In vitro immuno-prevention of nitration/dysfunction of myogenic stem cell activator HGF, towards developing a strategy for age-related muscle atrophy

Abstract

In response to peroxynitrite (ONOO^-) generation, myogenic stem satellite cell activator HGF (hepatocyte growth factor) undergoes nitration of tyrosine residues (Y198 and Y250) predominantly on fast IIa and IIx myofibers to lose its binding to the signaling receptor c-met, thereby disturbing muscle homeostasis during aging. Here we show that rat anti-HGF monoclonal antibody (mAb) 1H41C10, which was raised in-house against a synthetic peptide FTSNPEVR_nitroY₁₉₈EV, a site well-conserved in mammals, functions to confer resistance to nitration dysfunction on HGF. 1H41C10 was characterized by recognizing both nitrated and non-nitrated HGF with different affinities as revealed by Western blotting, indicating that the paratope of 1H41C10 may bind to the immediate vicinity of Y198. Subsequent experiments showed that 1H41C10-bound HGF resists peroxynitrite-induced nitration of Y198. A companion mAb-1H42F4 presented similar immuno-reactivity, but did not protect Y198 nitration, and thus served as the control. Importantly, 1H41C10-HGF also withstood Y250 nitration to retain c-met binding and satellite cell activation functions in culture. The Fab region of 1H41C10 exerts resistivity to Y250 nitration possibly due to its localization in the immediate vicinity to Y250, as supported by an additional set of experiments showing that the 1H41C10-Fab confers Y250-nitration resistance which the Fc segment does not. Findings highlight the in vitro preventive impact of 1H41C10 on HGF nitration-dysfunction that strongly impairs myogenic stem cell dynamics, potentially pioneering cogent strategies for counteracting or treating age-related muscle atrophy with fibrosis (including sarcopenia and frailty) and the therapeutic application of investigational HGF drugs.

Keywords: c‐met binding; hepatocyte growth factor (HGF); monoclonal antibody; muscle atrophy; peroxynitrite; rat; resident myogenic stem satellite cell; tyrosine nitration.

