Metabolic selection of glycosylation defects in human cells (original) (raw)

References

  1. Durand, G. & Seta, N. Protein glycosylation and diseases: blood and urinary oligosaccharides as marker for diagnosis and therapeutic monitoring. Clin. Chem. 46, 795–805 (2000).
    CAS PubMed Google Scholar
  2. Orntoft, T.F. & Vestergaard, E.M. Clinical aspects of altered glycosylation of glycoproteins in cancer. Electrophoresis 20, 362–371 (1999).
    Article CAS Google Scholar
  3. Axford, J.S. Glycosylation and rheumatic disease. Biochim. Biophys. Acta 1455, 219–229 (1999).
    Article CAS Google Scholar
  4. Mackiewicz, A. & Mackiewicz, K. Glycoforms of serum alpha 1-acid glycoprotein as markers of inflammation and cancer. Glycoconj. J. 12, 241–247 (1995).
    Article CAS Google Scholar
  5. Lemyre, E. et al. Clinical spectrum of infantile free sialic acid storage disease. Am J. Med. Genet. 82, 385–391 (1999).
    Article CAS Google Scholar
  6. Sell, S. Cancer-associated carbohydrates identified by monoclonal antibodies. Hum. Pathol. 21, 1003–1019 (1990).
    Article CAS Google Scholar
  7. Kukuruzinska, M.A. & Lennon, K. Protein _N_-glycosylation: molecular genetics and functional significance. Crit. Rev. Oral Biol. Med. 9, 415–448 (1998).
    Article CAS Google Scholar
  8. Stanley, P. & Ioffe, E. Glycosyltransferase mutants: key to new insights in glycobiology. FASEB J. 9, 1436–1444 (1995).
    Article CAS Google Scholar
  9. Dennis, J.W., Granovsky, M. & Warren, C.E. Protein glycosylation in development and disease. BioEssays 21, 412–421 (1999).
    Article CAS Google Scholar
  10. Stark, G.R. & Gudkov, A.V. Forward genetics in mammalian cells: functional approaches to gene discovery. Hum. Mol. Genet. 8, 1925–1938 (1999).
    Article CAS Google Scholar
  11. Rutishauser, U. Polysialic acid at the cell surface: biophysics in service of cell interactions and tissue plasticity. J. Cell. Biochem. 70, 304–312 (1998).
    Article CAS Google Scholar
  12. Sillanaukee, P., Ponnio, M. & Jaaskelainen, I.P. Occurrence of sialic acids in healthy humans and different disorders. Eur. J. Clin. Invest. 29, 413–425 (1999).
    Article CAS Google Scholar
  13. Mahal, L.K., Yarema, K.J. & Bertozzi, C.R. Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 276, 1125–1128 (1997).
    Article CAS Google Scholar
  14. Yarema, K.J., Mahal, L.K., Bruehl, R.E., Rodriguez, E.C. & Bertozzi, C.R. Metabolic delivery of ketone groups to sialic acid residues. Application to cell surface glycoform engineering. J. Biol. Chem. 273, 31168–31179 (1998).
    Article CAS Google Scholar
  15. Bellgard, M.I., Itoh, T., Watanabe, H., Imanishi, T. & Gojobor, T. Dynamic evolution of genomes and the concept of genome space. Ann. NY Acad. Sci. 870, 293–300 (1999).
    Article CAS Google Scholar
  16. Fabb, S.A. & Ragoussis, J. High-efficiency human B-cell cloning using hygromycin B-resistant feeder cells. Biotechniques 22, 814–822 (1997).
    Article CAS Google Scholar
  17. Jalanko, A., Kallio, A., Ruohonin-Lehto, M., Soderlund, H. & Ulmanen, I. An EBV-based mammalian cell expression vector for efficient expression of cloned coding sequences. Biochim. Biophys. Acta 949, 206–212 (1988).
    Article CAS Google Scholar
  18. Wang, W.-C. & Cummings, R.D. The immobilized leukoagglutinin from the seeds of Maackia amurensis binds with high affinity to complex-type Asn-linked oligosaccharides containing terminal sialic acid-linked α-2,3 to penultimate galactose residues. J. Biol. Chem. 263, 4576–4585 (1988).
    CAS PubMed Google Scholar
  19. Taatjes, D.J., Roth, J., Peumans, W. & Goldstein, I.J. Elderberry bark lectin–gold techniques for the detection of Neu5Ac (α2,6) Gal/GalNAc sequences: applications and limitations. Histochem. J. 20, 478–490 (1988).
    Article CAS Google Scholar
  20. Jourdian, G.W., Dean, L. & Roseman, S. The sialic acids. XI. A periodate–resorcinol method for the quantitative estimation of free sialic acids and their glycosides. J. Biol. Chem. 246, 430–435 (1971).
    CAS PubMed Google Scholar
  21. Hale, L.P., van de Ven, C.J., Wenger, D.A., Bradford, W.D. & Kahler, S.G. Infantile sialic acid storage disease: a rare cause of cytoplasmic vacuolation in pediatric patients. Pediatr. Pathol. Lab. Med. 15, 443–453 (1995).
    Article CAS Google Scholar
  22. Thomas, G.H., Scocca, J., Miller, C.S. & Reynolds, L. Evidence for non-lysosomal storage of _N_-acetylneuraminic acid (sialic acid) in sialuria fibroblasts. Clin. Genet. 36, 242–249 (1989).
    Article CAS Google Scholar
  23. Seppala, R. et al. Sialic acid metabolism in sialuria fibroblasts. J. Biol. Chem. 266, 7456–7461 (1991).
    CAS PubMed Google Scholar
  24. Lucka, L., Krause, M., Reutter, W. & Horstkorte, R. Primary structure and expression analysis of human UDP-_N_-acetyl-glucosamine-2-epimerase/_N_-acetylmannosamine kinase, the bifunctional enzyme in neuraminic acid biosynthesis. FEBS Lett. 454, 341–344 (1999).
    Article CAS Google Scholar
  25. Seppala, R., Lehto, V.P. & Gahl, W.A. Mutations in the human UDP-_N_-acetylglucosamine 2-epimerase gene define the disease sialuria and the allosteric site of the enzyme. Am. J. Hum. Genet. 64, 1563–1569 (1999).
    Article CAS Google Scholar
  26. Perera, A.D., Lagenaur, C.F. & Plant, T.M. Postnatal expression of polysialic acid–neural cell adhesion molecule in the hypothalamus of the male rhesus monkey (Macaca mulatta). Endocrinology 133, 2729–2735 (1993).
    Article CAS Google Scholar
  27. Ronn, L.C., Berezin, V. & Bock, E. The neural cell adhesion molecule in synaptic plasticity and ageing. Int. J. Dev. Neurosci. 18, 193–199 (2000).
    Article CAS Google Scholar
  28. Nakayama, J., Angata, K., Ong, E., Katwuyama, T. & Fukuda, M. Polysialic acid, a unique glycan that is developmentally regulated by two polysialyltransferases, PST and STX, in the central nervous system from biosynthesis to function. Pathol. Int. 48, 665–677 (1998).
    Article CAS Google Scholar
  29. Kameda, K. et al. Expression of highly polysialylated neural cell adhesion molecule in pancreatic cancer neural invasive lesion. Cancer Lett. 137, 201–207 (1999).
    Article CAS Google Scholar
  30. Hang, H. & Bertozzi, C.R. Ketone isoesters of 2-_N_-acetaminosugars as substrates for metabolic cell surface engineering. J. Am. Chem. Soc. 123, 1242–1243 (2001).
    Article CAS Google Scholar
  31. Keppler, O.T. et al. UDP-GlcNAc 2-epimerase: a regulator of cell surface sialylation. Science 284, 1372–1376 (1999).
    Article CAS Google Scholar
  32. Foley, K.P., Leonard, M.W. & Engel, J.D. Quantitation of RNA using the polymerase chain-reaction. Trends Genet. 9, 380–385 (1993).
    Article CAS Google Scholar
  33. Zimmermann, K. & Mannhalter, J.W. Technical aspects of quantitative competitive PCR. Biotechniques 21, 268 (1996).
  34. Halford, W.P., Falco, V.C., Gebhardt, B.M. & Carr, D.J.J. The inherent quantitative capacity of the reverse transcription of polymerase chain reaction. Anal. Biochem. 266, 181–191 (1999).
    Article CAS Google Scholar

