the highly purified signal peptidase preparation provided by Wickner. These data clearly indicate that prolipoprotein signal peptidase is distinct from the M13 procoat protein signal peptidase.
Receptors for a cytotoxic lectin, abrin and their role in cell intoxication.
The nature of binding of abrin to Chinese hamster ovary cells was examined in relation to the ensuing intoxication of the treated cells. Approx. 20% of [125I] abrin bound to CHO cells at 37 degree C was found to be resistant to the addition or presence of 0.1 M lactose. The extent of lactose-resistant binding depended inversely upon the temperature of incubation. Among various proteins, lectins and sugars, only non-labeled abrin could strongly inhibit the lactose-resistant binding of [125I] abrin. Lactose-resistant binding could lead to an inhibition of cellular protein synthesis and to a loss of cell viability. Abrin molecules bound at the lactose-sensitive and lactose-resistant binding sites apparently have an equal probability of being internalized by CHO cells. Binding of approx. 3.10(3) abrin molecules per CHO cell was required to elicit 50% loss of cell viability regardless of whether the binding occurs in the presence or absence of lactose. The result of a cross-linking experiment suggested that a membrane protein with an Mr of about 45 000 may be responsible for the lactose-resistant binding of abrin.
Temporal behavior of abrin in the intoxication of Chinese hamster cells (line CHO).
Abrin, a potent cytotoxin, was utilized as a probe to elucidate the mechanism by which external proteins are delivered to the cytoplasm of mammalian cells. Abrin bound rapidly to the surface receptors of the Chinese hamster cells (line CHO) and appeared to be internalized immediately without any significant lag. The maximum level of abrin internalization was achieved within eight minutes, based on both biochemical and electron microscopic autoradiographic studies with [125I]abrin. About 10% of the silver grains of internalized [125I] abrin were associated with vesicular structures, irrespective of the incubation time. Inhibition of protein synthesis began 30 minutes postincubation, and this latent period was not dependent on extracellular toxin concentration. SDS-polyacrylamide gel electrophoresis of the internalized [125I]abrin indicated that internalized abrin molecules remained intact even after two hours of incubation.
Biogenesis of membrane lipoproteins in Escherichia coli.
Globomycin-resistant mutants of Escherichia coli have been isolated and partially characterized. Approximately 2-5% of these mutants synthesize structurally altered Braun's lipoprotein. The majority of these mutants contain unprocessed and unmodified prolipoprotein. One mutant is found to contain modified, processed, but structurally altered lipoprotein. Mutants containing lipid-deficient prolipoprotein or lipoprotein also show increased resistance to globomycin. These results suggest that the inhibition of processing of modified prolipoprotein by globomycin may require fully modified prolipoprotein as the biochemical target of this novel antibiotic. Our failure to isolate mutant containing cleaved but unmodified lipoprotein among globomycin-resistant mutants is consistent with the possibility that modification of prolipoprotein precedes the removal of signal sequence by a unique signal peptidase. Recent evidence indicates that the minor lipoproteins in the cell envelope of E. coli are also synthesized as lipid-containing prolipoproteins and the processing of these prolipoproteins is inhibited by globomycin. These results suggest the existence of modifying enzymes in E. coli which would transfer glyceryl and fatty acyl moieties to cysteine residues located in the proper sequences of the precursor proteins. This speculation is confirmed by our demonstration that Bacillus licheniformis penicillinase synthesized in E. coli as well as in B. licheniformis is a lipoprotein containing glyceride-cysteine at its NH2-terminus.
Post-translational modification and processing of Escherichia coli prolipoprotein in vitro.
Escherichia coli strain MM18 cells containing malE-lacZ hybrid protein was reported to accumulate prolipoprotein when they were induced with maltose [Ito, K., Bassford, P. J. & Beckwith, J. (1981) Cell 24, 707-717]. We have shown that the prolipoprotein accumulated in maltose-induced MM18 cells is not modified, lacking covalently linked glyceride. When the cell envelope of MM18 containing unmodified prolipoprotein was incubated in the presence of detergent with [2-3H]glycerol-labeled cell envelope of strain JE5505 lacking murein lipoprotein, incorporation of [2-3H]glycerol radioactivity into both prolipoprotein and processed mature lipoprotein was observed. Likewise, when [3H]-palmitate-labeled JE5505 cell envelope was incubated with the MM18 cell envelope containing unmodified prolipoprotein in the presence of detergent, [3H]palmitate radioactivity was incorporated into prolipoprotein by ester linkage and into mature lipoprotein by both ester and amide linkages. These results indicate that our in vitro system contains activities of prolipoprotein modification and processing enzymes, including glyceryltransferase, O-acyltransferase, signal peptidase, and N-acyltransferase. The signal peptidase activity in our in vitro system was completely inhibited by globomycin. At pH 5.0, glyceryltransferase was inactive. Signal peptidase was active at pH 5.0, provided that prolipoprotein had been modified by glyceryltransferase (O-acyl-transferase) during a prior incubation at pH 9.1. These results strongly suggest that the modification of prolipoprotein by glyceryltransferase (and O-acyltransferase) precedes, and may in fact be a prerequisite for, the processing of prolipoprotein by signal peptidase.
