Epitope mapping of a monoclonal antibody specific to bovine dry milk: involvement of residues 66-76 of strand D in thermal denatured beta-lactoglobulin.
beta-Lactoglobulin (beta-LG) is a bovine milk protein sensitive to thermal denaturation. Previously, we demonstrated that such structural change can be detected by a monoclonal antibody (mAb) specific to denatured beta-LG. In the present study, we show a dramatic increase in beta-LG immunoreactivity when heating raw milk between 70 and 80 degrees C. To map out the specific epitope of beta-LG recognized by this mAb, we used a combined strategy including tryptic and CNBr fragments, chemical modifications (acetylation and carboxymethylation), peptide array containing in situ synthesized peptides, and a synthetic soluble peptide for immunoassays. The antigenic determinant we defined was exactly located within the D strand (residues 66-76) of beta-LG. Circular dichroic spectral analysis shows that carboxymethylation on beta-LG not only resulted in a substantial loss of beta-configuration but also exerted a 10 times increase in immunoreactivity as compared with heated beta-LG. The result suggests that a further disordered structure occurred in beta-LG and thus rendered the mAb recognition. Mutations on each charged residue (three Lys and one Glu) revealed that Lys-69 and Glu-74 were extremely essential in maintaining the antigenic structure. We also show an inverse relationship between the immunoreactivity in heated beta-LG and its binding to retinol or palmitic acid. Most interestingly, pH 9-10, which neutralizes the Lys groups of beta-LG, not only reduced its immunoreactivity but also its binding to palmitic acid implicating a role of Lys-69. Taken together, we concluded that strand D of beta-LG participated in the thermal denaturation between 70 and 80 degrees C and the binding to retinol and palmitic acid. The antigenic and biochemical roles of mAb specific to D strand are discussed in detail.
Song
Chun Ying
CY
Research Institute of Biochemical Engineering, Department of Biological Science and Technology, National Chiao Tung University, Hsinchu and Genesis Biotech Inc., 131, Lane 235, Bao-Chiao Road, Hsintien, Taipei, Taiwan, Republic of China.
Chen
Wen Liang
WL
Yang
Ming Chi
MC
Huang
Jen Pin
JP
Mao
Simon J T
SJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002417:Cattle; D002942:Circular Dichroism; D003890:Desiccation; D018604:Epitope Mapping; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D008892:Milk / Q000276:immunology*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D000911:Antibodies, Monoclonal; D007782:Lactoglobulins