# Minimal Collagen-Binding Epitope of Glycoprotein VI in Human and Mouse Platelets
## 3 Results  
### Collagen and CRP-Binding with GPVI Monomer in Mammalian Species  
Site-directed mutagenesis studies have identified several important amino acids located in the D1/D2 domain of human GPVI, which are involved in collagen- or CRP-binding (green box, Figure 1A). Using the online computational tool BLAST for multiple sequence alignment, we further analyzed the D1/D2 domain of human GPVI and compared it to that of other mammalians. Interestingly, we found that many investigated key amino acid residues, important for collagen- or CRP-binding, are not fully conserved between mammalian species (yellow box, Figure 1A). Definitively, an ancient mammalian species _Sorex araneus_ showed the most variable amino acid residues within the D1/D2 domain. However, important amino acid sequences of the D1/D2 domain, such as cysteine residues, which determine the secondary structure of the protein, showed high conservative sequences between species (Figure 1A). Next, we used 3D homology models for _Homo sapiens_, _Mus musculus_, _Bos Taurus_, and _Sorex araneus_, constructed using a SWISS-MODEL online tool ([[https://swissmodel.expasy.org](https://swissmodel.expasy.org)]([https://swissmodel.expasy.org](https://swissmodel.expasy.org)); accessed 27 September 2022). Similar to the multiple alignment data, the following amino acid residues were indicated as probably covering the collagen or CRP-binding surface: K79, R80, and R186 for _Homo sapiens_ (human GPVI); E80, R81, and S187 for _Mus musculus_ (mouse GPVI); E80, R81, and A187 for _Bos Taurus_ (bovine GPVI); and T80, L81, and S187 for _Sorex araneus_ (sorex GPVI) (Figure 1B). Analysis of the 3D structure of the GPVI monomer showed that the main structure of GPVI is similar between species, but amino acid changes are observed on the collagen- and CRP-binding surface, which clearly changes the electrostatic surface potentials (red color, negative; blue color, positive; white color, neutral residues) (Figure 1B). This result suggests that the biochemical properties of GPVI that allow binding of collagen or CRP may be different between mammalian species. Next, the phylogenetic relationship between mammalian GPVI isoforms was functionally characterized and placed in a single clade related to order. Over all, this result showed an evolutionally conserved relation between GPVI across numerous mammalian species (Figure 1C; Supplementary Figures S2 and S3).  
Figure 1: Conserved regions of GPVI in mammalian species. (**A**) The multiple sequence alignment of mammalian GPVI. D1/D2 domain of human GPVI was compared to other mammalians. The conserved amino acid residues involved in collagen- and CRP-binding are indicated in green. The amino acid residues involved in collagen- and CRP-binding which are not fully conserved between mammalian species are indicated in yellow. (**B**) 3D homology models for _Homo sapiens_, _Mus musculus_, _Bos Taurus_, and _Sorex aranneus_. The conserved amino acid residues of human (_Homo sapiens_ K79, R80, and R186) and mouse GPVI (_Mus musculus_ E80, R81, and S187) are shown as sticks; those of bovine (_Bos Taurus_ E80, R81, and A187) and sorex GPVI (_Sorex aranneus_ T80, L81, and S187) are highlighted in cyan and orange, respectively. Electrostatic surface potentials are shown in red and blue for negative and positive charges, respectively, and white for neutral residues. (**C**) The neighbor joining tree of the GPVI D1 domain indicating the phylogenetic relationship between mammalian GPVI isoforms.