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Abstract

Recently, asparagine-linked oligosaccharides (N-glycans) have been found to play a pivotal role in glycoprotein quality control in the endoplasmic reticulum (ER). In order to screen proteins interacting with N-glycans, we developed affinity chromatography by conjugating synthetic N-glycans on sepharose beads. Using the affinity beads with the dodecasaccharide Glc1Man9GlcNAc2, one structure of the N-glycans, a 75-kDa protein, was isolated from the membranous fraction including the ER in Aspergillus oryzae. By LC-MS/MS analysis using the A. oryzae genome database, the protein was identified as one (AO090009000313) sharing similarities with calnexin. Further affinity chromatographic experiments suggested that the protein specifically bound to Glc1Man9GlcNAc2, similarly to mammalian calnexins. We designated the gene AoclxA and expressed it as a fusion gene with egfp, revealing the ER localization of the AoClxA protein. Our results suggest that our affinity chromatography with synthetic N-glycans might help in biological analysis of glycoprotein quality control in the ER.

N-glycan, quality control, Aspergillus oryzae, calnexin, lectin
Issue Section:
Research Articles

Reference

1) Kitamoto, K., Molecular biology of the Koji molds. Adv. Appl. Microbiol., 51, 129–153 (2002).

2) Gouka, R. J., Punt, P. J., and van den Hondel, C. A., Efficient production of secreted proteins by Aspergillus: progress, limitations and prospects. Appl. Microbiol. Biotechnol., 47, 1–11 (1997).

3) Archer, D. B., Filamentous fungi as microbial cell factories for food use. Curr. Opin. Biotechnol., 11, 478–483 (2000).

4) Punt, P. J., van Biezen, N., Conesa, A., Albers, A., Mangnus, J., and van den Hondel, C. A., Filamentous fungi as cell factories for heterologous protein production. Trends Biotechnol., 20, 200–206 (2002).

5) Helenius, A., and Aebi, M., Roles of N-linked glycans in the endoplasmic reticulum. Annu. Rev. Biochem., 73, 1019–1049 (2004).

6) Williams, D. B., Beyond lectins: the calnexin/calreticulin chaperone system of the endoplasmic reticulum. J. Cell Sci., 119, 615–623 (2006).

7) Paquet, M. E., Leach, M. R., and Williams, D. B., In vitro and in vivo assays to assess the functions of calnexin and calreticulin in ER protein folding and quality control. Methods, 35, 338–347 (2005).

8) Leach, M. R., Cohen-Doyle, M. F., Thomas, D. Y., and Williams, D. B., Localization of the lectin, ERp57 binding, and polypeptide binding sites of calnexin and calreticulin. J. Biol. Chem., 277, 29686–29697 (2002).

9) Trombetta, E. S., and Parodi, A. J., Glycoprotein reglucosylation. Methods, 35, 328–337 (2005).

10) Oda, Y., Hosokawa, N., Wada, I., and Nagata, K., EDEM as an acceptor of terminally misfolded glycoproteins released from calnexin. Science, 299, 1394–1397 (2003).

11) Molinari, M., Calance, V., Galli, C., Lucca, P., and Paganetti, P., Role of EDEM in the release of misfolded glycoproteins from the calnexin cycle. Science, 299, 1397–1400 (2003).

12) Szathmary, R., Bielmann, R., Nita-Lazar, M., Burda, P., and Jakob, C. A., Yos9 protein is essential for degradation of misfolded glycoproteins and may function as lectin in ERAD. Mol. Cell, 19, 765–775 (2005).

13) Matsuo, I., and Ito, Y., Synthesis of an octamannosyled glycan chain, the key oligosaccharide structure in ER-associated degradation. Carbohydr. Res., 338, 2163–2168 (2003).

