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Binding of heparin to human thyroglobulin (Tg) involves multiple binding sites including a region corresponding to a binding site of rat Tg.

Abstract：

OBJECTIVE:Binding of thyroglobulin (Tg) to heparin allows efficient Tg interaction with its endocytic receptor, megalin. Rat Tg (rTg) binds to heparin using an exposed carboxyl terminal region (RELPSRRLKRPLPVK, Arg2489-Lys2503) rich in positively charged residues which is, however, not entirely conserved in human Tg (hTg) (Arg2489-Glu2503, REPPARALKRSLWVE). Here, we investigated whether and how this difference affects binding of heparin. DESIGN:To compare binding of heparin to rTg and hTg. To investigate the role of the sequence 2489-2503 using a peptide-based approach. METHODS:Binding of biotin-labeled heparin to rTg, hTg and to Tg peptides was measured in solid phase assays. RESULTS:Heparin bound to rTg with moderately high affinity (K(d): 34.2 nmol/l, K(i): 37.6 nmol/l) and to hTg with lower affinity (K(d): 118 nmol/l, K(i): 480 nmol/l) and to a lower extent. Binding was dose-dependent and saturable, and was reduced by several specific competitors (Tg itself, unlabeled heparin, lactoferrin). Heparin bound to synthetic peptides corresponding to the rat (rTgP) and to the human (hTgP) Tg sequence 2489-2503. Heparin bound to rTgP to a greater extent and with greater affinity than to hTgP. An antibody against hTgP reduced binding of heparin to intact hTg by 30%, suggesting that in hTg this region is, in part, involved in heparin binding, but also that other regions account for most of the binding. Starting from the sequence of rTgP, we designed 6 synthetic 'mutant' peptides by replacing one amino acid residue of rTgP with the corresponding residue of the sequence of hTgP. Heparin bound to 5 of 6 mutant peptides to a lower extent and with lower affinity than to rTgP. CONCLUSIONS:In spite of a reduced binding ability of the sequence 2489-2503, hTg binds to heparin, in part, using alternative, as yet unidentified, binding sites. Substitution of both positive and neutral residues within the sequence 2489-2503 reduced heparin-binding, suggesting that not only charge, but also sequence and/or conformation, may account for the heparin-binding ability of this region of Tg.

journal_name

Eur J Endocrinol

journal_title

European journal of endocrinology

authors

Lisi S,Pinchera A,McCluskey RT,Chiovato L,Marinò M

doi

10.1530/eje.0.1460591

subject

Has Abstract

pub_date

2002-04-01 00:00:00

pages

591-602

issue

eissn

0804-4643

issn

1479-683X

pii

146591

journal_volume

146

pub_type

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Binding of heparin to human thyroglobulin (Tg) involves multiple binding sites including a region corresponding to a binding site of rat Tg.