Skip to main content
SpringerLink
Log in

Expression of human chorionic gonadotropin uniformly labeled with NMR isotpes in Chinese hamster ovary cells: An advance toward rapid determination of glycoprotein structures

  • Research Papers
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

Summary

Most secreted eukaryotic proteins are modified by glycosylation, and it has been difficult to solve their structures by crystallographic or NMR techniques because of problems posed by the presence of the carbohydrate. The structure of a chemically deglycosylated form of the human pregnancy hormone, human chorionic gonadotropin (hCG), has been solved by crystallographic methods. Since chemical deglycosylation may have induced changes in the structure, and since it is known that deglycosylated hCG is biologically inactive, the crystallographic structure requires confirmation by NMR techniques. Also, it has not been possible to determine the structures of the isolated subunits, nor the nature of interactions between the carbohydrate side chains and the protein back bone by crystallographic methods. Structural information via NMR techniques can be obtained from proteins in solution if they can be uniformly labeled with 13C and 15N isotopes. We report the first such uniform labeling of a glycoprotein using a universal 13C-and 15N-labeling medium to express 13C, 15N-labeled hCG, suitable for solving the structure in solution of the native, biologically active form of hCG as well as that of its free subunits. The 13C, 15N-labeled recombinant hCG and its separated subunits are shown to be nearly identical to urinary hCG reference preparations on the basis of protein chemical studies, immunochemistry, biological activity, and the capability of isolated hormone subunits to recombine to form biologically active hormone. Mass spectrometric analysis and preliminary NMR studies indicate that the isotopic labeling is uniform and greater than 90% after only two growth passages in the labeling media. One unexpected finding during subunit purification was that lyophilization of glycoproteins from trifluoroacetic acid HPLC buffers may result in the loss of a significant portion of sialic acid.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Bellisario, R., Carlsen, R.B. and Bahl, O.P. (1973) J. Biol. Chem., 248, 6796–6809.

    Google Scholar 

  • Birken, S. and Canfield, R.E. (1982a) In Tumor Imaging: The Radioimmunological Detection of Cancer (Eds, Burchiel, S.W. and Rhodes, B.A.), Masson Publishing USA, Inc., New York, NY, pp. 41–52.

    Google Scholar 

  • Birken, S., Canfield, R.E., Agosto, G. and Lewis, J. (1982) Endocrinology, 110, 1555–1563.

    Google Scholar 

  • Birken, S., Gawinowicz-Kolks, M.A., Amr, S., Nisula, B. and Puett, D. (1986) J. Biol. Chem., 261, 10719–10727.

    Google Scholar 

  • Birken, S., Krichevsky, A., O'Connor, J., Lustbader, J. and Canfield, R. (1990) In Glycoprotein Hormones (Eds, Chin, W.W. and Boime, I.), Serono Symposia, Norwell, MA, pp. 45–61.

  • Birken, S., Gawinowicz, M.A., Kardana, A. and Cole, L.A. (1991) Endocrinology, 129, 1551–1558.

    Google Scholar 

  • Birken, S., Chen, Y., Gawinowicz, M.A., Lustbader, J.W., Pollak, S., Agosto, G., Buck, R. and O'Connor, J. (1993) Endocrinology, 133, 1390–1397.

    Google Scholar 

  • Carlsen, R.B., Bahl, O.P. and Swaminathan, N. (1973) J. Biol. Chem., 248, 6810–6827.

    Google Scholar 

  • Chen, H.-C., Shimohigashi, Y., Dufau, M.L. and Catt, K.J. (1982) J. Biol. Chem., 257, 14446–14452.

    Google Scholar 

  • Chowdhury, S.K., Katta, V. and Chait, B.T. (1990) Rapid Commun. Mass Spectrom., 4, 81–87.

    Google Scholar 

  • Daopin, S., Piez, K.A., Ogawa, Y. and Davies, D.R. (1992) Science, 257, 369–373.

    Google Scholar 

  • DeLean, A., Munson, P.J. and Rodbard, D. (1978) Am. J. Physiol., 235, E97–102.

    Google Scholar 

  • Fogh, R.H., Schipper, D., Boelens, B. and Kaptein, R. (1994) J. Biomol. NMR, 4, 123–128.

    Google Scholar 

  • Grzesiek, S. and Bax, A. (1992) J. Magn. Reson., 96, 432–440.

    Google Scholar 

  • Hancock, W.S. (Ed.) (1977) Specifications of Amino Acids, 4th ed., Ajinomoto, Japan, p. 70.

    Google Scholar 

  • Hansen, A.P., Petros, A.M., Mazar, A.P., Pederson, T.M., Rueter, A. and Fesik, S.W. (1992) Biochemistry, 31, 12713–12718.

    Google Scholar 

  • Keene, J.L., Matzuk, M.M., Otani, T., Fauser, B.C.J.M., Galway, A.B., Hseuh, A.J.W. and Boime, I. (1989) J. Biol. Chem., 264, 4769–4775.

    Google Scholar 

  • Keutmann, H.T., Jognson, L. and Ryan, R.J. (1985) Febs Lett., 185, 333–338.

    Google Scholar 

  • Laemmli, U.K. (1970) Nature, 227, 680–685.

