Abstract
Filamentous fungi are capable of secreting relatively large amounts of heterologous recombinant proteins. Recombinant human glycoproteins expressed in this system, however, carry only carbohydrates of the oligomannose type limiting their potential use in humans. One approach to the problem is genetic engineering of the fungal host to permit production of complex and hybrid N-glycans. UDP-GlcNAc:α3-d-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT I) is essential for the conversion of oligomannose to hybrid and complex N-glycans in higher eukaryotic cells. Since GnT I is not produced by fungi, we have introduced into the genome ofAspergillus nidulans the gene encoding full-length rabbit GnT I and demonstrated the expression of GnT I enzyme activity at levels appreciably higher than occurs in most mammalian tissues. All the GnT I activity in theAspergillus transformants remains intracellular suggesting that the rabbit trans-membrane sequence may be capable of targeting GnT I to the fungal Golgi apparatus.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Abbreviations
- CM:
-
complete medium
- Gal-T:
-
UDP-Gal:GlcNAc β-1,4-galactosyltransferase (EC 2.4.1.38/90)
- GnT I:
-
UDP-GlcNAc:α3-d-mannoside β-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101)
- HPLC:
-
high performance liquid chromatography
- M3-octyl:
-
Manα1-6[Manα1-3]Manβ-octyl
- PAGE:
-
polyacrylamide gel electrophoresis
- MES:
-
2-(N-morpholino)ethane sulfonate
- PCR:
-
polymerase chain reaction
- PEG:
-
polyethylene glycol
- PMSF:
-
phenyl methyl sulfonyl fluoride
- SDS:
-
sodium dodecyl sulfate
- SSC (1×):
-
0.15m NaCl/0.015m sodium citrate (pH 7.0)
- STC:
-
1.2m sorbitol, 100mm Tris-HCl, pH 7.4, and 10mm CaCl2
- STET:
-
0.1m NaCl, 10mm Tris-HCl, pH 8.0, 1mm EDTA, pH 8.0, 5% Triton-X-100
References
Gold L (1990)Methods Enzymol. 185: 11–14.
Kohno T, Carmichael DF, Sommer A, Thompson RC (1990)Methods Enzymol. 185: 187–95.
Emr SD (1990)Methods Enzymol 185:231–33.
Gwynne DI, Devchand M (1992)Biotechnology 23:203–14.
van den Hondel CAMJJ, Punt PJ, van Gorcom RFM (1992)Antonie Van Leeuwenhoek 61:153–60.
Jennes DJ, Mackenzie DA, Roberts IN, Archer DB (1991)Biotechnol Gene. Eng Rev9:327–67.
Punt PJ, Zegers ND, Busscher M, Pouwels PH, Van den Hondel CAMJJ (1991)J Biotechnol 17:19–34.
Schechtman MG (1992)Biotechnology 23:271–96.
Cullen D, Gray GL, Wilson LJ, Hayenga KJ, Lamsa MH, Rey MW, Norton S, Berka RM (1987)Bio/technology 5:369–76.
Ward M, Wilson LJ, Kodama KH, Rey MW, Berka RM (1990)Bio/technology 8:435–40.
Ward PP, May GS, Headon DR, Conneely OM (1992)Gene 122:219–23.
Carrez D, Janssens W, Degrave P, van den Hondel CAMJJ, Kinghorn JR, Fiers W, Contreras R (1990)Gene 94:147–54.
Contreras R, Carrez D, Kinghorn JR, van den Hondel CAMJJ, Fiers W (1991)Bio/technology 9:378–81.
Upshall A, Kumar AA, Bailey MC, Parker MD, Favreau MA, Lewison KP, Joseph ML, Maraganore JM, McKnight GL (1987)Bio/technology 5:1301–04.
Davidson DJ, Castellino FJ (1991)Biochemistry 30:6167–74.
Flores-Carreón A, Hixson SH, Gómez A, Shao MC, Krudy G, Rosevear PR, Wold F (1990)J Biol Chem 265:754–59.
Hernandez LM, Ballou L, Ballou CE (1990)Carbohydr Res 203:1–11.
Herscovics A, Orlean P (1993)Faseb J 7:540–50.
Jarvis DL, Summers MD (1989)Mol Cell Biol 9:214–23.
Khanh NQ, Ruttkowski E, Leidinger K, Albrecht H, Gottschalk M (1991)Gene 106:71–77.
Knepper TP, Arbogast B, Schreurs J, Deinzer ML (1992)Biochemistry 31:11651–59.
Kuroda K, Geyer H, Geyer R, Doerfler W, Klenk H-D (1990)Virology 174:418–29.
Lehle L (1992)Antonie Van Leeuwenhoek 61:133–34.
Livi GP, Ferrara A, Roskin R, Simon PL, Young PR (1990)Gene 88:297–301.
Svoboda M, Przybylski M, Schreurs J, Miyajima A, Hogeland K, Deinzer M (1991)J Chromatogr Biomed Appl 562:403–19.
Tanner W, Lehle L (1987)Biochim Biophys Acta 906:81–99.
Townsend RR, Atkinson PH, Trimble RB (1991)Carbohydr Res 215:211–17.
Turnbull IF, Smith DR, Sharp PJ, Cobon GS, Hynes MJ (1990)Appl. Environ. Microbiol. 56:2847–52
Wathen MW, Aeed PA, Elhammer AP (1991)Biochemistry 30:2863–68.
