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. 1986 Apr 1;102(4):1284–1297. doi: 10.1083/jcb.102.4.1284

Posttranslational processing of concanavalin A precursors in jackbean cotyledons

PMCID: PMC2114185  PMID: 3958046

Abstract

Metabolic labeling of immature jackbean cotyledons with 14C-amino acids was used to determine the processing steps involved in the assembly of concanavalin A. Pulse-chase experiments and analyses of immunoprecipitated lectin forms indicated a complex series of events involving seven distinct species. The structural relatedness of all of the intermediate species was confirmed by two-dimensional mapping of 125I-tryptic peptides. An initial glycosylated precursor was deglycosylated and cleaved into smaller polypeptides, which subsequently reannealed over a period of 10-27 h. NH2-terminal sequencing of the abundant precursors confirmed that the intact subunit of concanavalin A was formed by the reannealing of two fragments, since the alignment of residues 1-118 and 119-237 was reversed in the final form of the lectin identified in the chase and the precursor first labeled. When the tissue was pulse-chased in the presence of monensin, processing of the glycosylated precursor was inhibited. The weak bases NH4Cl and chloroquine were without effect. Immunocytochemical studies showed that monensin treatment caused the accumulation of immunoreactive material at the cell surface and indicated that the ionophore had induced the secretion of a component normally destined for deposition within the protein bodies. Consideration of the tertiary structure of the glycosylated precursor and mature lectin showed that the entire series of processing events could occur without significant refolding of the initial translational product. Proteolytic events included removal of a peptide from the surface of the precursor molecule that connected the NH2- and COOH-termini of the mature protein. This processing activated the carbohydrate-binding activity of the lectin. The chase data suggest the occurrence of a simultaneous cleavage and formation of a peptide bond, raising the possibility that annealment of the fragments to give rise to the mature subunit involves a transpeptidation event rather than cleavage and subsequent religation.

