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. 1986 Dec 15;240(3):777–782. doi: 10.1042/bj2400777

Biosynthesis of intestinal microvillar proteins. Processing of N-linked carbohydrate is not required for surface expression.

E M Danielsen, G M Cowell
PMCID: PMC1147486  PMID: 2881540

Abstract

Castanospermine, an inhibitor of glucosidase I, the initial enzyme in the trimming of N-linked carbohydrate, was used to study the importance of carbohydrate processing in the biosynthesis of microvillar enzymes in organ-cultured pig intestinal explants. For aminopeptidase N (EC 3.4.11.2), aminopeptidase A (EC 3.4.11.7), sucrase-isomaltase (EC 3.2.1.48-10) and maltase-glucoamylase (EC 3.2.1.20), castanospermine caused the formation of novel transient forms of higher Mr than corresponding controls, indicating a blocked removal of glucose residues. For the first three enzymes, the 'mature' (Golgi-processed) forms were similar in size to or slightly smaller than corresponding controls and were, as shown for aminopeptidase N, endoglycosidase-H-sensitive, evidence of a blocked attachment of complex sugars. Maltase-glucoamylase did not undergo conversion into a 'mature' form, suggesting that, unlike other microvillar enzymes, it does not receive post-translational O-linked carbohydrate. Castanospermine suppressed the synthesis of the four enzymes, but did not block their transport to the microvillar membrane, showing that processing of N-linked carbohydrate is not required for microvillar expression. The proteinase inhibitor leupeptin partially restored the suppressed synthesis, indicating that the majority of the wrongly processed enzymes, probably because of conformational instability, become degraded soon after synthesis rather than being transported to the microvillar membrane.

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Selected References

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

  1. Atkinson P. H., Lee J. T. Co-translational excision of alpha-glucose and alpha-mannose in nascent vesicular stomatitis virus G protein. J Cell Biol. 1984 Jun;98(6):2245–2249. doi: 10.1083/jcb.98.6.2245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blobel G. Intracellular protein topogenesis. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1496–1500. doi: 10.1073/pnas.77.3.1496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  4. Booth A. G., Kenny A. J. A rapid method for the preparation of microvilli from rabbit kidney. Biochem J. 1974 Sep;142(3):575–581. doi: 10.1042/bj1420575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Danielsen E. M. Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on aminopeptidase N and sucrase-isomaltase. Biochem J. 1982 Jun 15;204(3):639–645. doi: 10.1042/bj2040639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Danielsen E. M., Cowell G. M. Biosynthesis of intestinal microvillar proteins. Further characterization of the intracellular processing and transport. FEBS Lett. 1984 Jan 23;166(1):28–32. doi: 10.1016/0014-5793(84)80038-1. [DOI] [PubMed] [Google Scholar]
  7. Danielsen E. M., Cowell G. M. Biosynthesis of intestinal microvillar proteins. The intracellular transport of aminopeptidase N and sucrase-isomaltase occurs at different rates pre-Golgi but at the same rate post-Golgi. FEBS Lett. 1985 Oct 7;190(1):69–72. doi: 10.1016/0014-5793(85)80429-4. [DOI] [PubMed] [Google Scholar]
  8. Danielsen E. M., Cowell G. M., Norén O., Sjöström H. Biosynthesis of microvillar proteins. Biochem J. 1984 Jul 1;221(1):1–14. doi: 10.1042/bj2210001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Danielsen E. M., Cowell G. M., Poulsen S. S. Biosynthesis of intestinal microvillar proteins. Role of the Golgi complex and microtubules. Biochem J. 1983 Oct 15;216(1):37–42. doi: 10.1042/bj2160037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Danielsen E. M., Norén O., Sjöström H. Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes. Biochem J. 1983 Apr 15;212(1):161–165. doi: 10.1042/bj2120161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Danielsen E. M., Sjöström H., Norén O. Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV. Biochem J. 1983 Feb 15;210(2):389–393. doi: 10.1042/bj2100389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Danielsen E. M., Sjöström H., Norén O., Bro B., Dabelsteen E. Biosynthesis of intestinal microvillar proteins. Characterization of intestinal explants in organ culture and evidence for the existence of pro-forms of the microvillar enzymes. Biochem J. 1982 Mar 15;202(3):647–654. doi: 10.1042/bj2020647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Elder J. H., Alexander S. endo-beta-N-acetylglucosaminidase F: endoglycosidase from Flavobacterium meningosepticum that cleaves both high-mannose and complex glycoproteins. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4540–4544. doi: 10.1073/pnas.79.15.4540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hubbard S. C., Ivatt R. J. Synthesis and processing of asparagine-linked oligosaccharides. Annu Rev Biochem. 1981;50:555–583. doi: 10.1146/annurev.bi.50.070181.003011. [DOI] [PubMed] [Google Scholar]
  15. Kenny A. J., Maroux S. Topology of microvillar membrance hydrolases of kidney and intestine. Physiol Rev. 1982 Jan;62(1):91–128. doi: 10.1152/physrev.1982.62.1.91. [DOI] [PubMed] [Google Scholar]
  16. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Massey D., Maroux S. The carbohydrate moiety of aminopeptidase N of rabbit intestinal brush-border membrane. FEBS Lett. 1985 Feb 25;181(2):207–210. doi: 10.1016/0014-5793(85)80261-1. [DOI] [PubMed] [Google Scholar]
  19. Olden K., Parent J. B., White S. L. Carbohydrate moieties of glycoproteins. A re-evaluation of their function. Biochim Biophys Acta. 1982 May 12;650(4):209–232. doi: 10.1016/0304-4157(82)90017-x. [DOI] [PubMed] [Google Scholar]
  20. Pan Y. T., Hori H., Saul R., Sanford B. A., Molyneux R. J., Elbein A. D. Castanospermine inhibits the processing of the oligosaccharide portion of the influenza viral hemagglutinin. Biochemistry. 1983 Aug 2;22(16):3975–3984. doi: 10.1021/bi00285a038. [DOI] [PubMed] [Google Scholar]
  21. Sabatini D. D., Kreibich G., Morimoto T., Adesnik M. Mechanisms for the incorporation of proteins in membranes and organelles. J Cell Biol. 1982 Jan;92(1):1–22. doi: 10.1083/jcb.92.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sasak V. W., Ordovas J. M., Elbein A. D., Berninger R. W. Castanospermine inhibits glucosidase I and glycoprotein secretion in human hepatoma cells. Biochem J. 1985 Dec 15;232(3):759–766. doi: 10.1042/bj2320759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Saul R., Chambers J. P., Molyneux R. J., Elbein A. D. Castanospermine, a tetrahydroxylated alkaloid that inhibits beta-glucosidase and beta-glucocerebrosidase. Arch Biochem Biophys. 1983 Mar;221(2):593–597. doi: 10.1016/0003-9861(83)90181-9. [DOI] [PubMed] [Google Scholar]
  24. Schlesinger S., Malfer C., Schlesinger M. J. The formation of vesicular stomatitis virus (San Juan strain) becomes temperature-sensitive when glucose residues are retained on the oligosaccharides of the glycoprotein. J Biol Chem. 1984 Jun 25;259(12):7597–7601. [PubMed] [Google Scholar]
  25. Schmitz J., Preiser H., Maestracci D., Ghosh B. K., Cerda J. J., Crane R. K. Purification of the human intestinal brush border membrane. Biochim Biophys Acta. 1973 Sep 27;323(1):98–112. doi: 10.1016/0005-2736(73)90434-3. [DOI] [PubMed] [Google Scholar]
  26. Spiro R. G. Glycoproteins. Annu Rev Biochem. 1970;39:599–638. doi: 10.1146/annurev.bi.39.070170.003123. [DOI] [PubMed] [Google Scholar]
  27. Tulsiani D. R., Harris T. M., Touster O. Swainsonine inhibits the biosynthesis of complex glycoproteins by inhibition of Golgi mannosidase II. J Biol Chem. 1982 Jul 25;257(14):7936–7939. [PubMed] [Google Scholar]
  28. Umezawa H. Structures and activities of protease inhibitors of microbial origin. Methods Enzymol. 1976;45:678–695. doi: 10.1016/s0076-6879(76)45058-9. [DOI] [PubMed] [Google Scholar]

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