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Binding of pertussis toxin to eucaryotic cells and glycoproteins
M H Witvliet1, D L Burns, M J Brennan
1Laboratory of Pertussis, Food and Drug Administration, Bethesda, Maryland 20892.
Abstract:
The binding of pertussis toxin and its subunits to cell surface receptors and purified glycoproteins was examined. The interaction of pertussis toxin with components of two variant Chinese hamster ovary (CHO) cell lines was studied. These cell lines are deficient in either sialic acid residues (LEC 2) or sialic acid and galactose residues (LEC 8) on cell surface macromolecules. The binding of pertussis toxin to components of these cells differed from the binding of the toxin to wild-type components. Although the toxin bound to a 165,000-dalton glycoprotein found in N-octylglucoside extracts of wild-type cells, it did not bind to components found in extracts of LEC 2 cells. In contrast, the toxin bound to components found in extracts of LEC 8 cells, which are variant cells that contain increased amounts of terminal N-acetylglucosamine residues on cell surface macromolecules. These results suggest that the receptor for pertussis toxin on CHO cells contains terminal acetamido-containing sugars. The cytopathic effect of the toxin on both types of variant cells was much reduced compared with its effects on wild-type cells. Thus, optimal functional binding of pertussis toxin appears to require a complete sialyllactosamine (NeuAc----Gal beta 4GlcNAc) sequence on surface macromolecules. In addition to studying the nature of the eucaryotic receptor for pertussis toxin, we examined corresponding binding sites for glycoproteins on the toxin molecule. Binding of both S2-S4 and S3-S4 dimers of the toxin to cellular components and purified glycoproteins was observed. The two dimers bound to a number of glycoproteins containing N-linked oligosaccharides but not O-linked oligosaccharides, and differences in the binding of the two dimers to some glycoproteins was noted. These data indicate that the holotoxin molecule contains at least two glycoprotein-binding sites which may have slightly different specificities for glycoproteins.
Insights
Pertussis toxin binding to Chinese hamster ovary (CHO) cells requires a complete sialyllactosamine sequence for optimal function. The toxin
Area of Science:
- Molecular Biology
- Cell Biology
- Glycobiology
Background:
- Pertussis toxin is a bacterial toxin that interacts with cell surface receptors.
- Understanding the molecular basis of pertussis toxin binding is crucial for elucidating its mechanism of action.
- Chinese hamster ovary (CHO) cells are widely used models for studying cellular interactions.
Purpose of the Study:
- To investigate the specific cell surface receptors and glycoproteins involved in pertussis toxin binding.
- To determine the structural requirements for pertussis toxin interaction with its cellular targets.
- To analyze the glycoprotein-binding sites on the pertussis toxin molecule.
Main Methods:
- Comparative analysis of pertussis toxin binding to wild-type and variant CHO cell lines (LEC 2 and LEC 8) with defined cell surface carbohydrate deficiencies.
- Biochemical examination of toxin interaction with N-octylglucoside extracts of cellular components and purified glycoproteins.
- Assessment of the cytopathic effects of pertussis toxin on different cell lines.
Main Results:
- Pertussis toxin binding was significantly altered in variant CHO cells lacking specific sialic acid and galactose residues.
- The toxin demonstrated differential binding to cell components, with a preference for those containing terminal acetamido-containing sugars.
- Optimal functional binding of pertussis toxin necessitates a complete sialyllactosamine sequence on cell surface macromolecules.
- The pertussis toxin holotoxin possesses at least two distinct glycoprotein-binding sites, with varying specificities for N-linked oligosaccharides.
Conclusions:
- The receptor for pertussis toxin on CHO cells involves terminal acetamido-containing sugars, specifically the sialyllactosamine sequence.
- The cytopathic effects of pertussis toxin are dependent on the integrity of this specific carbohydrate structure.
- Pertussis toxin subunits exhibit distinct binding capabilities to different glycoproteins, suggesting complex interactions.