Additional file 1: Figure S1. The N-terminal Ab lock and VpreB were unable to mask the binding activity of CTLA4Ig. (A) Binding activity of Ab lock-CTLA4Ig (0.5 μg/ml, blue line) and conventional CTLA4Ig (0.5 μg/ml, black line) to HEK-293 cells overexpressing CD80 (CD80 cells), detected by FITC-conjugated goat anti-mouse Fcγ in flow cytometry. Gray line: unstained cells. (B) Binding activity of VpreB-CTLA4Ig (0.5 μg/ml, blue line) and conventional CTLA4Ig (0.5 μg/ml, black line) to CD80 cells, detected by FITC-conjugated goat anti-mouse Fcγ antibodies by flow cytometry. Gray line: unstained cells. VpreB: immunoglobulin iota chain. (C) Simulation of Ab lock-mCTLA4Ig by the computer software BIOVIA Discovery Studio 2019 (Discovery Studio v19.1.0.18287). The structures of CTLA-4, the CDR3-like domain and the Ab lock are shown in magenta, yellow and light blue, respectively. Figure S2. Full recovery of the binding activity of mAlb-CTLA4Ig after MMP2/9 digestion. Nondigested, MMP-digested mAlb-CTLA44Ig, and conventional mCTLA4Ig (all at 1 nM) were added to the ELISA. Binding of the fusion proteins on the plate was detected by an HRP-conjugated anti-mouse IgG Fcγ secondary antibody. Figure S3. Characterization of an alternative Alb-CTLA4Ig with MMP substrate linker between albumin and CTLA4Ig (mAlb-MMP-CTLA4Ig). (A) Schematic representations of mAlb-MMP-CTLA4Ig constructs. MMP: MMP substrate sequence (GPLGMWSR) linker, eCTLA4: extracellular domain of CTLA4. P: promoter in the expression vector. (B) Reducing SDS-PAGE (left) and western blot analysis (right) of purified mAlb-MMP-CTLA4Ig. (C) The stability of mAlb-MMP-CTLA4Ig in DMEM containing 10% fetal bovine sera for seven days. (D) mAlb-MMP-CTLA4Ig were digested with the indicated amount of MMP2/9 and analyzed by western blot. (E) mAlb-MMP-CTLA4Ig were subjected to varying degrees of digestion by MMP2/9. Part of the digestion was analyzed by western blot to determine the degree of cleavage. The percent (%) cleaved Alb-MMP-CTLA4Ig was quantitated and is indicated below each lane. The digestion was added to the cell-based ELISA. The binding of CD80 by 1 nM conventional mCTLA4Ig was used as a positive control. (F) Binding kinetics of mAlb-MMP-CTLA4Ig before (blue curve) and after MMP digestion (orange curve). The binding kinetics of conventional mCTLA4Ig are shown in the black curve. Figure S4. Lacking an N-terminal albumin and a C-terminal Fc decreases masking efficiency and stability. (A) Competitive binding of Ig-CTLA4 ECD (0.5 μg/ml, blue line) or conventional CTLA4Ig (0.5 μg/ml, black line) against PE-conjugated anti-mouse CD80 antibodies (1 μg/ml) to HEK-293 cells overexpressing CD80 (CD80 cells). Red line: CD80-expressing cells stained with PE-conjugated anti-CD 80 antibodies alone (1 μg/ml). Gray line: unstained cells. Digestion products of the IgG1 Fc-CTLA4 ECD by the indicated amounts of MMP2/9 are shown in the western blot using anti-mouse Fcγ antibodies (right panel). (B) Competitive binding activity of Alb-CTLA4 ECD (0.5 μg/ml, blue line) and conventional CTLA4Ig (0.5 μg/ml, black line) to CD80 cells in the presence of PE-conjugated anti-mouse CD80 antibodies (1 μg/ml) by flow cytometry. Red line: CD80 cells stained with PE-conjugated anti-CD80 antibody alone (1 μg/ml). Gray line: unstained cells. Digestion products of the Alb-CTLA4 ECD by the indicated amounts of MMP2/9 are shown in the western blot using anti-mouse CTLA4 antibodies (right panel). (C) Digestion products of conventional human CTLA4Ig (3.6 picomole) by 2 units of MMP2/9 is shown in the western blot using anti-human Fcγ antibodies. (D) Overdigestion of hAlb-CTLA4Ig (1.35 picomole) in higher amounts (9 units) of MMP2/9. Figure S5. The stability of hAlb-CTLA4Ig in sera. hAlb-CTLA4Ig incubated in RPMI 1640 containing 10% fetal bovine sera for seven days was analyzed by western blot using an anti-human Fcγ secondary antibody. Med: medium alone. Figure S6. Differential levels of MMP9 and MMP3 in normal control mice and CIA mice. Protein levels of MMP9 (A) and MMP3 (B) in the synovial fluid