Yu Ip CC, Miller WJ, Kubek DJ, Strang AM, Van Halbeek H, Piesecki SJ, Alhadeff JA (1992)Biochemistry 31:285–295.
Schachter H (1986) Biochem.Cell Biol. 64:163–81
Schachter H (1991)Glycobiology 1:453–61
Sarkar M, Hull E, Simpson RJ, Moritz RL, Dunn R, Schachter H (1990)Glycoconjugate J. 7:380.
Sarkar M, Hull E, Nishikawa Y, Simpson RJ, Moritz RL, Dunn R, Schachter H (1991)Proc NatlAcad Sci USA 88:234–38.
Kumar R, Yang J, Larsen RD, Stanley P (1990)Proc Natl Acad Sci USA 87:9948–52
Hull E, Sarkar M, Spruijt MPN, Höppener JWM, Dunn R, Schachter H (1991)Biochem Biophys Res Commun 176:608–15.
Pownall S, Kozak CA, Schappert K, Sarkar M, Hull E, Schachter H, Marth JD (1992)Genomics 12:699–704.
Kumar R, Yang J, Eddy RL, Byers MG, Shows TB, Stanley P (1992)Glycobiology 2:383–93.
Fukada T, Iida K, Kioka N, Sakai H, Komano T (1994)Biosci Biotechnol Biochem 58:200–1.
Warren CE, Cummings L, Narasimhan S, Schachter H, Dennis JW (1992)Glycobiology 2:488
Burke J, Pettitt JM, Schachter H, Sarkar M, Gleeson PA (1992)J Biol Chem 267:24433–40
Burke J, Pettitt JM, Humphris D, Gleeson PA (1994)J Biol Chem 269:12049–59.
Paulson JC, Colley KJ (1989)J Biol Chem 264:17615–18.
Schachter H (1991)Current Opinion in Structural Biology 1:755–65.
Brockhausen I, Matta KL, Orr J, Schachter H (1985)Biochemistry 24:1866–74.
Paulsen H, Meinjohanns E (1992)Tetrahedron Lett 33:7327–30.
Paulsen H, Reck F, Brockhausen I (1992)Carbohydr Res 236:39–71.
Gwynne DI, Buxton FP, Williams SA, Garven S, Davies RW (1987)Biotechnology 5:713–19.
Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, van den Hondel CA (1987)Gene 56:117–24.
Scharf SJ (1990) InPCR Protocols: A Guide to Methods and Applications (Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds) San Diego, CA: Academic Press.
Sambrook J, Fritsch EF, Maniatis T (1989)Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Sanger F, Nicklen S, Coulson AR (1977)Proc Natl Acad Sci USA 74:5463–67.
Möller G, Reck F, Paulsen H, Kaur KJ, Sarkar M, Schachter H, Brockhausen I (1992)Glycoconjugate J 9:180–90
Palcic MM, Heerze LD, Pierce M, Hindsgaul O (1988)Glycoconjugate J 5:49–63.
Nishikawa Y, Pegg W, Paulsen H, Schachter H (1988)J Biol Chem 263:8270–81
Laemmli UK (1970)Nature 227:681–85.
Cummings RD (1993) InGlycobiology. A Practical Approach (Fukuda M, Kobata A, eds.) pp 243–89 New York, NY: Oxford University Press.
Datti A, Orlacchio A, Siminovitch KA, Dennis JW (1992)Anal Biochem 206:262–66
Ballance DJ, Buxton FP, Turner G (1983)Biochem Biophys Res Commun 112:284–89.
Vella GJ, Paulsen H, Schachter H (1984)Can J Biochem Cell Biol 62:409–17.
Brockhausen I, Carver J, Schachter H (1988)Biochem. Cell Biol 66:1134–1151.
Velardo MA, Bretthauer RK, Boutaud A, Reinhold B, Reinhold VN, Castellino FJ (1993)J Biol Chem 268:17902–7.
Chrispeels MJ (1985)Plant Physiol 78:835–38.
Johnson KD, Chrispeels MJ (1987)Plant Physiol 84:1301–8.
Szumilo T, Kaushal GP, Elbein AD (1986)Biochem. Biophys. Res. Communs. 134:1395–403.
Vitale A, Chrispeels MJ (1984)J Cell Biol 99:133–40.
Saunders G, Pickett TM, Tuite MF, Ward M (1989)Trends in Biotechnol. 7:283–87.
Cumming DA (1991)Glycobiology 1:115–30.
Cumming DA (1992)Dev Biol Stand 76:83–94.
Furukawa K, Kobata A (1992)Curr Opin Biotechnol 3:554–59.
Kobata A (1992)Eur J Biochem 209:483–501.
Sarkar M, Schachter H (1992)Glycobiology 2:483.
Author information
Authors and Affiliations
Additional information
Deceased. This paper is dedicated to the memory of Lorne S. Reid.
Rights and permissions
About this article
Cite this article
Kalsner, I., Hintz, W., Reid, L.S. et al. Insertion intoAspergillus nidulans of functional UDP-GlcNAc: α3-d-mannoside β-1,2-N-acetylglucosaminyltransferase I, the enzyme catalysing the first committed step from oligomannose to hybrid and complex N-glycans. Glycoconjugate J 12, 360–370 (1995). https://doi.org/10.1007/BF00731339
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00731339