Comparison of the diphtheria mutant toxin, CRM197, with a Haemophilus influenzae type-b polysaccharide-CRM197 conjugate by optical spectroscopy.
A Haemophilus influenzae type-b capsular polysaccharide-CRM197 protein conjugate vaccine was compared with unconjugated CRM197 and diphtheria toxin, its parent molecule. Using CD and fluorescence spectroscopy, it has been possible to observe differences in structure and stability to pH and temperature due to the G52-->E mutation in CRM197 and the 'glycosylation' of CRM197 in the conjugate. CRM197 resembles the 'open' conformation of diphtheria toxin [Blewitt, M. G., Chung, L. A. & London, E. (1985) Biochemistry 24, 5458-5464] and the attachment of poly(ribosyl-ribitol phosphate) carbohydrate chains results in a still 'more open' state, although only a small decrease in the amount of ordered structure was observed. Fluorescence spectra of gel-filtration column fractions of the conjugate suggest that material of higher apparent molecular size is in the 'more open' conformation. Conjugated CRM197 begins unfolding at slightly lower temperatures (25-35 degrees C) than native material (> 35 degrees C). In the conjugate, tryptophan residues are more accessible to the non-ionic fluorescence quencher acrylamide at 35 degrees C. The conformational change observed at pH4-6 for diphtheria toxin is also observed for CRM197, but in the conjugate begins at higher pH. This may result from the presence of charged oligosaccharide residues on the surface or the conjugation methods used. The consequences of these changes in conformation and solution behaviour of the carrier protein in terms of its ability to induce a protective, T-cell-dependent response to H. influenzae polysaccharide remain to be determined.
Crane
D T
DT
Laboratory for Molecular Structure, National Institute for Biological Standards and Control, South Mimms, Herts, UK.
Bolgiano
B
B
Jones
C
C
D001426:Bacterial Proteins / Q000737:chemistry*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000235:genetics; D018073:Haemophilus Vaccines / Q000737:chemistry*; D009154:Mutation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014614:Vaccines, Synthetic / Q000737:chemistry*
D001426:Bacterial Proteins; D004167:Diphtheria Toxin; D018073:Haemophilus Vaccines; D014614:Vaccines, Synthetic; C061963:HibTITER protein, Haemophilus influenzae