Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 Sep;84(17):6184–6188. doi: 10.1073/pnas.84.17.6184

Use of a synthetic peptide antigen to generate antisera reactive with a proteolytic processing site in native human proinsulin: demonstration of cleavage within clathrin-coated (pro)secretory vesicles.

D F Steiner, J Michael, R Houghten, M Mathieu, P R Gardner, M Ravazzola, L Orci
PMCID: PMC299034  PMID: 3306670

Abstract

Polyclonal antibodies reactive with a cleavage site in human proinsulin (HPI) (C-peptide-A-chain junction) have been raised (rabbit, guinea pig) using a synthetic peptide antigen coupled with keyhole limpet hemocyanin. These antisera recognize native HPI and des-31,32-HPI equally well but react 20-50 times less well with des-64,65-HPI, the intermediate cleaved at the C-peptide-A-chain junction and lacking the Lys-Arg pair. The guinea pig antisera did not recognize insulin but reacted weakly with C peptide at high concentrations; the rabbit antisera reacted with neither insulin nor C peptide. Immunocytochemical studies with human islet tissue localized the immunoreactivity of these antisera to clathrin-coated (pro)secretory vesicles derived from the trans Golgi, indicating that cleavage of the C-peptide-A-chain junction of proinsulin occurs mainly, if not exclusively, in this compartment of the beta cell.

