Abstract
The endoplasmic reticulum-resident chaperone heat-shock protein 47 (HSP47) plays an essential role in procollagen biosynthesis. The function of HSP47 relies on its specific interaction with correctly folded triple-helical regions comprised of Gly-Xaa-Yaa repeats, and Arg residues at Yaa positions have been shown to be important for this interaction. The amino acid at the Yaa position (Yaa(-3)) in the N-terminal-adjoining triplet containing the critical Arg (defined as Arg(0)) was also suggested to be directly recognized by HSP47 (Koide, T., Asada, S., Takahara, Y., Nishikawa, Y., Nagata, K., and Kitagawa, K. (2006) J. Biol. Chem. 281, 3432-3438). Based on this finding, we examined the relationship between the structure of Yaa(-3) and HSP47 binding using synthetic collagenous peptides. The results obtained indicated that the structure of Yaa(-3) determined the binding affinity for HSP47. Maximal binding was observed when Yaa(-3) was Thr. Moreover, the required relative spatial arrangement of these key residues in the triple helix was analyzed by taking advantage of heterotrimeric collagen-model peptides, each of which contains one Thr(-3) and one Arg(0). The results revealed that HSP47 recognizes the Yaa(-3) and Arg(0) residues only when they are on the same peptide strand. Taken together, the data obtained led us to define the HSP47-binding structural epitope in the collagen triple helix and also define the HSP47-binding motif in the primary structure. A motif search against human protein database predicted candidate clients for this molecular chaperone. The search result indicated that not all collagen family proteins require the chaperoning by HSP47.
Highlights
Heat-shock protein 47 (HSP47)3 is an essential molecular chaperone for normal procollagen biosynthesis in mammals
We focused on the structure of YaaϪ3, which was expected to be another structural determinant for HSP47 binding, together with the side chain structure of Arg0
The peptides were immobilized onto N-hydroxysuccinimide (NHS)-activated beads, and their interactions with recombinant HSP47 were examined by peptide-pull-down assays
Summary
Synthetic Peptides—Peptide chains were constructed manually based on the N-(9-fluorenyl)methoxycarbonyl-based solid-phase method on APRIL 21, 2006 VOLUME 281 NUMBER 16. Escherichia coli lysates containing either recombinant mouse [11] or human [12] HSP47 protein (70 l) were mixed with 130 l of binding buffer (50 mM HEPES-Na (pH 7.5), 150 mM NaCl, 1 mM EDTA) and 25-l bed of the affinity beads. Cell lysates were obtained by resuspension of collected cells into lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40), followed by the removal of insoluble materials by centrifugation at 13,000 ϫ g for 10 min. After washing the beads with TBST buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.005% Tween 20) three times, proteins retained on the beads were separated by SDS-PAGE using 4 –20% gradient gels and transferred to nitrocellulose membranes. (Nacalai Tesque, Kyoto, Japan) or an alkaline phosphatase substrate kit (Bio-Rad) as appropriate
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