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J Biol Chem, Vol. 274, Issue 43, 30377-30386, October 22, 1999
From the Pathology Research Laboratory. Massachusetts General
Hospital, Harvard Medical School,
Charlestown, Massachusetts 02129
We recently showed that thyroglobulin (Tg) is a
heparin-binding protein and that heparin inhibits binding of Tg to its
endocytic receptor megalin (gp330). Here we have identified a
heparin-binding region in the carboxyl-terminal portion of rat Tg and
have studied its involvement in megalin binding. Rat thyroid extracts,
obtained by ammonium sulfate precipitation, were separated by column
fractionation into four Tg polypeptides, with apparent masses of 660, 330, 210, and 50 kDa. As assessed by enzyme-linked immunoadsorbent
assays and ligand blot binding assays, megalin bound to intact Tg (660 and 330 kDa) and, to a even greater extent, to the 210-kDa Tg polypeptide. Furthermore, the 210-kDa Tg polypeptide inhibited megalin
binding to intact Tg by ~70%. Solid phase assays showed binding of
biotin-labeled heparin to intact Tg and to the 210-kDa Tg polypeptide.
We characterized the 210-kDa Tg polypeptide by matrix-assisted laser
desorption/ionization mass spectrometry analysis and found that it
corresponds to the carboxyl-terminal portion of rat Tg. We developed a
synthetic peptide corresponding to a 15-amino acid sequence in the
carboxyl-terminal portion of rat Tg
(Arg689-Lys703), containing a
heparin-binding consensus sequence (SRRLKRP) and demonstrated heparin
binding to this peptide. A rabbit antibody raised against the peptide
recognized intact Tg in its native conformation and under denaturing
conditions. This antibody markedly reduced heparin-binding to intact
Tg, indicating that the region of native Tg corresponding to the
peptide is involved in heparin binding. Furthermore, the anti-Tg
peptide antibody almost completely inhibited binding of megalin to Tg,
suggesting that the Tg region containing the peptide sequence is
required for megalin binding. Physiologically, Tg binding to megalin on
thyroid cells may be facilitated by Tg interaction with heparin-like
molecules (heparan sulfate proteoglycans) via adjacent binding sites.
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