lavages, sera, and paws of normal control mice and CIA mice were measured by ELISA. Protein levels are expressed as ng/ml in synovial fluid lavages and sera, ng/mg protein in protein extracts of the paws. Figure S7. Histopathological microphotographs of the digits of a normal mouse or CIA mice. The area enclosed by red rectangles at low magnification (5X objective, upper row) is shown at higher magnification (20X objective, lower row) for inflammatory cells. Figure S8. Splenocyte proliferation in response to different concentrations of M. tuberculosis restimulation. Splenocytes from a normal (preimmune) mouse and a CIA mouse were stimulated with 0 μg/ml 5μg/ml or 25 μg/ml M. tuberculosis extracts for 72 h. BrdU was added at the final 2 hours of stimulation. Proliferation (BrdU incorporation) of the CD45+ splenocytes was analyzed by flow cytometry.
A helical epitope-peptide (lle85-Gly94) was selected from the α-helix structure of the HIV protease (PR) as the template, which represents an intricate interplay between structure conformation and dimerization. The peptide template was mixed with water, trifluoroethanol (TFE), and acetonitrile (ACN) at a certain ratio to enlarge the helical conformation in the solution for the fabrication of helical epitope-mediated molecularly imprinted polymers (HEMIPs) on a quartz crystal microbalance (QCM) chip. The template molecules were then removed under equilibrium batch rebinding conditions involving 5% acetic acid/water. The resulting HEMIPs chip exhibited a high affinity toward template peptide HIV PR85–94, His-tagged HIV PR, and HIV PR, with dissociation constants (Kd) as 160, 43.3, and 78.5 pM, respectively. The detection limit of the developed HIV PR85–94 QCM sensor is 0.1 ng/mL. The HEMIPs chip exhibited a high affinity and selectivity to bind HIV PR and subsequently to an inhibitor of HIV PR (nelfinavir). The HIV PR binding site was properly oriented on the HEMIPs-chip to develop a HIV PR/HEMIPs chip, which can effectively bind nelfinavir to establish a sandwich assay. The nelfinavir then attached to the HIV PR/HEMIPs chip, which can be easily removed involving 0.8% acetic acid/water. Therefore, HIV PR/HEMIPs chip can be useful to screen for other HIV PR inhibitors. This technique may improve drug targeting for HIV therapy and also strengthen investigations into other virus assays.
Abstract Background CTLA4Ig is a dimeric fusion protein of the extracellular domain of cytotoxic T-lymphocyte protein 4 (CTLA4) and an Fc (Ig) fragment of human IgG 1 that is approved for treating rheumatoid arthritis. However, CTLA4Ig may induce adverse effects. Developing a lesion-selective variant of CTLA4Ig may improve safety while maintaining the efficacy of the treatment. Methods We linked albumin to the N-terminus of CTLA4Ig (termed Alb-CTLA4Ig) via a substrate sequence of matrix metalloproteinase (MMP). The binding activities and the biological activities of Alb-CTLA4Ig before and after MMP digestion were analyzed by a cell-based ELISA and an in vitro Jurkat T cell activation assay. The efficacy and safety of Alb-CTLA4Ig in treating joint inflammation were tested in mouse collagen-induced arthritis. Results Alb-CTLA4Ig is stable and inactive under physiological conditions but can be fully activated by MMPs. The binding activity of nondigested Alb-CTLA4Ig was at least 10,000-fold weaker than that of MMP-digested Alb-CTLA4Ig. Nondigested Alb-CTLA4Ig was unable to inhibit Jurkat T cell activation, whereas MMP-digested Alb-CTLA4Ig was as potent as conventional CTLA4Ig in inhibiting the T cells. Alb-CTLA4Ig was converted to CTLA4Ig in the inflamed joints to treat mouse collagen-induced arthritis, showing similar efficacy to that of conventional CTLA4Ig. In contrast to conventional CTLA4Ig, Alb-CTLA4Ig did not inhibit the antimicrobial responses in the spleens of the treated mice. Conclusions Our study indicates that Alb-CTLA4Ig can be activated by MMPs to suppress tissue inflammation in situ. Thus, Alb-CTLA4Ig is a safe and effective treatment for collagen-induced arthritis in mice.