14) Matsuo, I., Wada, M., Manabe, S., Yamaguchi, Y., Otake, K., Kato, K., and Ito, Y., Synthesis of monoglucosylated high-mannose-type dodecasaccharide, a putative ligand for molecular chaperone, calnexin, and calreticulin. J. Am. Chem. Soc., 125, 3402–3403 (2003).

15) Matsuo, I., Kashiwagi, T., Totani, K., and Ito, Y., First chemical synthesis of triglucosylated tetradecasaccharide (Glc3Man9GlcNAc2), a common precursor of asparagine-linked oligosaccharides. Tetrahedron Lett., 46, 4197–4200 (2005).

16) Matsuo, I., Totani, K., Tatami, A., and Ito, Y., Comprehensive synthesis of ER related high-mannose-type sugar chains by convergent strategy. Tetrahedron, 62, 8262–8277 (2006).

17) Totani, K., Ihara, Y., Matsuo, I., Koshino, H., and Ito, Y., Synthetic substrates for an endoplasmic reticulum protein-folding sensor, UDP-glucose: glycoprotein glucosyltransferase. Angew. Chem. Int. Ed. Engl., 44, 7950–7954 (2005).

18) Totani, K., Ihara, Y., Matsuo, I., and Ito, Y., Substrate specificity analysis of endoplasmic reticulum glucosidase II using synthetic high mannose-type glycans. J. Biol. Chem., 281, 31502–31508 (2006).

19) Conesa, A., Jeenes, D., Archer, D. B., van den Hondel, C. A., and Punt, P. J., Calnexin overexpression increases manganese peroxidase production in Aspergillus niger. Appl. Environ. Microbiol., 68, 846–851 (2002).

20) Geysens, S., Pakula, T., Uusitalo, J., Dewerte, I., Penttila, M., and Contreras, R., Cloning and characterization of the glucosidase II alpha subunit gene of Trichoderma reesei: a frameshift mutation results in the aberrant glycosylation profile of the hypercellulolytic strain Rut-C30. Appl. Environ. Microbiol., 71, 2910–2924 (2005).

21) Totani, K., Matsuo, I., Ihara, Y., and Ito, Y., High-mannose-type glycan modifications of dihydrofolate reductase using glycan-methotrexate conjugates. Bioorg. Med. Chem., 14, 5220–5229 (2006).

22) Ellman, G. L., Tissue sulfhydryl groups. Arch. Biochem. Biophys., 82, 70–77 (1959).

23) Shevchenko, A., Wilm, M., Vorm, O., and Mann, M., Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem., 68, 850–858 (1996).

24) Perkins, D. N., Pappin, D. J., Creasy, D. M., and Cottrell, J. S., Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis, 20, 3551–3567 (1999).

25) Mabashi, Y., Kikuma, T., Maruyama, J., Arioka, M., and Kitamoto, K., Development of a versatile expression plasmid construction system for Aspergillus oryzae and its application to visualization of mitochondria. Biosci. Biotechnol. Biochem., 70, 1882–1889 (2006).

26) Yamada, O., Lee, B. R., and Gomi, K., Transformation system for Aspergillus oryzae with double auxotrophic mutations, niaD and sC. Biosci. Biotechnol. Biochem., 61, 1367–1369 (1997).

27) Ware, F. E., Vassilakos, A., Peterson, P. A., Jackson, M. R., Lehrman, M. A., and Williams, D. B., The molecular chaperone calnexin binds Glc1Man9GlcNAc2 oligosaccharide as an initial step in recognizing unfolded glycoproteins. J. Biol. Chem., 270, 4697–4704 (1995).

28) Maruyama, J., Kikuchi, S., and Kitamoto, K., Differential distribution of the endoplasmic reticulum network as visualized by the BipA-EGFP fusion protein in hyphal compartments across the septum of the filamentous fungus, Aspergillus oryzae. Fungal Genet. Biol., 43, 642–654 (2006).

29) Arai, M. A., Matsuo, I., Hagihara, S., Totani, K., Maruyama, J., Kitamoto, K., and Ito, Y., Design and synthesis of oligosaccharides that interfere with glycoprotein quality-control systems. Chembiochem, 6, 2281–2289 (2005).