    PubMed  Google Scholar 

  • Lapthorn, A.J., Harris, D.C., Littlejohn, A., Lustbader, J.W., Canfield, R.E., Machin, K.J., Morgan, F.J. and Isaacs, N.W. (1994) Nature, 369, 455–461.

    Google Scholar 

  • Lustbader, J.W., Wu, H., Birken, S., Pollak, S., Gawinowicz Kolks, M.A., Pound, A.M., Austen, D., Hendrickson, W.A. and Canfield, R.E. (1995) Endocrinology, 136, 640–650.

    Google Scholar 

  • Matzuk, M., Keene, J.L. and Boime, I. (1989) J. Biol. Chem., 264, 2409–2414.

    Google Scholar 

  • McDonald, N.Q., Lapatto, R., Murray-Rust, J., Gunning, J., Wlodawer, A., Blundell, T.L. (1991) Nature, 354, 411–414.

    Google Scholar 

  • Mise, T. and Bahl, O.P. (1980) J. Biol. Chem., 255, 8516–8522.

    Google Scholar 

  • Mise, T. and Bahl, O.P. (1981) J. Biol. Chem., 256, 6587–6592.

    Google Scholar 

  • Morgan, F.J., Birken, S. and Canfield, R.E. (1973) Mol. Cell. Biochem., 2, 97–99.

    Google Scholar 

  • Morgan, F.J., Birken, S. and Canfield, R.E. (1975) J. Biol. Chem., 250, 5247–5258.

    Google Scholar 

  • Manjunath, P. and Sairam, M.R. (1983) J. Biol. Chem.,258, 3554–3558.

    Google Scholar 

  • O'Connor, J.F., Birken, S., Lustbader, J.W., Krichevsky, A., Chen, Y. and Canfield, R.E. (1994) Endocr. Rev., 15, 650–683.

    Google Scholar 

  • Oefner, C., D'Arcy, A., Winkler, F.K., Eggimann, B. and Hosang, M. (1992) Embo J., 11, 3921–3926.

    Google Scholar 

  • Osborn, B.L. and Abramson, F.P. (1995) Anal. Biochem., 229, 347–350.

    Google Scholar 

  • Remerowski, M.L., Domke, T., Groenewegen, A., Pepermans, H.A.M., Hilbers, C.W. and Van deVen, F.J.M. (1994) J. Biomol. NMR 4, 257–278.

    Google Scholar 

  • Sairam, M.R. and Manjunath, P. (1982) Int. J. Pept. Protein Res., 19, 315–320.

    Google Scholar 

  • Sairam, M.R. and Manjunath, P. (1983) J. Biol. Chem., 258, 445–449.

    Google Scholar 

  • Sairam, M.R., Linggen, J. and Bhargavi, G.N. (1988) Biosci. Rep., 8, 271–278.

    Google Scholar 

  • Sairam, M.R., Linggen, J., Sairam, J. and Bhargavi, G.N. (1990) Biochem. Cell Biol., 68, 889–893.

    Google Scholar 

  • Schlunegger, M.P., Grutter, M.G. (1992) Nature, 358, 430–434.

    Google Scholar 

  • Strickland, T.W. and Puett, D. (1981) Endocrinology, 109, 1933–1942.

    Google Scholar 

  • Weisshaar, G., Hiyama, J. and Renwick, G.C. (1991) Glycobiology, 1, 393–404.

    Google Scholar 

  • Wold, F. (1981) Annu. Rev. Biochem., 50, 783–814.

    Google Scholar 

  • Wold, F. and Moldave, K. (1984) Methods Enzymol., 106, 29–58.

    Google Scholar 

  • Wray, W., Bolikas, T., Wray, V.P. and Hancock, R. (1981) Anal. Biochem., 118, 197–203.

    Google Scholar 

  • Wu, H., Lustbader, J.W., Liu, Y., Canfield, R.E. and Hendrickson, W.A. (1994) Structure, 2, 545–558.

    Google Scholar 

  • YMC Application No. 10132 Technical Report (1994), YMC Inc., Wilmington, NC.

Download references

Author information

Authors and Affiliations

  1. Department of Medicine, Columbia University, 630 West 168th Street, 10032, New York, NY, USA

    Joyce W. Lustbader, Steven Birken, Susan Pollak, Shakuntala Ramnarain & Robert E. Canfield

  2. Protein Chemistry Core Laboratory, Columbia University, 630 West 168th Street, 10032, New York, NY, USA

    Andrew Pound

  3. Rockefeller University, 1230 York Avenue, 10021, New York, NY, USA

    Brian T. Chait & Urooj A. Mirza

  4. Martek Biosciences Inc., 6480 Dobben Road, 21045, Columbia, MD, USA

    Jonathan Miles Brown

Authors
  1. Joyce W. Lustbader
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Steven Birken
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Susan Pollak
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andrew Pound
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Brian T. Chait
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Urooj A. Mirza
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shakuntala Ramnarain
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Robert E. Canfield
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jonathan Miles Brown
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

To whom correspondence should be addressed.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lustbader, J.W., Birken, S., Pollak, S. et al. Expression of human chorionic gonadotropin uniformly labeled with NMR isotpes in Chinese hamster ovary cells: An advance toward rapid determination of glycoprotein structures. J Biomol NMR 7, 295–304 (1996). https://doi.org/10.1007/BF00200431

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00200431

Keywords

Search

Navigation