Identification of a homologous heparin binding peptide sequence present in fibronectin and the 70 kDa family of heat-shock proteins.
This study was undertaken to characterize the potential heparin affinity of an amino-acid sequence within the 70 kDa heat-shock family of proteins (HSPs) that shares homology with a heparin-binding sequence present in the carboxy-terminus of fibronectin (FN), defined by the synthetic peptide, FN-C/H-II (KNNQKSEPLIGRKKT). To first define the heparin binding sequence within FN-C/H-II, solid phase binding assays were performed using overlapping, short (7 amino acids) synthetic peptides corresponding to the amino-acid sequence within FN-C/H-II. Only the sequence LIGRKKT bound [3H] heparin, and the LIGRKKT peptide blocked heparin binding to intact fibronectin by 47% (+/- 0.4, p < 0.001). A computer-generated homology search revealed that two members of the 70 kDa HSP family, HSP70 and HSC70, contain the sequences LIGRK and LIGRR, respectively. Examination of heparin binding using affinity chromatography indicated that while native HSC70 binds heparin, native HSP70 does not. Treatment of the heparin-unbound fraction with heat or urea led to enhanced HSP70 binding to heparin affinity columns. Soluble LIGRKKT peptide or anti-FN-C/H-II IgG also significantly inhibited heparin binding to HSC70 that had been purified by heparin affinity chromatography. Finally, Western blot analysis of HSC70 purified by heparin affinity chromatography demonstrated that polyclonal anti-FN-C/H-II IgG could recognize HSC70. These data demonstrate that LIGRK or LIGRR represent a a common heparin binding motif in fibronectin, HSP70, and HSC70, and are consistent with a proposed role for heparin or similar polyanionic structures in the function of the 70 kDa heat-shock proteins.
Hansen
L K
LK
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 5545, USA.
O'Leary
J J
JJ
Skubitz
A P
AP
Furcht
L T
LT
McCarthy
J B
JB
D000595:Amino Acid Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D005353:Fibronectins / Q000737:chemistry*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D005353:Fibronectins; D018840:HSP70 Heat-Shock Proteins; D006493:Heparin