## 3 Results
### Glycan-Independent Digestion of DC-SIGN by MSPL  
Next, to evaluate the effect of the MSPL-DC-SIGN interaction on MSPL protease activity, we performed a co-incubation study. When MSPL-ECD and DC-SIGN-ECD were co-incubated in the presence of Ca\({}^{2+}\), the band at 45 kDa in the DC-SIGN-ECD sample disappeared and three new bands were detected (Figure 4a, lane 3). This result indicates that DC-SIGN-ECD was degraded upon incubation with MSPL-ECD. To determine the optimal conditions for DC-SIGN digestion, we tested various MSPL-ECD concentrations (Figure 4b) and incubation times (Figure 4c). The results showed that DC-SIGN digestion was dependent on MSPL-ECD dose and time and incubation with 1 \(\upmu\)g MSPL for 6 h was the optimal condition. We next performed a co-incubation study in the presence of EDTA (Figure 4d,e). The digested fragment of DC-SIGN-ECD showed a different pattern from that observed in the presence of Ca\({}^{2+}\), and one DC-SIGN-ECD band at 33 kDa overlapped with the MSPL band (Figure 4d, green and purple arrowheads). Notably, the full-length DC-SIGN-ECD band at 45 kDa disappeared with even a low level (0.5 \(\upmu\)g) of MSPL-ECD, indicating that DC-SIGN-ECD degraded more efficiently when treated with EDTA. To confirm the DC-SIGN-ECD cleavage sites, we performed N-terminal amino acid sequencing analysis (Figure 4f). We identified the AAVGE sequence, which frequently appears in the DC-SIGN-Fc repeat domain, the SNRFTW sequence of the CRD, and the DYKDD sequence of the N-terminal FLAG-tag. These results clearly showed different cutting patterns of DC-SIGN-ECD in the presence or absence of Ca\({}^{2+}\). Given that DC-SIGN  
Figure 2: Expression of MSPL/TMPRSS13 in human colorectal carcinoma tissues.  
binds to MSPL in a Ca2+-dependent manner, the results of these co-incubation studies suggest that glycan-mediated MSPL recognition by DC-SIGN interferes with DC-SIGN digestion by MSPL (see Figure S1).  
Figure 4: Proteolytic activity of MPSL in the presence of Ca2+ and EDTA. (**a**) The purified MSPL–ECD and DC-SIGN–ECD were separated via SDS–PAGE under reducing conditions and detected with CBB staining (lanes 1 and 2). The two molecules were co-incubated in the presence of Ca2+ at 37 \({}^{\circ}\)C for 6 h (lane 3). The colored arrowheads indicate bands of full-length (red) and digested DC-SIGN–ECD (purple), and MSPL–ECD (green). (**b–e**) The MSPL–ECD dose- (**b**,**d**) and time-dependency (**c**,**e**) of DC-SIGN degradation in the presence of Ca2+ (**b**,**c**) and EDTA (**d**,**e**) were tested. (**f**) DC-SIGN–ECD cleavage sites were identified through N-terminal amino acid sequencing (BIOSUMS, Shiga, Japan). The amino acid sequences obtained are shown using one-letter codes. CRD: Carbohydrate recognition domain.  
Figure 3: Ca2+-dependent recognition of MSPL by DC-SIGN. (**a**) The extracellular domain of human MSPL (MSPL–ECD(extracellular domain)) with a C-terminal FLAG-tag was expressed in human hepatoma HLF cells. The secreted MSPL was purified and separated through sodium dodecyl sulfate–polyacrylamideamide gel electrophoresis (SDS–PAGE) under reducing conditions and detected through Coomassieie Brilliant Blue (CBB) staining. (**b**) The extracellular domain of DC-SIGN (DC-SIGN–ECD) was prepared and applied to DC-SIGN lectin blotting. MSPL–ECD blotted on a nitrocellulose membrane was reacted with DC-SIGN–ECD in the presence of Ca2+ or EDTA. (**c**) The purified MSPL–ECD was pretreated with PNGase F for 24 h at 37 \({}^{\circ}\)C and then subjected to DC-SIGN lectin blotting.