Partial characterization of the human serum transferrin epitope reactive with the monoclonal antibody TRC-2.
A murine monoclonal antibody (MAb) (TRC-2) specific for human serum transferrin (Tf(h)) was developed. This antibody was depressive on cell growth in serum-free medium in the presence of limiting amounts of Tf(h), but it did not inhibit the binding of Tf(h)-alkaline phosphatase (AP) conjugate to the Tf-receptor (TfR) in a cellular enzyme-linked immunosorbent assay (CELISA) system. On the other hand, the immune complex Tf(h)-TRC-2 was implicated to bind to the receptor in indirect CELISA. Moreover, the detectability of Tf(h)-TfR on the cell surface via Tf-bound TRC-2 suggested that the antibody may inhibit the rapid internalization of this complex. To map the TRC-2-specific epitope, Tf(h) was subjected to proteolytic degradation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The treatment with trypsin gave rise to, among others, a fragment of about 42 kDa, which was reactive with TRC-2. Through sequence analysis by automated Edman degradation, the N-terminal sequence of the 42 kDa-tryptic fragment was aligned to the N-terminus of mature transferrin (VPDKTVR). The N-terminal sequence of an immunoreactive CNBr-fragment of about 13 kDa was, in turn, identical with the sequence (NQLRGKK) corresponding to the residues 110-116 on Tf(h).
Oztürk
S
S
Research Institute for Genetic Engineering and Biotechnology, 41470 Gebze-Kocaeli, Turkey. selma@rigeb.gov.tr
Cirakoglu
B
B
Bermek
E
E
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D015153:Blotting, Western; D002455:Cell Division; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D020014:K562 Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014168:Transferrin / Q000737:chemistry* / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D016895:Culture Media, Serum-Free; D000939:Epitopes; D014168:Transferrin; D000469:Alkaline Phosphatase; D014357:Trypsin