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Selected References

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  1. Barton K. A., Thompson J. F., Madison J. T., Rosenthal R., Jarvis N. P., Beachy R. N. The biosynthesis and processing of high molecular weight precursors of soybean glycinin subunits. J Biol Chem. 1982 Jun 10;257(11):6089–6095. [PubMed] [Google Scholar]
  2. Bauer K., Lipmann F. Attempts toward biosynthesis of the thyrotropin-releasing hormone and studies on its breakdown in hypothalamic tissue preparations. Endocrinology. 1976 Jul;99(1):230–242. doi: 10.1210/endo-99-1-230. [DOI] [PubMed] [Google Scholar]
  3. Becker J. W., Reeke G. N., Jr, Wang J. L., Cunningham B. A., Edelman G. M. The covalent and three-dimensional structure of concanavalin A. III. Structure of the monomer and its interactions with metals and saccharides. J Biol Chem. 1975 Feb 25;250(4):1513–1524. [PubMed] [Google Scholar]
  4. Bowles D. J., Chaplin M. F., Marcus S. E. Interaction of concanavalin A with native and denatured forms of jackbean alpha-D-mannosidase. Eur J Biochem. 1983 Feb 15;130(3):613–618. doi: 10.1111/j.1432-1033.1983.tb07193.x. [DOI] [PubMed] [Google Scholar]
  5. Carrington D. M., Auffret A., Hanke D. E. Polypeptide ligation occurs during post-translational modification of concanavalin A. Nature. 1985 Jan 3;313(5997):64–67. doi: 10.1038/313064a0. [DOI] [PubMed] [Google Scholar]
  6. Chrispeels M. J., Higgins T. J., Craig S., Spencer D. Role of the endoplasmic reticulum in the synthesis of reserve proteins and the kinetics of their transport to protein bodies in developing pea cotyledons. J Cell Biol. 1982 Apr;93(1):5–14. doi: 10.1083/jcb.93.1.5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cunningham B. A., Hemperly J. J., Hopp T. P., Edelman G. M. Favin versus concanavalin A: Circularly permuted amino acid sequences. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3218–3222. doi: 10.1073/pnas.76.7.3218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cunningham B. A., Wang J. L., Waxdal M. J., Edelman G. M. The covalent and three-dimensional structure of concanavalin A. II. Amino acid sequence of cyanogen bromide fragment F3. J Biol Chem. 1975 Feb 25;250(4):1503–1512. [PubMed] [Google Scholar]
  9. Dean R. T., Jessup W., Roberts C. R. Effects of exogenous amines on mammalian cells, with particular reference to membrane flow. Biochem J. 1984 Jan 1;217(1):27–40. doi: 10.1042/bj2170027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Edmundson A. B., Ely K. R., Sly D. A., Westholm F. A., Powers D. A., Liener I. E. Isolation and characterization of concanavalin A polypeptide chains. Biochemistry. 1971 Sep 14;10(19):3554–3559. doi: 10.1021/bi00795a010. [DOI] [PubMed] [Google Scholar]
  11. Elder J. H., Pickett R. A., 2nd, Hampton J., Lerner R. A. Radioiodination of proteins in single polyacrylamide gel slices. Tryptic peptide analysis of all the major members of complex multicomponent systems using microgram quantities of total protein. J Biol Chem. 1977 Sep 25;252(18):6510–6515. [PubMed] [Google Scholar]
  12. Ereken-Tumer N., Richter J. D., Nielsen N. C. Structural characterization of the glycinin precursors. J Biol Chem. 1982 Apr 25;257(8):4016–4018. [PubMed] [Google Scholar]
  13. Foriers A., Lebrun E., Van Rapenbusch R., de Neve R., Strosberg A. D. The structure of the lentil (Lens culinaris) lectin. Amino acid sequence determination and prediction of the secondary structure. J Biol Chem. 1981 Jun 10;256(11):5550–5560. [PubMed] [Google Scholar]
  14. Foriers A., de Neve R., Kanarek L., Strosberg A. D. Common ancestor for concanavalin A and lentil lectin? Proc Natl Acad Sci U S A. 1978 Mar;75(3):1136–1139. doi: 10.1073/pnas.75.3.1136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Glickman J., Croen K., Kelly S., Al-Awqati Q. Golgi membranes contain an electrogenic H+ pump in parallel to a chloride conductance. J Cell Biol. 1983 Oct;97(4):1303–1308. doi: 10.1083/jcb.97.4.1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hardman K. D., Ainsworth C. F. Structure of the concanavalin A-methyl alpha-D-mannopyranoside complex at 6-A resolution. Biochemistry. 1976 Mar 9;15(5):1120–1128. doi: 10.1021/bi00650a026. [DOI] [PubMed] [Google Scholar]
  17. Hemperly J. J., Mostov K. E., Cunningham B. A. In vitro translation and processing of a precursor form of favin, a lectin from Vicia faba. J Biol Chem. 1982 Jul 10;257(13):7903–7909. [PubMed] [Google Scholar]
