Linked Life Data

8820657

Source:http://linkedlifedata.com/resource/pubmed/id/8820657

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
4
pubmed:dateCreated
1996-12-16
pubmed:abstractText
The homopentameric B subunit of verotoxin 1 (VT1) binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). We produced mutants with alanine substitutions for residues found near the cleft between adjacent subunits. Substitution of alanine for phenylalanine 30 (Phe-30) resulted in a fourfold reduction in B subunit binding affinity for Gb3 and a 10-fold reduction in receptor density in a solid-phase binding assay. The interaction of wild-type and mutant B subunits with Pk trisaccharide in solution was examined by titration microcalorimetry. The carbohydrate binding of the mutant was markedly impaired compared with that of the wild type and was too weak to allow calculation of a binding constant. These results demonstrate that the mutation significantly impaired the carbohydrate-binding function of the B subunit. To ensure that the mutation had not caused a significant change in structure, the mutant B subunit was crystallized and its structure was determined by X-ray diffraction. Difference Fourier analysis showed that its structure was identical to that of the wild type, except for the substitution of alanine for Phe-30. The mutation was also produced in the VT1 operon, and mutant holotoxin was purified to homogeneity. The cytotoxicity of the mutant holotoxin was reduced by a factor of 10(5) compared to that of the wild type in the Vero cell cytotoxicity assay. The results suggest that the aromatic ring of Phe-30 plays a major role in binding of the B subunit to the Galalpha1-4Galbeta1-4Glc trisaccharide portion of Gb3. Examination of the VT1 B crystal structure suggests two potential carbohydrate-binding sites which lie on either side of Phe-30.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0950-382X
pubmed:author
pubmed:issnType
Print
pubmed:volume
19
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
891-9
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Phenylalanine 30 plays an important role in receptor binding of verotoxin-1.
pubmed:affiliation
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't