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J. Biol. Chem., Vol. 275, Issue 38, 29458-29465, September 22, 2000
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From the G domains of the mouse laminin
High and Low Affinity Heparin-binding Sites in the G Domain of
the Mouse Laminin
4 Chain*
§,§,,
**, and
Graduate Course for Regulation of Biological
Signals, Graduate School of Bioagricultural Sciences and
Bioscience Center, Nagoya University, Nagoya 464-8601, Japan and
the ¶ Graduate School of Environmental Earth Science, Hokkaido
University, Sapporo 060-0810, Japan
1 and 4
chains consisting of its five subdomains LG1-LG5 were overexpressed in
Chinese hamster ovary cells and purified by heparin
chromatography. 1LG1-LG5 and 4LG1-LG5 eluted at NaCl
concentrations of 0.30 and 0.47 M, respectively. In
solid phase binding assays with immobilized heparin, half-maximal
concentrations of 14 (1LG1-LG5) and 1.4 nM
(4LG1-LG5) were observed. N-Glycan cleavage of
4LG1-LG5 did not affect affinity to heparin. The affinity of
4LG1-LG5 was significantly reduced upon denaturation with 8 M urea but could be recovered by removing urea.
Chymotrypsin digestion of 4LG1-LG5 yielded high and low heparin
affinity fragments containing either the 4LG4-LG5 or 4LG2-LG3
modules, respectively. Trypsin digestion of heparin-bound 4LG1-LG5
yielded a high affinity fragment of about 190 residues corresponding to
the 4LG4 module indicating that the high affinity binding site is
contained within 4LG4. Competition for heparin binding of synthetic
peptides covering the 4LG4 region with complete 4LG1-LG5
suggests that the sequence AHGRL1521 is crucial for high affinity
binding. Introduction of mutation of H1518A or R1520A in glutathione
S-transferase fusion protein of the 4LG4 module produced
in Escherichia coli markedly reduced heparin binding activity of the wild type. When compared with the known structure of
2LG5, this sequence corresponds to the turn connecting strands E and
F of the 14-stranded 
-sheet sandwich, which is opposite to the
proposed binding sites for calcium ion, -dystroglycan, and heparan sulfate.
*
This work was supported by Grant-in-Aids 09460046 and
11460154 for Scientific Research from the Ministry of Education,
Science, Culture and Sports of Japan (to Y. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Graduate School of
Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya-shi 464-8601, Japan. Tel./Fax: 81-52-789-5227; E-mail: i45073a@nucc.cc.nagoya-u.ac.jp.
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