30) Jannatipour, M., and Rokeach, L. A., The Schizosaccharomyces pombe homologue of the chaperone calnexin is essential for viability. J. Biol. Chem., 270, 4845–4853 (1995).

31) Parlati, F., Dignard, D., Bergeron, J. J., and Thomas, D. Y., The calnexin homologue cnx1  + in Schizosaccharomyces pombe, is an essential gene which can be complemented by its soluble ER domain. EMBO J., 14, 3064–3072 (1995).

32) Wang, H., Entwistle, J., Morlon, E., Archer, D. B., Peberdy, J. F., Ward, M., and Jeenes, D. J., Isolation and characterisation of a calnexin homologue, clxA, from Aspergillus niger. Mol. Genet. Genomics, 268, 684–691 (2003).

33) Spiro, R. G., Zhu, Q., Bhoyroo, V., and Soling, H. D., Definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver Golgi. J. Biol. Chem., 271, 11588–11594 (1996).

34) Vassilakos, A., Michalak, M., Lehrman, M. A., and Williams, D. B., Oligosaccharide binding characteristics of the molecular chaperones calnexin and calreticulin. Biochemistry, 37, 3480–3490 (1998).

35) Schrag, J. D., Bergeron, J. J., Li, Y., Borisova, S., Hahn, M., Thomas, D. Y., and Cygler, M., The Structure of calnexin, an ER chaperone involved in quality control of protein folding. Mol. Cell, 8, 633–644 (2001).

36) Corbett, E. F., and Michalak, M., Calcium, a signaling molecule in the endoplasmic reticulum? Trends Biochem. Sci., 25, 307–311 (2000).

37) Gurr, S. J., Unkles, S. E., and Kinghorn, J. R., The structure and organisation of nuclear genes in filamentous fungi. In “Gene Structure in Eukaryotic Microbes,” ed. Kinghorn, J. R., IRL Press, Oxford, pp. 93–139 (1987).

38) Mulder, H. J., Nikolaev, I., and Madrid, S. M., HACA, the transcriptional activator of the unfolded protein response (UPR) in Aspergillus niger, binds to partly palindromic UPR elements of the consensus sequence 5′-CAN(G/A)NTGT/GCCT-3′. Fungal Genet. Biol., 43, 560–572 (2006).

39) Ito, Y., Hagihara, S., Matsuo, I., and Totani, K., Structural approaches to the study of oligosaccharides in glycoprotein quality control. Curr. Opin. Struct. Biol., 15, 481–489 (2005).

40) Hagihara, S., Totani, K., Matsuo, I., and Ito, Y., Thermodynamic analysis of interactions between N-linked sugar chains and F-box protein Fbs1. J. Med. Chem., 48, 3126–3129 (2005).

41) Xu, X., Azakami, H., and Kato, A., P-domain and lectin site are involved in the chaperone function of Saccharomyces cerevisiae calnexin homologue. FEBS Lett., 570, 155–160 (2004).

42) Song, Y., Azakami, H., Shamima, B., He, J., and Kato, A., Different effects of calnexin deletion in Saccharomyces cerevisiae on the secretion of two glycosylated amyloidogenic lysozymes. FEBS Lett., 512, 213–217 (2002).

43) Song, Y., Sata, J., Saito, A., Usui, M., Azakami, H., and Kato, A., Effects of calnexin deletion in Saccharomyces cerevisiae on the secretion of glycosylated lysozymes. J. Biochem., 130, 757–764 (2001).

44) Parodi, A. J., Reglucosylation of glycoproteins and quality control of glycoprotein folding in the endoplasmic reticulum of yeast cells. Biochim. Biophys. Acta, 1426, 287–295 (1999).

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© Japan Society for Bioscience, Biotechnology, and Agrochemistry 2007
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