  18. Herbert E., Uhler M. Biosynthesis of polyprotein precursors to regulatory peptides. Cell. 1982 Aug;30(1):1–2. doi: 10.1016/0092-8674(82)90002-2. [DOI] [PubMed] [Google Scholar]
  19. Higgins T. J., Chandler P. M., Zurawski G., Button S. C., Spencer D. The biosynthesis and primary structure of pea seed lectin. J Biol Chem. 1983 Aug 10;258(15):9544–9549. [PubMed] [Google Scholar]
  20. Higgins T. J., Chrispeels M. J., Chandler P. M., Spencer D. Intracellular sites of synthesis and processing of lectin in developing pea cotyledons. J Biol Chem. 1983 Aug 10;258(15):9550–9552. [PubMed] [Google Scholar]
  21. Izaki K., Matsuhashi M., Strominger J. L. Biosynthesis of the peptidoglycan of bacterial cell walls. 8. Peptidoglycan transpeptidase and D-alanine carboxypeptidase: penicillin-sensitive enzymatic reaction in strains of Escherichia coli. J Biol Chem. 1968 Jun 10;243(11):3180–3192. [PubMed] [Google Scholar]
  22. Jacobs S., Kull F. C., Jr, Cuatrecasas P. Monensin blocks the maturation of receptors for insulin and somatomedin C: identification of receptor precursors. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1228–1231. doi: 10.1073/pnas.80.5.1228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Joseph T., Higgins V., Spencer D. Precursor Forms of Pea Vicilin Subunits: MODIFICATION BY MICROSOMAL MEMBRANES DURING CELL-FREE TRANSLATION. Plant Physiol. 1981 Feb;67(2):205–211. doi: 10.1104/pp.67.2.205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Julius D., Schekman R., Thorner J. Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathway. Cell. 1984 Feb;36(2):309–318. doi: 10.1016/0092-8674(84)90224-1. [DOI] [PubMed] [Google Scholar]
  25. Kreibich G., Ojakian G., Rodriguez-Boulan E., Sabatini D. D. Recovery of ribophorins and ribosomes in "inverted rough" vesicles derived from rat liver rough microsomes. J Cell Biol. 1982 Apr;93(1):111–121. doi: 10.1083/jcb.93.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  27. Laursen R. A. Solid-phase Edman degradation. An automatic peptide sequencer. Eur J Biochem. 1971 May 11;20(1):89–102. doi: 10.1111/j.1432-1033.1971.tb01366.x. [DOI] [PubMed] [Google Scholar]
  28. Lesk A. M., Hardman K. D. Computer-generated schematic diagrams of protein structures. Science. 1982 Apr 30;216(4545):539–540. doi: 10.1126/science.7071602. [DOI] [PubMed] [Google Scholar]
  29. Lord J. M. Precursors of ricin and Ricinus communis agglutinin. Glycosylation and processing during synthesis and intracellular transport. Eur J Biochem. 1985 Jan 15;146(2):411–416. doi: 10.1111/j.1432-1033.1985.tb08667.x. [DOI] [PubMed] [Google Scholar]
  30. Lord J. M. Synthesis and intracellular transport of lectin and storage protein precursors in endosperm from castor bean. Eur J Biochem. 1985 Jan 15;146(2):403–409. doi: 10.1111/j.1432-1033.1985.tb08666.x. [DOI] [PubMed] [Google Scholar]
  31. Lotan R., Lis H., Sharon N. Aggregation and fragmentation of soybean agglutinin. Biochem Biophys Res Commun. 1975 Jan 6;62(1):144–150. doi: 10.1016/s0006-291x(75)80416-5. [DOI] [PubMed] [Google Scholar]
  32. Lowe D. A., Richardson N. P., Taylor P., Donatsch P. Increasing intracellular sodium triggers calcium release from bound pools. Nature. 1976 Mar 25;260(5549):337–338. doi: 10.1038/260337a0. [DOI] [PubMed] [Google Scholar]
  33. Marcus S. E., Burgess J., Maycox P. R., Bowles D. J. A study of maturation events in jackbeans (Canavalia ensiformis). Biochem J. 1984 Aug 15;222(1):265–268. doi: 10.1042/bj2220265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Meehan E. J., Jr, McDuffie J., Einspahr H., Bugg C. E., Suddath F. L. The crystal structure of pea lectin at 6-A resolution. J Biol Chem. 1982 Nov 25;257(22):13278–13282. [PubMed] [Google Scholar]
  35. Meiri H., Erulkar S. D., Lerman T., Rahamimoff R. The action of the sodium ionophore, monensin, or transmitter release at the frog neuromuscular junction. Brain Res. 1981 Jan 5;204(1):204–208. doi: 10.1016/0006-8993(81)90665-x. [DOI] [PubMed] [Google Scholar]
  36. Perlman R. L., Cossi A. F., Role L. W. Mechanisms of ionophore-induced catecholamine secretion. J Pharmacol Exp Ther. 1980 May;213(2):241–246. [PubMed] [Google Scholar]
  37. Pohlmann R., Krüger S., Hasilik A., von Figura K. Effect of monensin on intracellular transport and receptor-mediated endocytosis of lysosomal enzymes. Biochem J. 1984 Feb 1;217(3):649–658. doi: 10.1042/bj2170649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Reeke G. N., Jr, Becker J. W., Edelman G. M. The covalent and three-dimensional structure of concanavalin A. IV. Atomic coordinates, hydrogen bonding, and quaternary structure. J Biol Chem. 1975 Feb 25;250(4):1525–1547. [PubMed] [Google Scholar]
