Epitope characterization of a supramolecular protein assembly with a collection of monoclonal antibodies: the case of casein micelle.
In milk, kappa-, beta-, alphas(1)- and alphas(2)-casein (CN) are associated into a supramolecular assembly, the micelle. In this work, CN micelles contained in fresh skim milk were used to produce over 100 monoclonal antibodies. The specificity of these probes was determined using libraries of synthetic peptides and peptides fractionated from tryptic hydrolysis of purified CNs. Although kappa-CN and alphas(2)-CN are minor proteins in the micelle (ratio 1:1:4:4 for kappa, alphas(2), alphas(1), beta) a proportionally high number of clones were produced towards these two proteins (32 for each), compared to 9 and 29 for alphas(1)-CN and beta-CN, respectively. Most of the beta-CN and kappa-CN epitopes were identified, while about 50% of alphas(1)-CN and alphas(2)-CN antibodies were suspected to react to conformational linear or discontinuous epitopes, since no peptide binding could be identified. Antibody binding to the phosphoserine rich regions of the three calcium sensitive CNs was weak or non-existing, suggesting them to be hidden in the micelle structure together with alphas(1)-CN. The C-terminal glycomacropeptide of kappa-CN and the C-terminal moiety of beta-CN were well exposed generating the majority of the antibodies specific for these two proteins. The two major antigenic sites of alphas(2) were alphas(2)-CN (f96-114) and (f16-35). Cross-reaction between alphas(2)-CN specific antibodies with alphas(1)-CN illustrated the tangled structure between the two proteins. Immuno-dominant epitopes identified in the present study totally differ from those known for the purified caseins suggesting they were specific for the micelle supramolecular structure.
Johansson
Annette
A
INRA-Unité de Recherches en Technologie et Analyses Latières, BP 20089, F-39800 Poligny, France.
Lugand
Damien
D
Rolet-Répécaud
Odile
O
Mollé
Daniel
D
Delage
Marie-Madeleine
MM
Peltre
Gabriel
G
Marchesseau
Sylvie
S
Léonil
Joëlle
J
Dupont
Didier
D
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D002364:Caseins / Q000276:immunology*; D002417:Cattle; D003429:Cross Reactions; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000276:immunology*; D005260:Female; D051379:Mice; D008823:Micelles; D008892:Milk / Q000276:immunology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D055503:Protein Multimerization
D000911:Antibodies, Monoclonal; D002364:Caseins; D000939:Epitopes; D008823:Micelles; D010455:Peptides