Mapping the functional domains of human transcobalamin using monoclonal antibodies.
Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA...TAAM(103-198) or the C-terminal peptide LEPA...LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS...QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA...HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment KRSN...RTVR(207-227), which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor.
Fedosov
Sergey N
SN
Protein Chemistry Laboratory, Department of Molecular Biology, University of Aarhus,Denmark. snf@mb.au.dk
Orning
Lars
L
Løvli
Trond
T
Quadros
Edward V
EV
Thompson
Keith
K
Berglund
Lars
L
Petersen
Torben E
TE
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D003488:Cyanogen Bromide / Q000737:chemistry; D003553:Cystine / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D020349:Surface Plasmon Resonance; D014155:Transcobalamins / Q000737:chemistry* / Q000276:immunology / Q000502:physiology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D014155:Transcobalamins; D003553:Cystine; D003488:Cyanogen Bromide