  39. Snell C. R., Smyth D. G. Proinsulin: a proposed three-dimensional structure. J Biol Chem. 1975 Aug 25;250(16):6291–6295. [PubMed] [Google Scholar]
  40. Spencer D., Higgins T. J., Button S. C., Davey R. A. Pulse-labeling Studies on Protein Synthesis in Developing Pea Seeds and Evidence of a Precursor Form of Legumin Small Subunit. Plant Physiol. 1980 Sep;66(3):510–515. doi: 10.1104/pp.66.3.510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Suchard S. J., Lattanzio F. A., Jr, Rubin R. W., Pressman B. C. Stimulation of catecholamine secretion from cultured chromaffin cells by an ionophore-mediated rise in intracellular sodium. J Cell Biol. 1982 Sep;94(3):531–539. doi: 10.1083/jcb.94.3.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tartakoff A. M. Perturbation of vesicular traffic with the carboxylic ionophore monensin. Cell. 1983 Apr;32(4):1026–1028. doi: 10.1016/0092-8674(83)90286-6. [DOI] [PubMed] [Google Scholar]
  43. Tartakoff A., Vassalli P. Plasma cell immunoglobulin M molecules. Their biosynthesis, assembly, and intracellular transport. J Cell Biol. 1979 Nov;83(2 Pt 1):284–299. doi: 10.1083/jcb.83.2.284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Van der Wilden W., Herman E. M., Chrispeels M. J. Protein bodies of mung bean cotyledons as autophagic organelles. Proc Natl Acad Sci U S A. 1980 Jan;77(1):428–432. doi: 10.1073/pnas.77.1.428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vitale A., Ceriotti A., Bollini R., Chrispeels M. J. Biosynthesis and processing of phytohemagglutinin in developing bean cotyledons. Eur J Biochem. 1984 May 15;141(1):97–104. doi: 10.1111/j.1432-1033.1984.tb08162.x. [DOI] [PubMed] [Google Scholar]
  46. Vitale A., Chrispeels M. J. Transient N-acetylglucosamine in the biosynthesis of phytohemagglutinin: attachment in the Golgi apparatus and removal in protein bodies. J Cell Biol. 1984 Jul;99(1 Pt 1):133–140. doi: 10.1083/jcb.99.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Vodkin L. O. Isolation and Characterization of Messenger RNAs for Seed Lectin and Kunitz Trypsin Inhibitor in Soybeans. Plant Physiol. 1981 Sep;68(3):766–771. doi: 10.1104/pp.68.3.766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wachter E., Machleidt W., Hofner H., Otto J. Aminopropyl glass and its p-phenylene diisothiocyanate derivative, a new support in solid-phase Edman degradation of peptides and proteins. FEBS Lett. 1973 Sep 1;35(1):97–102. doi: 10.1016/0014-5793(73)80585-x. [DOI] [PubMed] [Google Scholar]
  49. Wang J. L., Cunningham B. A., Edelman G. M. Unusual fragments in the subunit structure of concanavalin A. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1130–1134. doi: 10.1073/pnas.68.6.1130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wang J. L., Cunningham B. A., Waxdal M. J., Edelman G. M. The covalent and three-dimensional structural of concanavalin A. I. Amino acid sequence of cyanogen bromide fragments F1 and F2. J Biol Chem. 1975 Feb 25;250(4):1490–1502. [PubMed] [Google Scholar]
  51. Watson E. L., Farnham C. J., Friedman J., Farnham W. Effects of monensin on amylase release from mouse parotid acini. Am J Physiol. 1981 May;240(5):C189–C192. doi: 10.1152/ajpcell.1981.240.5.C189. [DOI] [PubMed] [Google Scholar]
  52. Waxdal M. J., Wang J. L., Pflumm M. N., Edelman G. M. Isolation and order of the cyanogen bromide fragments of concanavalin A. Biochemistry. 1971 Aug 31;10(18):3343–3347. doi: 10.1021/bi00794a004. [DOI] [PubMed] [Google Scholar]
  53. Wright H. T. Comparison of the crystal structures of chymotrypsinogen-A and alpha-chymotrypsin. J Mol Biol. 1973 Sep 5;79(1):1–11. doi: 10.1016/0022-2836(73)90265-9. [DOI] [PubMed] [Google Scholar]
  54. Zhang F., Schneider D. L. The bioenergetics of Golgi apparatus function: evidence for an ATP-dependent proton pump. Biochem Biophys Res Commun. 1983 Jul 29;114(2):620–625. doi: 10.1016/0006-291x(83)90825-2. [DOI] [PubMed] [Google Scholar]
  55. Zimmerman C. L., Appella E., Pisano J. J. Rapid analysis of amino acid phenylthiohydantoins by high-performance liquid chromatography. Anal Biochem. 1977 Feb;77(2):569–573. doi: 10.1016/0003-2697(77)90276-7. [DOI] [PubMed] [Google Scholar]

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