## 3 Results and Discussion  
### Binding of HSP47 to the Collagen Toolkit Peptides  
To identify the HSP47 binding site on native collagens II and III, we screened HSP47 binding sites on collagen using the collagen toolkits II and III, respectively. These kits contained sets of overlapping triple-helical peptides (THPs), as briefly described above [22]. Each peptide contains a guest sequence of 27 amino acids, the C-terminal 9 amino acids of which form the first 9 guest amino acids of the next peptide; thus, the toolkit advances 18 amino acids along the triple-helical sequence of human collagen II or III with each successive peptide, and a 9-amino acid overlap is included between adjacent peptides. These kits have been successfully used to identify many ligand-binding motifs on collagen [23; 30; 31]. Recombinant HSP47 with a C-terminal His-tag was incubated in wells of Immunol 2 HB 96-well plates coated with toolkit peptides. Certain integrin-binding collagen peptides (GX\({}^{-4}\)O\({}^{-3}\)GE\({}^{-1}\)R\({}^{0}\)) with an Arg at position Y\({}^{0}\) and different residues at position X\({}^{-4}\) were selected as control peptides [32; 33; 34].  
Significant binding of HSP47 could be detected from 12 peptides, 9 derived from collagen II and 3 from collagen III (Table 1, Figure 1). Amongst them, four collagen II peptides (II-13, II-14, II-20, II-26) and one collagen III peptide (III-5) showed stronger binding signals. Interestingly, most of these peptides are located near the N-terminus of collagen. As the triple helix assembly of procollagen occurs from the C-terminus to the N-terminus, HSP47 is likely to play a stabilization role only at the late stage of procollagen triple helix assembly. All of these identified HSP47-binding peptides contain the Gly-Xaa-Arg or GXR motif, which is consistent with previous findings that the arginine residue in these THPs is critical for HSP47 binding; however, there are more than 40 GXR motifs in both collagens II and III, only 12 of which showed appreciable HSP47 binding (Figures S1 and S2). Notably, four of the five integrin-binding peptides containing GXOGER motifs showed strong HSP47 binding, while HSP47 binding on the fifth peptide with R at the X position was relatively weak (Table 1). Together, these observations indicate that sequences adjacent to the GXR motif also play an important role in defining an efficient HSP47 binding site. This is consistent with previous observations that residues located at position -3 of collagen also affected the binding of HSP47 [31]. It should also be noted that the parent toolkit peptides that contain the same HSP47-positive, integrin-binding GXOGER motifs do not generally support HSP47 binding. Peptides II-7/8, II-18/19, II-28, and II-31 contain GLOER, GAOGER, GFOGER, and GMOGER, respectively, but show negligible HSP47 binding. The same applies to peptides III-4, III-8, III-29, and III-31. This indicates that nearby residues (-6 and -7) may restrict HSP47 binding, as discussed further below.  
\begin{table}
\begin{tabular}{l c c} \hline \hline
**Peptide Name** & **Sequence** & **Mean A\({}_{450}\)** \\ \hline
**Toolkit II** & & \\ \hline
14 & _GPP_**GRPG**O**GP**G**G**AT**G**PL**GP**KG**QT**GEO**G**G**G**G**G**G**P** & \textgreater{}2 \\
20 & _GPP_**GANGD**O**G**ROGEO**G**G**G**G**G**L**G**RG**D**AG