Identification of a heparin-binding region of rat thyroglobulin involved in megalin binding

Michele Marinò, Joel A. Friedlander, Robert T. McCluskey, David Andrews

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)30377-30386
Number of pages10
JournalJournal of Biological Chemistry
Volume274
Issue number43
DOIs
StatePublished - Oct 22 1999
Externally publishedYes

Fingerprint

Low Density Lipoprotein Receptor-Related Protein-2
Thyroglobulin
Heparin
Rats
Peptides
Assays
Antibodies
Thyroid Gland

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of a heparin-binding region of rat thyroglobulin involved in megalin binding. / Marinò, Michele; Friedlander, Joel A.; McCluskey, Robert T.; Andrews, David.

In: Journal of Biological Chemistry, Vol. 274, No. 43, 22.10.1999, p. 30377-30386.

Research output: Contribution to journalArticle

Marinò, Michele ; Friedlander, Joel A. ; McCluskey, Robert T. ; Andrews, David. / Identification of a heparin-binding region of rat thyroglobulin involved in megalin binding. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 43. pp. 30377-30386.
@article{c2b21bf6ea874a33975b0cba48aeab37,
title = "Identification of a heparin-binding region of rat thyroglobulin involved in megalin binding",
abstract = "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.",
author = "Michele Marin{\`o} and Friedlander, {Joel A.} and McCluskey, {Robert T.} and David Andrews",
year = "1999",
month = "10",
day = "22",
doi = "10.1074/jbc.274.43.30377",
language = "English",
volume = "274",
pages = "30377--30386",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "43",

}

TY - JOUR

T1 - Identification of a heparin-binding region of rat thyroglobulin involved in megalin binding

AU - Marinò, Michele

AU - Friedlander, Joel A.

AU - McCluskey, Robert T.

AU - Andrews, David

PY - 1999/10/22

Y1 - 1999/10/22

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0032742534&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032742534&partnerID=8YFLogxK

U2 - 10.1074/jbc.274.43.30377

DO - 10.1074/jbc.274.43.30377

M3 - Article

C2 - 10521414

AN - SCOPUS:0032742534

VL - 274

SP - 30377

EP - 30386

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 43

ER -