Fig 2: (a) Results of SDS-PAGE analysis: a band of the dimer at ∼44 kDa is observed under non-reduced conditions. Under reduced conditions, only a single ∼29 kDa band corresponding to the monomer is detected. Bands other than the monomer of the light chains were essentially absent under either reducing or non-reducing conditions. (b) Cleavage reaction for FRET substrates: the cleavage reaction was performed with 5 μM H34 using 25 μM of FRET-PD1 (PD-1; aa 123–140), FRET-Aβ (Aβ; aa 26–33) or FRET-Tau (Tau; aa 391–408). H34 exhibited strong catalytic activity for FRET-PD1, but low activity on FRET-Aβ or FRET-Tau. All reactions were carried out in duplicate. (c) Bond cleavage for FRET-PD1: the H34 reaction products were analyzed by HPLC and MS. The fragments were identified as 7-MCA-GAISLAPKAQ-OH (31.7 min) and NH2-IKESLRAEK(DNP)-NH2 (29.6 min), indicating cleavage of the peptide bond between Gln132 and Ile133. (d) Kinetic analysis: [S]: concentration of the FRET-PD1 substrate. [V]: initial rate of the cleavage reaction. The Hanes–Woolf plot demonstrates that cleavage by the H34 light chain fits Michaelis–Menten kinetics, indicating that the reaction is enzymatic. kcat and Km were 1.3 × 10−2 min−1 and 3.32 × 10−6 M, respectively.

Fig 3: Interrelations of features of H34wt and the mutants. Mutagenesis in this study clarified the amino acid residues constructing the catalytic site, which is composed of Arg96, Thr93, and Gln89, revealing a new catalytic site for the cleavage of PD-1, which is different from the serine protease triad. The figure shows the interrelation among H34wt and the mutants; in addition to structural changes, several crucial changes are detected in terms of chemical and immunological characters. The number indicates the distance between the Arg and Thr residues. The figure shows the interrelation among H34wt and the mutants; in addition to structural changes, several crucial changes are detected in terms of chemical and immunological characters. The number indicates the distance between the Arg and Thr residues.

Fig 4: (a and b) SDS-PAGE analysis with silver staining under reduced conditions: (a) cleavage of recombinant PD-1 molecules: recombinant PD1 molecules (rPD1; 1 μM) were incubated with H34 (0.5 μM) at 37 °C for 72 h. The rPD1 bands are around 38 kDa and 43 kDa, whereas H34 is a single band at 30 kDa. After 24 h, bands at 28 kDa and around 17 kDa are visible and become gradually more prominent from 24 h to 72 h. In contrast, the 38–42 kDa PD-1 bands gradually fade with time. (b) Irrelevant protein HSA: HSA is an irrelevant protein used as a control. Only the band at ∼66 kDa and the rPD-1 band were observed. No fragments were detected from H34, PD-1, HSA and a mixture of H34 and HSA up to 72 h. Thus, H34 cleaved the rPD-1 molecule in a time-dependent manner. (c) ELISA-1: first, rPD-1 alone, H34 alone or a mixture of both was incubated in TGT buffer (pH 7.7) at 37 °C for 48 h. Next, 50 μL of each sample was assayed by ELISA on rPD-L1-coated wells at room temperature. The reaction was detected using the HRP-conjugated anti-V5 tag epitope antibody. For rPD-1 alone, the absorbance at 492 nm was 0.569, and 0.003 for H34 alone, suggesting no reaction of H34 with rPD-L1. In contrast, the absorbance for the mixture of rPD-1 and H34 was 0.151. This is lower than the value of rPD-1 alone by a factor of four. It is clear that H34 diminished the binding interaction between PD-1 and PD-L1 by degrading the PD-1 molecule. (d) ELISA-2: a mixture of rPD-1 (0.18 μM) and H34 (0.5 μM) was incubated up to 48 h. A sample at 0, 6, 24, and 48 h was taken and we measured the binding of rPD-1/PD-L1 by ELISA.

Fig 5: (a) Time course of the cleavage reaction for FRET-PD1 peptide by mutants. FRET-PD1: 25 µM. H34wt & mutants: 5 µM. In this experiment, H34wt was tested together with the mutants for ensuring accurate comparison among the samples. H34wt and the mutants of D1A and T97A show high catalytic activity. In contrast, three mutants of R95P, R95A, and T93A exhibit low catalytic activity. The catalytic activity of P95(+) is substantially suppressed. (b) Cleavage of recombinant PD-1 molecule. Recombinant PD-1 (rPD-1): 1 µM. H34wt, D1A, R96A and T97A: 0.5 µM. Reaction temperature: 37 °C. The SDS-PAGE (12% gel) experiment was performed under reduced condition, and the visualization was performed by silver staining. The cleavage reaction by H34wt and the several mutants were carried out over 48 h of incubation. The robust bands at approximately 29-kDa represent the monomer band of H34wt and/or the mutants. In H34wt light chain, a band at 17.5 kDa, which corresponds to the fragment of rPD1 (see ref.), is detected at 48 h of incubation; however, it is not detected at 0 h of incubation. Similar results are observed for D1A mutant, where the same band at 17.5 kDa is detected. In contrast, no band at 17.5 kDa is detected in the case of the mutant R96A, indicating that R96A does not cleave rPD-1. These results are consistent with the cleavage activity for FRET-PD1. Both H34wt and D1A, but not R96A, cleaves FRET-PD1 peptide with high activity.