Download references

Acknowledgements

The authors gratefully acknowledge Lara K. Mahal for contributions of ManLev, and P. Schow and H. Nolla (Flow Cytometry Laboratory, Center for Cancer Research, Molecular and Cell Biology, University of California, Berkeley) for helpful discussions and technical assistance. This work was supported by the director, Office of Science, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering, and the Office of Energy Biosciences of the US Department of Energy under Contract No. DE-AC03-76SF00098 and the National Institutes of Health (GM58867-01).

Author information

Author notes

  1. Kevin J. Yarema
    Present address: Department of Biomedical Engineering, G.W.C. Whiting School of Engineering, Johns Hopkins University, Baltimore, MD, 21218

Authors and Affiliations

  1. Department of Chemistry, University of California, Berkeley, 94720, CA
    Kevin J. Yarema, Scarlett Goon & Carolyn R. Bertozzi
  2. Department of Molecular and Cell Biology, University of California, Berkeley, 94720, CA
    Carolyn R. Bertozzi
  3. Materials Sciences Division, Center for Advanced Materials, Lawrence Berkeley National Laboratory, Berkeley, 94720, CA
    Kevin J. Yarema & Carolyn R. Bertozzi
  4. Howard Hughes Medical Institute, Berkeley, 94720, CA
    Carolyn R. Bertozzi

Authors

  1. Kevin J. Yarema
  2. Scarlett Goon
  3. Carolyn R. Bertozzi

Corresponding author

Correspondence toCarolyn R. Bertozzi.