FIGURE 1

Effect of pretreatment with anti‐Y198‐HGF mAbs 1C10 and 2F4 on HGF nitration. (a) In vitro experimental design for the preparation of nitrated and non‐nitrated HGF. Two‐step dilution protocol for the peroxynitrite stock (0.1 M ONOO⁻ on ice) was optimized for the exposure of recombinant HGF (carrier protein‐free; disulfide‐linked heterodimer of 60‐kDa α‐chain and 30‐kDa β‐chain as the major form in the product) to active peroxynitrite at 1:2000 (mole ratio relative to HGF), pH 7.4, 25°C, for 30 min, in order to induce nitration of Y198 and Y250 under physiological conditions, as described previously (Elgaabari et al., 2024). Control HGF (non‐nitrated HGF), incubated in treatment buffer at pH 7.4 only, without peroxynitrite for 30 min. Lower column is a supplemental sketch of tyrosine residue nitration, peroxynitrite‐induced nitrotyrosine formation by introduction of a nitro group (‐NO₂) into a Cε atom in aromatic ring of the side chain. (b) Characterization of anti‐Y198‐HGF mAbs 1C10 and 2F4. Visualization of immuno‐reactivity of anti‐Y198‐HGF mAbs IH41C10 and IH42F4 (abbreviated as 1C10 and 2F4, respectively) by ECL Western blotting of non‐nitrated control HGF (lanes a’ and a”) and nitrated HGF (lanes b’ and b”). Blots were first treated with 1C10/2F4 and HRP‐conjugated anti‐rat IgG Abs (upper column), followed by re‐probing with HRP‐labeled anti‐HGF α‐chain mAb after stripping with SDS‐βME solution (lower column). Hybridoma clone 3A11C6 (abbreviated as 1C6; raised and characterized by Elgaabari et al., 2024) served as a control and displayed high immuno‐specificity to nitroY198‐HGF (lanes a and b). A wide molecular weight range (30–120 kDa) covers the 90‐kDa HGF proform and the 60‐kDa HGF α‐chain (including the NK2 segment responsible for c‐met binding) was displayed. MW‐STD, MagicMark molecular weight standards. (c) Experimental design for evaluating the effect of pretreatment with anti‐Y198‐HGF mAbs 1C10 and 2F4 on HGF nitration. Recombinant HGF was incubated with 1C10 and 2F4 in a range of 1:0–1:0.1 (mole ratios relative to HGF) for 30 min prior to peroxynitrite protocol (at 1:500 relative to HGF, pH 7.4, 25°C, for 30 min). (d) Evaluation of nitration levels of HGF pretreated with 1C10 (lanes a–d) and 2F4 (lanes a’–c’). Assayed by HRPO‐labeled anti‐nitrotyrosine (anti‐nitroY) mAb in Western blotting format normalized by total HGF α‐chain (detected with HRP‐labeled anti‐HGF α‐chain mAb after the stripping step as described in Figure 1b), followed by re‐probing with anti‐nitroY198‐HGF mAb (clone 1C6) and anti‐nitroY250‐HGF mAb (clone 2C3); clones 1C6 and 2C3 mAbs displayed high immuno‐specificity to nitroY198‐HGF and nitroY250‐HGF, respectively, as described previously (Elgaabari et al., 2024). Densitometric analysis of panel d, normalized with total HGF α‐chain (positive for anti‐HGF α‐chain mAb) is shown in Figure S2. (e) Evaluation of nitration levels of HGF pretreated with control IgG (1:0.4 mole ratio relative to HGF). Assayed by western blotting normalized by whole HGF as same as panel d (lanes a” and e”). MW‐STD, MagicMark molecular weight standards. Note that there was no detectable effect of control IgG even at 1:0.4 that is higher mole ratio than the 1C10/2F4 treatments (see panel d). (f) Evaluation of preventive effect on HGF‐Y198/Y250 nitration (Fab vs. Fc fragments of 1C10 IgG). Nitration levels of HGF pretreated with 1C10‐Fab (lanes a–e) and 1C10‐Fc (lanes a’, b’, and d). Assayed by anti‐nitroY198‐HGF mAb (1C6; first row) and anti‐nitroY250‐HGF mAb (2C3; second row) and HRP‐labeled anti‐rat IgG‐Fc secondary Ab, and then by HRPO‐labeled anti‐nitrotyrosine mAb (anti‐nitroY; third row) in Western blotting format normalized by total HGF α‐chain (detected with HRP‐labeled anti‐HGF α‐chain mAb; fourth row) as described in Figure 1d. MW‐STD, ProteinLadder molecular weight standards.

