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
. 1990 Mar;87(5):1879–1883. doi: 10.1073/pnas.87.5.1879

Modular mutagenesis of human placental ribonuclease inhibitor, a protein with leucine-rich repeats.

F S Lee 1, B L Vallee 1
PMCID: PMC53587  PMID: 2408043

Abstract

Human placental ribonuclease inhibitor (PRI) is a potent protein inhibitor of pancreatic ribonucleases and the homologous blood vessel-inducing protein angiogenin. Although inhibition by PRI occurs with a 1:1 stoichiometry, its primary structure is composed predominantly of seven internal leucine-rich repeats. These internal repeats were systematically deleted either singly or in combination by "modular" mutagenesis. Deletion of repeat units 3 plus 4 or repeat unit 6 results in mutants that both bind to and inhibit ribonuclease A. Therefore, the angiogenin/ribonuclease binding site in PRI must reside primarily or entirely in repeats 1, 2, 5, or 7, the short N- or C-terminal segments, or a combination of these. Deletion of repeat units 3-5, 5-6, or 5 alone results in mutants that exhibit only binding activity. Hence, the binding site cannot reside exclusively in repeat 5. Other internal deletions or N- or C-terminal deletions of 6-86% of the protein all abolish activity. These results suggest that PRI has a modular structure, with one primary structural repeat constituting one module. The approach taken may be applicable to other proteins with repeat structures.

