Protein Protocols Protein Protocols

Microassay of Protein Glycosylation 849
GNA should stain positive for RNase B and ovalbumin but not the other glycoproteins/
proteins; however, negative results with RNase B were shown even after repeated tests
such as using increased concentrations of RNase B (up to 20 µg). The sugar of RNase B
was analyzed by hexose assay and shown to contain the expected 3%; thus the specificity
of GNA is not as published, and the lectin can distinguish between ovalbumin and RNase
B glycosylation.
17. Table 3 shows the binding preferences of the lectins used. From the results obtained, the
sensitivity of lectins can detect protein concentrations down to 10 ng. The choice between
the commercial digoxigenin-labeled and biotinylated lectins used relate to their recognition
of a range of defined and limited sugar structures depending on the samples tested.
One drawback to the procedure is that because a number of structurally distinct oligosaccharides
interact identically with certain lectins, this suggests that the structural heterogeneity
frequently encountered with glycoproteins may not always be reflected in the
interactions of these moieties with lectins.
18. For pigeon intestinal mucin (2) 8 of the 10 lectins (AAL, LEL, LTL, MAL-1, MPL, RCA-1,
SBA, SJA, UEA-II, and WGA) reacted specifically with a different spectrum of PLLoligosaccharide
conjugates. LTL stained the entire blot, suggesting that this lectin
interacted with the nitrocellulose. SJA reacted with all of the fractions and controls,
indicative of nonspecific interaction with PLL itself. For the other lectins there was a wide
range of activities with the various HPLC fractions. Difference in staining patterns
between the lectins demonstrated that oligosaccharides of varying structure have been
released from pigeon mucin by hydrazinolysis and have successfully been coupled to PLL,
thus allowing binding to the NCM. Optimization of conditions at the macro-level included
both neutral and sialylated oligosaccharides, with the chosen conditions giving maximum
yield of both. The principle of this assay is derived essentially from enzyme-linked
immunosorbent assay (ELISA) and commercial kits are available, where lectins are
already linked to antibodies or other methods for detection.
Acknowledgments
The authors wish to thank Erminia Barboni for collaboration in a larger study that
furnished the analytical amount of Thy-1 used in this study and to the following for
support: E. B. PRIN97 program grant, University for Scientific and Technological
Research, Rome, Italy; N. K. C. W., EU-BIOTECH research grant PL9765055; G. J.
R., EU-HCM grant; C. I. B., Wellcome Trust grants 042462 and 052676; and E. F. H.
and D. V. R., the UK Medical Research Council.
References
1. Sharon, N. (1998) Lectins: from obscurity into the limelight. Protein Sci. 7, 2042–2048.
2. Baldwin, C. I., Calvert, J. E., Renouf, D. V., Kwok, C., and Hounsell, E. F. (1999) Analysis of
pigeon intestinal mucin allergens using a novel dot blot assay. Carbohydr. Res., in press.
3. Barboni, E., Pliego Rivero, B., George, A. J. T., Martin, S. R., Renouf, D. V., Hounsell,
E. F., et al. (1995) The glycophosphatidylinositol anchor affects the conformation of the
Thy-1 protein. J. Cell Sci. 108, 487–497.
4. Baldwin, C. I., Todd, A., Bourke, S. J., Allen, A., and Clavert, J. E. (1998) IgG subclass
responses to pigeon intestinal mucin are related to development of pigeon fanciers’ lung.
Clin. Exp. Allergy 28, 349–357.
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liquid chromatography of oligosaccharide alditols and glycopeptides on a graphitised carbon
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