Chinese hamster ovary cell mutants defective in the internalization of ricin.
Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin.
Bacillus licheniformis penicillinase synthesized in Escherichia coli contains covalently linked fatty acid and glyceride.
DNA sequence analysis of the structural gene for Bacillus licheniformis penicillinase has revealed a tetrapeptide sequence of Leu-Ala-Gly-Cys within the NH2-terminal part of the precursor form of penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6). The same tetrapeptide occurs in the signal sequence of the prolipoprotein of Escherichia coli, and the cysteine residue in the tetrapeptide of prolipoprotein is modified to form glyceride-cysteine which becomes the NH2 terminus of Braun's lipoprotein. On the basis of labeling, with [2-3H]glycerol, [3H]palmitate, [35S]methionine, and [35S]sulfuric acid, of an E. coli strain lysogenic for a lambda vector containing the penicillinase gene from B. licheniformis and of immunoprecipitation with rabbit antisera against purified B. licheniformis penicillinase, we conclude that B. licheniformis penicillinase synthesized in E. coli contains covalently linked glyceride and fatty acid. These results strongly suggest the operation of a modification system in E. coli, and presumably in other Gram-negative bacteria, which results in the formation of a glyceride-cysteine residue if the proper peptide sequence is present in the signal sequence of membrane proteins.
Globomycin sensitivity of Escherichia coli and Salmonella typhimurium: effects of mutations affecting structures of murein lipoprotein.
The sensitivity of strains of Escherichia coli and Salmonella typhimurium to globomycin is increased in mutants defective in the lipopolysaccharide structure. E. coli mutants altered in the structures or biosynthesis of murein lipoprotein are more resistant to globomycin than the parental strains.
Internalization of ricin in Chinese hamster ovary cells.
Internalization of ricin into Chinese hamster ovary cells has been investigated. Combined treatment with galactose and pronase at 0 degrees C resulted in a complete release of surface-bound [125I]ricin into the media. Galactose-pronase-resistant cell-bound [125I]ricin represents internalized ricin molecules inside the cells. The internalization process is time, temperature, and concentration dependent. The pH optimum of internalization of ricin is about pH 7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has revealed that intact ricin molecules are internalized. Neither reduction nor proteolytic processing of ricin is required for the entry of ricin into Chinese hamster ovary cells.
Enhancement of cytotoxicities of ricin and Pseudomonas toxin in Chinese hamster ovary cells by nigericin.
Nigericin and monensin, ionophores for Na+ and K+, have been found to enhance the cytotoxicities of abrin, ricin, and Pseudomonas aeruginosa exotoxin A in Chinese hamster ovary (CHO) cells. They do not affect the cytotoxicity of diphtheria toxin in the same cell line. Maximal sensitization of the CHO cells toward ricin and Pseudomonas toxin requires preculture of CHO cells in the presence of nigericin. Inhibition of protein synthesis in CHO cells by ricin or Pseudomonas toxin is also enhanced by preculture of CHO cells in the presence of nigericin. These results suggest a common step in the intoxication process of ricin and Pseudomonas toxin, the rate of which is facilitated by pretreatment with nigericin. This step is, however, not shared by the intoxication of CHO cells with diphtheria toxin.
Enhanced internalization of ricin in nigericin-pretreated Chinese hamster ovary cells.
Biochemical and electron microscopic autoradiographic studies with [125I] ricin have revealed that nigericin-pretreated Chinese hamster ovary cells are more efficient than untreated cells in the internalization of the toxin into the cells. These results suggest that the enhanced rate of internalization of ricin in nigericin-pretreated cells may account for the enhancement of cytotoxicity of ricin in Chinese hamster ovary cells by nigericin.
Acyl moieties in phospholipids are the precursors for the fatty acids in murein lipoprotein of Escherichia coli.
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Purification and characterization of the outer membrane lipoprotein from an Escherichia coli mutant altered in the signal sequence of prolipoprotein.
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Assembly of membrane proteins in Escherichia coli.
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Assembly of the outer membrane lipoprotein in Escherichia coli.
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Characterization of murein-bound lipoprotein in an Excherichia coli mutant altered in the signal sequence of prolipoprotein.
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Biosynthesis and assembly of murein lipoprotein in Escherichia coli.
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