In a recent directed-evolution study, Escherichia coli D-sialic acid aldolase was converted by introducing eight point mutations into a new enzyme with relaxed specificity, denoted RS-aldolase (also known formerly as L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase), which showed a preferred selectivity toward L-KDO. To investigate the underlying molecular basis, we determined the crystal structures of D-sialic acid aldolase and RS-aldolase. All mutations are away from the catalytic center, except for V251I, which is near the opening of the (α/β)(8)-barrel and proximal to the Schiff base-forming Lys-165. The change of specificity from D-sialic acid to RS-aldolase can be attributed mainly to the V251I substitution, which creates a narrower sugar-binding pocket, but without altering the chirality in the reaction center. The crystal structures of D-sialic acid aldolase·l-arabinose and RS-aldolase·hydroxypyruvate complexes and five mutants (V251I, V251L, V251R, V251W, and V251I/V265I) of the D-sialic acid aldolase were also determined, revealing the location of substrate molecules and how the contour of the active site pocket was shaped. Interestingly, by mutating Val251 alone, the enzyme can accept substrates of varying size in the aldolase reactions and still retain stereoselectivity. The engineered D-sialic acid aldolase may find applications in synthesizing unnatural sugars of C(6) to C(10) for the design of antagonists and inhibitors of glycoenzymes.
The antitumor effects elicited by immune checkpoint inhibitors (ICIs) have transformed cancer treatments. However, severe immune-related adverse events (irAEs) resulting from these treatments have restricted the application of ICIs. To overcome the adverse events, we developed a tumor lesion-selective pro-PD-1Ig that is activated by proteases overexpressed in tumors. We genetically linked albumin to the N-terminus of a modified PD-1Ig (termed mutPD-1Ig hereafter) via an MMP substrate sequence to form Alb-hinge-mutPD-1Ig. We demonstrate that the binding activity of nondigested Alb-hinge-mutPD-1Ig is approximately 11-folds lower than mutPD-1Ig. However, digestion by type IV collagenase restored the binding activity of Alb-hinge-mutPD-1Ig to a level comparable to that of native mutPD-1Ig. In order to enhance the masking efficiency of Alb-mutPD-1Ig, we simulated the effects of diverse MMP substrate linkers for connecting albumin and PD-1 at various starting positions by bioinformatics tools. Our validation experiments indicate Alb-hinge-mutPD-1Ig displayed the best masking efficiency among all simulated constructs. Our study suggests that albumin may be best applicable to mask a target protein whose binding motif is centralized and in the proximity of the N-terminus of the protein.
Large momentum effective theory allows extraction of hadron parton distribution functions in lattice QCD by matching them to quark bilinear matrix elements of hadrons with large momenta. We calculate the matching kernels for the unpolarized, helicity, and transversity isovector parton distribution functions and skewless generalized parton distributions of all hadrons in the hybrid-RI/MOM scheme. This renormalization scheme uses RI/MOM when the Wilson line length is less then $z_s$, otherwise a mass subtraction scheme is used. By design, the non-hybrid scheme is recovered as $z_s \to \infty$. In the opposite limit, $z \to 0$, the self renormalization scheme is obtained. When the parameters $p_z^R=0$ and $\mu^R z_s \ll 1$, the hybrid-RI/MOM scheme coincides with the hybrid-ratio scheme times the charge of the PDF. We also discuss the subtlety related to the commutativity of Fourier transform and $\epsilon$ expansion in the $\bar{\text{MS}}$ scheme.
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