Supplementary information

Supplementary Text (PDF 5 kb) (download PDF )

Supplementary Figure 1. (download JPG )

Strategy for reconstituting the sialuria phenotype in wild-type Jurkat cells by introduction of mutant UDP-GlcNAc 2-epimerase. Jurkat cells (colored) harboring mutant epimerase genes were isolated from a large population of wild-type cells (black) by ManLev-based metabolic selection methods. Using the methods described in the main text, mRNA was (1) isolated from each of these cell types, (2) used for cDNA synthesis, (3) subjected to PCR amplification, (4) DNA sequencing, and subsequently (5) cloned into the expression vector pREP9. Note that pREP9(WT), black, contained the wild-type gene; pREP9(R263Q), red, and pREP9(R266W), purple, contained the indicated mutant gene. These plasmids were transfected into wild-type Jurkat cells by the LipofectAMINE method (see main text). The cells were incubated with 300 mg/ml G418 (selection agent for the neomycin-resistance gene of pREP9) for two weeks. The concentration of G418 was then doubled to 600 mg/ml and maintained at this concentration. The cells were periodically analyzed to determine internal sialic acid levels and cell surface SiaLev expression by the methods described in the main text. (JPG 52 kb)

Supplementary Figure 2. (download JPG )

Reconstitution of the low-SiaLev phenotype in wild-type Jurkat cells by expression of mutant UDP-GlcNAc 2-epimerase genes. SiaLev expression was determined under standard conditions after incubation with ManLev as described in the main text following transfection of wild-type Jurkat cells with the wild-type, R263Q, or R266W form of UDP-GlcNAc 2-epimerase. SiaLev expression analysis for the epimerase-transfected cell lines are shown in black superimposed on similar data for negative (ManLev (-), blue) and positive control (ManLev (+), green) parent cells. (A) Two weeks after transfection and subsequent incubation with a low level of selection agent (300 mg/ml G418), the wild-type epimerase gene had no effect on SiaLev expression in Jurkat cells (left, cell population I). By contrast, a subpopulation of cells with reduced SiaLev expression was developing in both the R263Q- and the R266W-transfected Jurkat cells (center and right, II). Both of these cultures also contained cells with wild-type SiaLev expression (III) possibly due to the survival of nontransformed cells in the presence of relatively low level of selection agent. Consequently G418 concentration was increased to 600 mg/ml to ensure removal of these cells. (B) The wild-type epimerase gene had no noticeable affect on SiaLev expression five weeks after transfection into Jurkat cells (left, cell population IV). The proportion of low-SiaLev cells in the R263Q- and R266W-transfected cultures (center and right, V) had increased relative to the number of cells with normal SiaLev expression (VI). A reasonable explanation for the two distinct cell populations is that the low-SiaLev cells expressed both genes situated on the engineered pREP9 plasmid (i.e., the mutant epimerase and neomycin resistance gene), whereas the cells with normal SiaLev expressed only the neomycin resistance gene. (C) Baseline separation of populations V and VI (panel B) allowed rapid sorting of the low-SiaLev cells into homogeneous cell populations (center and right, VII) by flow cytometry. Similar sorting for the culture transfected with the wild-type epimerase gene yielded no cells (panel C, left), verifying the causal role that the mutant epimerase gene plays in the development of the low-SiaLev phenotype. (JPG 83 kb)

Supplementary Figure 3. (download JPG )

Analysis of Jurkat cells expressing the wild-type and mutant UDP-GlcNAc 2-epimerase genes. SiaLev expression of various Jurkat subpopulations was determined by biotin hydrazide labeling, FITC-avidin staining, and flow cytometry analysis of the cells. Sialic acid levels were determined by the periodate resorcinol assay. In (A), data labeled (+) and (-) indicate wild-type Jurkat cells incubated with and without ManLev. These cells function as positive and negative control populations, respectively. In both panels data labeled WT represent cells transfected with the wild-type epimerase (population IV, Supplementary Fig. 2B, left) and data labeled R263Q and R266W represent cells transfected with either form of the mutant epimerase (population VII, Supplementary Fig. 2C, center and right, respectively). Each data point is the average of five determinations. (A) Jurkat cells transfected with the wild-type epimerase show a level of SiaLev expression similar to nontransformed, positive control cells (two lefthand bars). By contrast, cells transfected with an epimerase containing either the R263Q or the R266W mutation have levels of SiaLev expression comparable to nontransformed, negative control (ManLev(-)) cells (three righthand bars). (B) Authentic sialuria cells are characterized by increased levels of sialic acid, particularly in the free monosaccharide form. Therefore the sialic acid levels of the transfected cells were determined to verify that the R263Q and R266W mutations fully reconstitute the sialuria phenotype. As shown, the wild-type epimerase has no effect on either total sialic acid levels or the free monosaccharide form of this sugar. By contrast, both of the mutant forms of the epimerase substantially increase the free sialic acid content of the transformed cells. (JPG 32 kb)

Rights and permissions

About this article

Cite this article

Yarema, K., Goon, S. & Bertozzi, C. Metabolic selection of glycosylation defects in human cells.Nat Biotechnol 19, 553–558 (2001). https://doi.org/10.1038/89305

Download citation