Full text

PDF
6184

Images in this article

6187Image
on p.6187

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Armbruster B. L., Carlemalm E., Chiovetti R., Garavito R. M., Hobot J. A., Kellenberger E., Villiger W. Specimen preparation for electron microscopy using low temperature embedding resins. J Microsc. 1982 Apr;126(Pt 1):77–85. doi: 10.1111/j.1365-2818.1982.tb00358.x. [DOI] [PubMed] [Google Scholar]
  2. Cohen R. M., Given B. D., Licinio-Paixao J., Provow S. A., Rue P. A., Frank B. H., Root M. A., Polonsky K. S., Tager H. S., Rubenstein A. H. Proinsulin radioimmunoassay in the evaluation of insulinomas and familial hyperproinsulinemia. Metabolism. 1986 Dec;35(12):1137–1146. doi: 10.1016/0026-0495(86)90027-2. [DOI] [PubMed] [Google Scholar]
  3. Houghten R. A. General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5131–5135. doi: 10.1073/pnas.82.15.5131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Kemmler W., Peterson J. D., Steiner D. F. Studies on the conversion of proinsulin to insulin. I. Conversion in vitro with trypsin and carboxypeptidase B. J Biol Chem. 1971 Nov 25;246(22):6786–6791. [PubMed] [Google Scholar]
  5. Leszczynski J. F., Rose G. D. Loops in globular proteins: a novel category of secondary structure. Science. 1986 Nov 14;234(4778):849–855. doi: 10.1126/science.3775366. [DOI] [PubMed] [Google Scholar]
  6. Madsen O. D., Cohen R. M., Fitch F. W., Rubenstein A. H., Steiner D. F. The production and characterization of monoclonal antibodies specific for human proinsulin using a sensitive microdot assay procedure. Endocrinology. 1983 Dec;113(6):2135–2144. doi: 10.1210/endo-113-6-2135. [DOI] [PubMed] [Google Scholar]
  7. Madsen O. D., Frank B. H., Steiner D. F. Human proinsulin-specific antigenic determinants identified by monoclonal antibodies. Diabetes. 1984 Oct;33(10):1012–1016. doi: 10.2337/diab.33.10.1012. [DOI] [PubMed] [Google Scholar]
  8. Mojsov S., Heinrich G., Wilson I. B., Ravazzola M., Orci L., Habener J. F. Preproglucagon gene expression in pancreas and intestine diversifies at the level of post-translational processing. J Biol Chem. 1986 Sep 5;261(25):11880–11889. [PubMed] [Google Scholar]
  9. Moore H. H., Kelly R. B. Re-routing of a secretory protein by fusion with human growth hormone sequences. Nature. 1986 May 22;321(6068):443–446. doi: 10.1038/321443a0. [DOI] [PubMed] [Google Scholar]
  10. Orci L., Halban P., Amherdt M., Ravazzola M., Vassalli J. D., Perrelet A. A clathrin-coated, Golgi-related compartment of the insulin secreting cell accumulates proinsulin in the presence of monensin. Cell. 1984 Nov;39(1):39–47. doi: 10.1016/0092-8674(84)90189-2. [DOI] [PubMed] [Google Scholar]
  11. Orci L., Ravazzola M., Amherdt M., Madsen O., Vassalli J. D., Perrelet A. Direct identification of prohormone conversion site in insulin-secreting cells. Cell. 1985 Sep;42(2):671–681. doi: 10.1016/0092-8674(85)90124-2. [DOI] [PubMed] [Google Scholar]
  12. Orci L., Ravazzola M., Storch M. J., Anderson R. G., Vassalli J. D., Perrelet A. Proteolytic maturation of insulin is a post-Golgi event which occurs in acidifying clathrin-coated secretory vesicles. Cell. 1987 Jun 19;49(6):865–868. doi: 10.1016/0092-8674(87)90624-6. [DOI] [PubMed] [Google Scholar]
  13. Pollack S. J., Jacobs J. W., Schultz P. G. Selective chemical catalysis by an antibody. Science. 1986 Dec 19;234(4783):1570–1573. doi: 10.1126/science.3787262. [DOI] [PubMed] [Google Scholar]
  14. Rholam M., Nicolas P., Cohen P. Precursors for peptide hormones share common secondary structures forming features at the proteolytic processing sites. FEBS Lett. 1986 Oct 20;207(1):1–6. doi: 10.1016/0014-5793(86)80002-3. [DOI] [PubMed] [Google Scholar]
  15. Roth J., Bendayan M., Orci L. Ultrastructural localization of intracellular antigens by the use of protein A-gold complex. J Histochem Cytochem. 1978 Dec;26(12):1074–1081. doi: 10.1177/26.12.366014. [DOI] [PubMed] [Google Scholar]
  16. Sando H., Borg J., Steiner D. F. Studies on the secretion of newly synthesized proinsulin and insulin from isolated rat islets of Langerhans. J Clin Invest. 1972 Jun;51(6):1476–1485. doi: 10.1172/JCI106944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schwartz T. W. The processing of peptide precursors. 'Proline-directed arginyl cleavage' and other monobasic processing mechanisms. FEBS Lett. 1986 May 5;200(1):1–10. doi: 10.1016/0014-5793(86)80500-2. [DOI] [PubMed] [Google Scholar]
  18. Shibasaki Y., Kawakami T., Kanazawa Y., Akanuma Y., Takaku F. Posttranslational cleavage of proinsulin is blocked by a point mutation in familial hyperproinsulinemia. J Clin Invest. 1985 Jul;76(1):378–380. doi: 10.1172/JCI111973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Steiner D. F., Docherty K., Carroll R. Golgi/granule processing of peptide hormone and neuropeptide precursors: a minireview. J Cell Biochem. 1984;24(2):121–130. doi: 10.1002/jcb.240240204. [DOI] [PubMed] [Google Scholar]
  20. Steiner D. F. The biosynthesis of insulin: genetic, evolutionary, and pathophysiologic aspects. Harvey Lect. 1982 1983;78:191–228. [PubMed] [Google Scholar]
  21. Taussig R., Scheller R. H. The Aplysia FMRFamide gene encodes sequences related to mammalian brain peptides. DNA. 1986 Dec;5(6):453–461. doi: 10.1089/dna.1.1986.5.453. [DOI] [PubMed] [Google Scholar]
  22. Thim L., Hansen M. T., Norris K., Hoegh I., Boel E., Forstrom J., Ammerer G., Fiil N. P. Secretion and processing of insulin precursors in yeast. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6766–6770. doi: 10.1073/pnas.83.18.6766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tramontano A., Janda K. D., Lerner R. A. Catalytic antibodies. Science. 1986 Dec 19;234(4783):1566–1570. doi: 10.1126/science.3787261. [DOI] [PubMed] [Google Scholar]
  24. Walker L. E., Ketler T. A., Houghten R. A., Schulz G., Chersi A., Reisfeld R. A. Human major histocompatibility complex class I antigens: residues 61-83 of the HLA-B7 heavy chain specify an alloreactive site. Proc Natl Acad Sci U S A. 1985 Jan;82(2):539–542. doi: 10.1073/pnas.82.2.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Welsh M., Hammer R. E., Brinster R. L., Steiner D. F. Stimulation of growth hormone synthesis by glucose in islets of Langerhans isolated from transgenic mice. J Biol Chem. 1986 Oct 5;261(28):12915–12917. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES