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. 1984 Feb 1;98(2):407–416. doi: 10.1083/jcb.98.2.407

Processing of MOPC 315 immunoglobulin A oligosaccharides: evidence for endoplasmic reticulum and trans Golgi alpha 1,2-mannosidase activity

PMCID: PMC2113121  PMID: 6420419

Abstract

The processing of asparagine-linked oligosaccharides on the alpha- chains of an immunoglobulin A (IgA) has been investigated using MOPC 315 murine plasmacytoma cells. These cells secrete IgA containing complex-type oligosaccharides that were not sensitive to endo-beta-N- acetylglucosaminidase H. In contrast, oligosaccharides present on the intracellular alpha-chain precursor were of the high mannose-type, remaining sensitive to endo-beta-N-acetylglucosaminidase H despite a long intracellular half-life of 2-3 h. The major [3H]mannose-labeled alpha-chain oligosaccharides identified after a 20-min pulse were Man8GlcNAc2 and Man9GlcNAc2. Following chase incubations, the major oligosaccharide accumulating intracellularly was Man6GlcNAc2, which was shown to contain a single alpha 1,2-linked mannose residue. Conversion of Man6GlcNAc2 to complex-type oligosaccharides occurred at the time of secretion since appreciable amounts of Man5GlcNAc2 or further processed structures could not be detected intracellularly. The subcellular locations of the alpha 1,2-mannosidase activities were studied using carbonyl cyanide m-chlorophenylhydrazone and monensin. Despite inhibiting the secretion of IgA, these inhibitors of protein migration did not effect the initial processing of Man9GlcNAc2 to Man6GlcNAc2. Furthermore, no large accumulation of Man5GlcNAc2 occurred, indicating the presence of two subcellular locations of alpha 1,2-mannosidase activity involved in oligosaccharide processing in MOPC 315 cells. Thus, the first three alpha 1,2-linked mannose residues were removed shortly after the alpha-chain was glycosylated, most likely in rough endoplasmic reticulum, since this processing occurred in the presence of carbonyl cyanide m-chlorophenylhydrazone. However, the removal of the final alpha 1,2-linked mannose residue as well as subsequent carbohydrate processing occurred just before IgA secretion, most likely in the trans Golgi complex since processing of Man6GlcNAc2 to Man5GlcNAc2 was greatly inhibited in the presence of monensin.

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Selected References

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  1. Bischoff J., Kornfeld R. Evidence for an alpha-mannosidase in endoplasmic reticulum of rat liver. J Biol Chem. 1983 Jul 10;258(13):7907–7910. [PubMed] [Google Scholar]
  2. 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]
  3. Dunphy W. G., Fries E., Urbani L. J., Rothman J. E. Early and late functions associated with the Golgi apparatus reside in distinct compartments. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7453–7457. doi: 10.1073/pnas.78.12.7453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Elting J. J., Chen W. W., Lennarz W. J. Characterization of a glucosidase involved in an initial step in the processing of oligosaccharide chains. J Biol Chem. 1980 Mar 25;255(6):2325–2331. [PubMed] [Google Scholar]
  5. Forsee W. T., Schutzbach J. S. Purification and characterization of a phospholipid-dependent alpha-mannosidase from rabbit liver. J Biol Chem. 1981 Jul 10;256(13):6577–6582. [PubMed] [Google Scholar]
  6. Godelaine D., Spiro M. J., Spiro R. G. Processing of the carbohydrate units of thyroglobulin. J Biol Chem. 1981 Oct 10;256(19):10161–10168. [PubMed] [Google Scholar]
  7. Griffiths G., Quinn P., Warren G. Dissection of the Golgi complex. I. Monensin inhibits the transport of viral membrane proteins from medial to trans Golgi cisternae in baby hamster kidney cells infected with Semliki Forest virus. J Cell Biol. 1983 Mar;96(3):835–850. doi: 10.1083/jcb.96.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Grinna L. S., Robbins P. W. Glycoprotein biosynthesis. Rat liver microsomal glucosidases which process oligosaccharides. J Biol Chem. 1979 Sep 25;254(18):8814–8818. [PubMed] [Google Scholar]
  9. Hakimi J., Atkinson P. H. Glycosylation of intracellular Sindbis virus glycoproteins. Biochemistry. 1982 Apr 27;21(9):2140–2145. doi: 10.1021/bi00538a024. [DOI] [PubMed] [Google Scholar]
  10. Harpaz N., Schachter H. Control of glycoprotein synthesis. Processing of asparagine-linked oligosaccharides by one or more rat liver Golgi alpha-D-mannosidases dependent on the prior action of UDP-N-acetylglucosamine: alpha-D-mannoside beta 2-N-acetylglucosaminyltransferase I. J Biol Chem. 1980 May 25;255(10):4894–4902. [PubMed] [Google Scholar]
  11. Hickman S., Kulczycki A., Jr, Lynch R. G., Kornfeld S. Studies of the mechanism of tunicamycin in hibition of IgA and IgE secretion by plasma cells. J Biol Chem. 1977 Jun 25;252(12):4402–4408. [PubMed] [Google Scholar]
  12. 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]
  13. Ichishima E., Arai M., Shigematsu Y., Kumagai H., Sumida-Tanaka R. Purification of an acidic alpha-D-mannosidase from Aspergillus saitoi and specific cleavage of 1,2-alpha-D-mannosidic linkage in yeast mannan. Biochim Biophys Acta. 1981 Mar 13;658(1):45–53. doi: 10.1016/0005-2744(81)90248-5. [DOI] [PubMed] [Google Scholar]
  14. Ito S., Muramatsu T., Kobata A. Endo-beta-N-acetylglucosaminidases acting on carbohydrate moieties of glycoproteins: purification and properties of the two enzymes with different specificities from Clostridium perfringens. Arch Biochem Biophys. 1975 Nov;171(1):78–86. doi: 10.1016/0003-9861(75)90009-0. [DOI] [PubMed] [Google Scholar]
  15. Ito S., Muramatsu T., Kobata A. Release of galactosyl oligosaccharides by endo-beta-N-acetylglucosaminidase D. Biochem Biophys Res Commun. 1975 Apr 21;63(4):938–944. doi: 10.1016/0006-291x(75)90659-2. [DOI] [PubMed] [Google Scholar]
  16. Ledger P. W., Nishimoto S. K., Hayashi S., Tanzer M. L. Abnormal glycosylation of human fibronectin secreted in the presence of monensin. J Biol Chem. 1983 Jan 10;258(1):547–554. [PubMed] [Google Scholar]
  17. Mellis S. J., Baenziger J. U. Separation of neutral oligosaccharides by high-performance liquid chromatography. Anal Biochem. 1981 Jul 1;114(2):276–280. doi: 10.1016/0003-2697(81)90480-2. [DOI] [PubMed] [Google Scholar]
  18. Pesonen M., Käriäinen L. Incomplete complex oligosaccharides in semliki forest virus envelope proteins arrested within the cell in the presence of monensin. J Mol Biol. 1982 Jun 25;158(2):213–230. doi: 10.1016/0022-2836(82)90430-2. [DOI] [PubMed] [Google Scholar]
  19. Pohlmann R., Waheed A., Hasilik A., von Figura K. Synthesis of phosphorylated recognition marker in lysosomal enzymes is located in the cis part of Golgi apparatus. J Biol Chem. 1982 May 25;257(10):5323–5325. [PubMed] [Google Scholar]
  20. Quinn P., Griffiths G., Warren G. Dissection of the Golgi complex. II. Density separation of specific Golgi functions in virally infected cells treated with monensin. J Cell Biol. 1983 Mar;96(3):851–856. doi: 10.1083/jcb.96.3.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rothman J. E. The golgi apparatus: two organelles in tandem. Science. 1981 Sep 11;213(4513):1212–1219. doi: 10.1126/science.7268428. [DOI] [PubMed] [Google Scholar]
  22. Ruddon R. W., Bryan A. H., Hanson C. A., Perini F., Ceccorulli L. M., Peters B. P. Characterization of the intracellular and secreted forms of the glycoprotein hormone chorionic gonadotropin produced by human malignant cells. J Biol Chem. 1981 May 25;256(10):5189–5196. [PubMed] [Google Scholar]
  23. Sitia R., Kikutani H., Rubartelli A., Bushkin Y., Stavnezer J., Hammerling U. Membrane-bound and secreted IgA contain structurally different alpha-chains. J Immunol. 1982 Feb;128(2):712–716. [PubMed] [Google Scholar]
  24. Tabas I., Kornfeld S. Purification and characterization of a rat liver Golgi alpha-mannosidase capable of processing asparagine-linked oligosaccharides. J Biol Chem. 1979 Nov 25;254(22):11655–11663. [PubMed] [Google Scholar]
  25. Tabas I., Kornfeld S. The synthesis of complex-type oligosaccharides. III. Identification of an alpha-D-mannosidase activity involved in a late stage of processing of complex-type oligosaccharides. J Biol Chem. 1978 Nov 10;253(21):7779–7786. [PubMed] [Google Scholar]
  26. Tartakoff A. M., Vassalli P. Plasma cell immunoglobulin secretion: arrest is accompanied by alterations of the golgi complex. J Exp Med. 1977 Nov 1;146(5):1332–1345. doi: 10.1084/jem.146.5.1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Tulsiani D. R., Hubbard S. C., Robbins P. W., Touster O. alpha-D-Mannosidases of rat liver Golgi membranes. Mannosidase II is the GlcNAcMAN5-cleaving enzyme in glycoprotein biosynthesis and mannosidases Ia and IB are the enzymes converting Man9 precursors to Man5 intermediates. J Biol Chem. 1982 Apr 10;257(7):3660–3668. [PubMed] [Google Scholar]
  29. Ugalde R. A., Staneloni R. J., Leloir L. F. Microsomal glucosidases of rat liver. Partial purification and inhibition by disaccharides. Eur J Biochem. 1980 Dec;113(1):97–103. doi: 10.1111/j.1432-1033.1980.tb06144.x. [DOI] [PubMed] [Google Scholar]
  30. Yamashita K., Ichishima E., Arai M., Kobata A. An alpha-mannosidase purified from Aspergillus saitoi is specific for alpha 1,2 linkages. Biochem Biophys Res Commun. 1980 Oct 16;96(3):1335–1342. doi: 10.1016/0006-291x(80)90097-2. [DOI] [PubMed] [Google Scholar]

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