Section 10
Chapter 9,915

Molecular recognition of a Salmonella trisaccharide epitope by monoclonal antibody Se155-4

Bundle, D.R.; Eichler, E.; Gidney, M.A.; Meldal, M.; Ragauskas, A.; Sigurskjold, B.W.; Sinnott, B.; Watson, D.C.; Yaguchi, M.; Young, N.M.

Biochemistry 33(17): 5172-5182


ISSN/ISBN: 0006-2960
PMID: 7513555
Accession: 009914249

The binding site of monoclonal antibody Se155-4, which has been the object of successful crystallographic and antibody-engineering studies, is shown by solid-phase immunoassays to be complementary to a branched trisaccharide, alpha-D-Galp(1 fwdarw 2) (alpha-D-Abep(1 fwdarw 3))-alpha-D-Manp(1, rather than to the tetrasaccharide repeating unit alpha-D-Galp(1 fwdarw 2) (alpha-D-Abep(1 fwdarw 3))-alpha-D-Manp(1 fwdarw 4)alpha-L-Rhap(1- of the bacterial antigen. Specificity for the 3,6-dideoxy-D-xylo-hexose (3,6-dideoxy-D-galactose) epitope present in Salmonella paratyphi B O-antigens was ensured by screening hybridoma experiments with glycoconjugates derived from synthetic oligosaccharides. Detailed epitope mapping of the molecular recognition by modified and monodeoxy oligosaccharide derivatives showed that complementary surfaces and three antibodysaccharide hydrogen bonds are essential for full binding activity. Both hydroxyl groups of the 3,6-dideoxy-D-galactose residue were obligatory for binding and consistent with the directional nature of their involvement in carbohydrate-protein hydrogen bonds; related tetrasaccharides built from the isomeric 3,6-dideoxyhexoses, 3,6-dideoxy-D-glucose, paratose, and 3,6-dideoxy-D-mannose, tyvelose were not bound by the antibody. Titration microcalorimetry measurements were consistent with the hydrogen-bonding map inferred from the crystal structure and suggest that the displacement of water molecules from the binding site accounts for the favorable entropy that accompanies binding of the native trisaccharide determinant. The protein sequences determined for the antibody V-L and V-H domains reveal somatic mutation of the V-L germ line gene, implying that this antibody-binding site results from a mature antibody response.

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