FIGURE 2

Prevention of HGF nitration‐dysfunction by interaction with anti‐Y198‐HGF mAb 1C10. (a) The experimental scheme for assays in culture. Primary cultures of rat satellite cells received 5 ng/mL HGF (control, peroxynitrite‐treated, and 1C10/2F4‐pretreated HGF prior to the peroxynitrite treatment, as shown in Figure 1c,d) in DMEM‐10% HS (pH 7.2) for 24 h beginning at 24‐h post‐plating followed by pulse‐labeling with BrdU for 2 h just prior to the 48‐h, time‐point of the BrdU‐incorporation assay for activation. (b) Cell activation response to HGF that was pretreated with 1C10 and 2F4 before exposure to peroxynitrite (ONOO⁻). Satellite cell cultures were assayed for cell activation (BrdU‐positive cell percentage) at 48‐h post‐plating. Open bars a, negative control untreated‐cultures in DMEM‐10% HS; black‐solid bars b, positive control cultures with 5 ng/mL recombinant HGF for 24 h beginning 24‐h post‐plating; gray bars c, cultures with 5 ng/mL nitrated HGF (peroxynitrite‐treated at 1:500 relative to HGF). Red‐gradient bars, cultures with 5 ng/mL HGF that received pretreatment with 1C10 and 2F4 (at 1:0, 1:0.5, 1:1, and 1:5 mole ratios relative to HGF) prior to the peroxynitrite treatment. Far‐right red‐bars, cultures with 5 ng/mL HGF that just received the 1C10/2F4 pretreatment (at 1:5, without peroxynitrite treatment). Bars represent mean ± SEM and significant differences from the nitrated HGF (bars c) at p < 0.05 and p < 0.01 are indicated by (*) and (**), respectively. NS, not significant at p < 0.05. (c) Sandwich ELISA‐like assay on recombinant c‐met‐Fc chimera (see photo of the triplicate assay and schematic diagram of the mechanism of action). (d) Receptor c‐met binding activity of HGF pretreated with mAb 1C10. Optical absorbance_{450‐535 nm} was presented by subtracting the value of the negative control without HGF (bar a). Black bar b, control HGF without any treatment served as a positive control; gray bar c, nitrated HGF (1:500 peroxynitrite treatment); red‐gradient bars (from left to right), HGF pretreated with 1C10 (at 1:0, 1:0.5, 1:1, and 1:5 mole ratios relative to HGF) prior to 1:500 peroxynitrite treatment. Far‐right red bar, HGF that just received the 1C10 pretreatment (at 1:5, without peroxynitrite). Bars represent mean ± SEM and significant differences from the nitrated HGF (bar c) at p < 0.05 and p < 0.01 are indicated by (*) and (**), respectively. (e) A possible model for interaction of HGF (NK2 region composed of N, K1, and K2 domains; RCSB PDB ID: 3HN4 (Tolbert et al., 2010) and 1C10 mAb. A paratope of 1C10 IgG2a(κ), also known as an antigen‐binding site (a small region at N‐terminal tips of the heavy (Hc) and light chains (Lc) in Fab region), binds to a Y198‐containing epitope in K1 domain to prevent peroxynitrite‐induced nitration of Y198 (indicated by red open‐circle). Fab may also contribute to the resistivity of Y250 to nitration, possibly by localizing very close to Y250 (non‐nitrated form; indicated by blue open circle); the area hidden behind the 1C10‐Fab is outlined with a yellow broken line. The three‐dimensional structure of IgG2a(κ) was re‐drawn from synchrotron diffraction data for mouse anti‐malignant canine lymphoma mAb (RCSB PDB ID: 1IGT, with 2.80 Å resolution (Harris et al., 1997) using the program PyMOL Molecular Graphics System (Version 2.6.0a0; copyright: Schrödinger, LLC., created by Dr. Warren L. DeLano). Crystal structure of human HGF‐NK2 segment, originally displayed based on RCSB PDB ID 3HN4 (Tolbert et al., 2010) and reproduced from Elgaabari et al. (2024) with pink‐highlighting all tyrosine residues including Y198 and Y250 indicated by red and blue circles, respectively. (f) Schematic presentation of a possible mechanism for satellite cell activation by 1C10‐bound HGF. Quiescent satellite cells are activated to re‐enter the cell cycle in response to mechanical perturbation of muscle tissue through a molecular cascade of events including calcium ion influx from extracellular compartment by functional coupling of a mechano‐sensitive cation channel (MS‐channel) and a long‐lasting type of voltage‐gated Ca²⁺ channel (L‐VGC channel), calcium‐calmodulin (Ca‐CaM) formation, NO radical production by activated constitutive NO synthase (cNOS; neuronal NOS and endothelial NOS), matrix metalloproteinase 2 (MMP2) activation, rapid release of HGF from its extracellular tethering (possibly with associated extracellular segment of proteoglycans) to give rise to ng/ml level of the growth factor, and the subsequent presentation to cell‐membrane receptor c‐met to generate a signal for satellite cell activation (reproduced from Tatsumi & Allen,  and Tatsumi, 2010) with some modifications). 1C10 binds to ECM‐bound HGF as revealed by immuno‐fluorescence staining of satellite cell culture (Figure S4 panel c, visualized at 24‐h and 48‐h post‐plating). The affinity of the HGF‐1C10 complex to c‐met is lower than that of HGF ligand alone because: (i) both Y198 and Y250 localize in the c‐met binding sites as revealed by the cryo‐EM structure of a complex of HGF and the receptor c‐met dimer (RCSB PDB ID: 7MO7) (Uchikawa et al., 2021); (ii) Y198 is occupied by 1C10‐Fab, making the K1 domain of HGF inaccessible to c‐met; and (iii) Y250 is prevented from nitration possibly through the steric hindrance action of IC10‐Fab as modeled in panel e and hence able to associate with c‐met monomer to generate an activation signal as a result (see a c‐met depicted by the red line in panel f, redrawn to focus on the binding of HGF‐1C10 complex to c‐met monomer in contrast to nitroY198, 250‐HGF that does not bind to c‐met).