Full text

PDF
1879

Images in this article

1881Image
on p.1881
1881Image
on p.1881
1881Image
on p.1881
1882Image
on p.1882

Selected References

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

  1. Blackburn P. Ribonuclease inhibitor from human placenta: rapid purification and assay. J Biol Chem. 1979 Dec 25;254(24):12484–12487. [PubMed] [Google Scholar]
  2. Blackburn P., Wilson G., Moore S. Ribonuclease inhibitor from human placenta. Purification and properties. J Biol Chem. 1977 Aug 25;252(16):5904–5910. [PubMed] [Google Scholar]
  3. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  4. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  5. Fett J. W., Strydom D. J., Lobb R. R., Alderman E. M., Bethune J. L., Riordan J. F., Vallee B. L. Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells. Biochemistry. 1985 Sep 24;24(20):5480–5486. doi: 10.1021/bi00341a030. [DOI] [PubMed] [Google Scholar]
  6. Goldenberg D. P., Creighton T. E. Circular and circularly permuted forms of bovine pancreatic trypsin inhibitor. J Mol Biol. 1983 Apr 5;165(2):407–413. doi: 10.1016/s0022-2836(83)80265-4. [DOI] [PubMed] [Google Scholar]
  7. Green N. M. Avidin. Adv Protein Chem. 1975;29:85–133. doi: 10.1016/s0065-3233(08)60411-8. [DOI] [PubMed] [Google Scholar]
  8. Hashimoto C., Hudson K. L., Anderson K. V. The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein. Cell. 1988 Jan 29;52(2):269–279. doi: 10.1016/0092-8674(88)90516-8. [DOI] [PubMed] [Google Scholar]
  9. Hofsteenge J., Kieffer B., Matthies R., Hemmings B. A., Stone S. R. Amino acid sequence of the ribonuclease inhibitor from porcine liver reveals the presence of leucine-rich repeats. Biochemistry. 1988 Nov 15;27(23):8537–8544. doi: 10.1021/bi00423a006. [DOI] [PubMed] [Google Scholar]
  10. Kataoka T., Broek D., Wigler M. DNA sequence and characterization of the S. cerevisiae gene encoding adenylate cyclase. Cell. 1985 Dec;43(2 Pt 1):493–505. doi: 10.1016/0092-8674(85)90179-5. [DOI] [PubMed] [Google Scholar]
  11. Kellis J. T., Jr, Nyberg K., Fersht A. R. Energetics of complementary side-chain packing in a protein hydrophobic core. Biochemistry. 1989 May 30;28(11):4914–4922. doi: 10.1021/bi00437a058. [DOI] [PubMed] [Google Scholar]
  12. Krusius T., Ruoslahti E. Primary structure of an extracellular matrix proteoglycan core protein deduced from cloned cDNA. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7683–7687. doi: 10.1073/pnas.83.20.7683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kurachi K., Rybak S. M., Fett J. W., Shapiro R., Strydom D. J., Olson K. A., Riordan J. F., Davie E. W., Vallee B. L. Expression of human angiogenin in cultured baby hamster kidney cells. Biochemistry. 1988 Aug 23;27(17):6557–6562. doi: 10.1021/bi00417a054. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Lee F. S., Auld D. S., Vallee B. L. Tryptophan fluorescence as a probe of placental ribonuclease inhibitor binding to angiogenin. Biochemistry. 1989 Jan 10;28(1):219–224. doi: 10.1021/bi00427a030. [DOI] [PubMed] [Google Scholar]
  16. Lee F. S., Fox E. A., Zhou H. M., Strydom D. J., Vallee B. L. Primary structure of human placental ribonuclease inhibitor. Biochemistry. 1988 Nov 15;27(23):8545–8553. doi: 10.1021/bi00423a007. [DOI] [PubMed] [Google Scholar]
  17. Lee F. S., Shapiro R., Vallee B. L. Tight-binding inhibition of angiogenin and ribonuclease A by placental ribonuclease inhibitor. Biochemistry. 1989 Jan 10;28(1):225–230. doi: 10.1021/bi00427a031. [DOI] [PubMed] [Google Scholar]
  18. Lee F. S., Vallee B. L. Expression of human placental ribonuclease inhibitor in Escherichia coli. Biochem Biophys Res Commun. 1989 Apr 14;160(1):115–120. doi: 10.1016/0006-291x(89)91628-8. [DOI] [PubMed] [Google Scholar]
  19. Lim W. A., Sauer R. T. Alternative packing arrangements in the hydrophobic core of lambda repressor. Nature. 1989 May 4;339(6219):31–36. doi: 10.1038/339031a0. [DOI] [PubMed] [Google Scholar]
  20. Lopez J. A., Chung D. W., Fujikawa K., Hagen F. S., Davie E. W., Roth G. J. The alpha and beta chains of human platelet glycoprotein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2135–2139. doi: 10.1073/pnas.85.7.2135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lopez J. A., Chung D. W., Fujikawa K., Hagen F. S., Papayannopoulou T., Roth G. J. Cloning of the alpha chain of human platelet glycoprotein Ib: a transmembrane protein with homology to leucine-rich alpha 2-glycoprotein. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5615–5619. doi: 10.1073/pnas.84.16.5615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Luger K., Hommel U., Herold M., Hofsteenge J., Kirschner K. Correct folding of circularly permuted variants of a beta alpha barrel enzyme in vivo. Science. 1989 Jan 13;243(4888):206–210. doi: 10.1126/science.2643160. [DOI] [PubMed] [Google Scholar]
  23. Matsumura M., Becktel W. J., Matthews B. W. Hydrophobic stabilization in T4 lysozyme determined directly by multiple substitutions of Ile 3. Nature. 1988 Aug 4;334(6181):406–410. doi: 10.1038/334406a0. [DOI] [PubMed] [Google Scholar]
  24. McLachlan A. D. Gene duplication and the origin of repetitive protein structures. Cold Spring Harb Symp Quant Biol. 1987;52:411–420. doi: 10.1101/sqb.1987.052.01.048. [DOI] [PubMed] [Google Scholar]
  25. Reinke R., Krantz D. E., Yen D., Zipursky S. L. Chaoptin, a cell surface glycoprotein required for Drosophila photoreceptor cell morphogenesis, contains a repeat motif found in yeast and human. Cell. 1988 Jan 29;52(2):291–301. doi: 10.1016/0092-8674(88)90518-1. [DOI] [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schneider R., Schneider-Scherzer E., Thurnher M., Auer B., Schweiger M. The primary structure of human ribonuclease/angiogenin inhibitor (RAI) discloses a novel highly diversified protein superfamily with a common repetitive module. EMBO J. 1988 Dec 20;7(13):4151–4156. doi: 10.1002/j.1460-2075.1988.tb03310.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shapiro R., Harper J. W., Fox E. A., Jansen H. W., Hein F., Uhlmann E. Expression of Met-(-1) angiogenin in Escherichia coli: conversion to the authentic less than Glu-1 protein. Anal Biochem. 1988 Dec;175(2):450–461. doi: 10.1016/0003-2697(88)90569-6. [DOI] [PubMed] [Google Scholar]
  29. Shapiro R., Vallee B. L. Human placental ribonuclease inhibitor abolishes both angiogenic and ribonucleolytic activities of angiogenin. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2238–2241. doi: 10.1073/pnas.84.8.2238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Strydom D. J., Fett J. W., Lobb R. R., Alderman E. M., Bethune J. L., Riordan J. F., Vallee B. L. Amino acid sequence of human tumor derived angiogenin. Biochemistry. 1985 Sep 24;24(20):5486–5494. doi: 10.1021/bi00341a031. [DOI] [PubMed] [Google Scholar]
  31. Takahashi N., Takahashi Y., Putnam F. W. Periodicity of leucine and tandem repetition of a 24-amino acid segment in the primary structure of leucine-rich alpha 2-glycoprotein of human serum. Proc Natl Acad Sci U S A. 1985 Apr;82(7):1906–1910. doi: 10.1073/pnas.82.7.1906. [DOI] [PMC free article] [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