Synthetic antibody-derived immunopeptide provides neuroprotection in glaucoma through molecular interaction with retinal protein histone H3.1

Abstract

Glaucoma is a group of optic neuropathies characterized by the progressive degeneration of retinal ganglion cells (RGCs) as well as their axons leading to irreversible loss of sight. Medical management of the intraocular pressure (IOP) still represents the gold standard in glaucoma therapy, which only manages a single risk factor and does not directly address the neurodegenerative component of this eye disease. Recently, our group showed that antibody-derived immunopeptides (encoding complementarity-determining regions, CDRs) provide attractive glaucoma medication candidates and directly interfere its pathogenic mechanisms by different modes of action. In accordance with these findings, the present study showed the synthetic complementary-determining region 2 (CDR2) peptide (INSDGSSTSYADSVK) significantly increased RGC viability in vitro in a concentration-dependent manner (p < 0.05 using a CDR2 concentration of 50 μg/mL). Employing state-of the-art immunoprecipitation experiments, we confirmed that synthetic CDR2 exhibited a high affinity toward the retinal target protein histone H3.1 (HIST1H3A) (p < 0.001 and log2-fold change > 3). Furthermore, molecular dynamics (MD) simulations along with virtual docking analyses predicted potential CDR2-specific binding regions of HIST1H3A, which might represent essential post-translational modification (PTM) sites for epigenetic regulations. Quantitative mass spectrometry (MS) analysis of retinas demonstrated 39 proteins significantly affected by CDR2 treatment (p < 0.05). An up-regulation of proteins involved in the energy production (e.g., ATP5F1B and MT-CO2) as well as the regulatory ubiquitin proteasome system (e.g., PSMC5) was induced by the synthetic CDR2 peptide. On the other hand, CDR2 reduced metabolic key enzymes (e.g., DDAH1 and MAOB) as well as ER stress-related proteins (e.g., SEC22B and VCP) and these data were partially confirmed by microarray technology. Our outcome measurements indicate that specific protein-peptide interactions influence the regulatory epigenetic function of HIST1H3A promoting the neuroprotective mechanism on RGCs in vitro. In addition to IOP management, such synthetic peptides as CDR2 might serve as a synergistic immunotherapy for glaucoma in the future.

Glaucoma-Associated CDR1 Peptide Promotes RGC Survival in Retinal Explants through Molecular Interaction with Acidic Leucine Rich Nuclear Phosphoprotein 32A (ANP32A)

“…Thereby, immunotherapeutics possess a wide variety of modes of action ranging from receptor blocking or inhibition of signaling transmission to much more regulatory functions such as immunomodulation or cell cycle coordination. Particularly, antibodies, as well as antibody-derived peptides (encoding the hypervariable complementaritydetermining region, CDR), offer promising therapeutic approaches for the treatment of glaucoma in vivo and in vitro by inducing direct RGC neuroprotection [8][9][10][11]. Recent findings of our group confirmed, for instance, that monoclonal anti-HMGB1 enhanced the viability of RGCs in vivo by modulating the retinal RNA metabolism as well as the cytokine secretion [9].…”

“…The remaining sequence encoded the CDR1 sequence motif (underlined part) flanked by 5 aa to each side representing the adjacent framework regions (FR1 and FR2). Due to experiences from previous studies [8,11], we supposed that particularly the CDR1 sequence motif determines the biological function and activity of the peptide. The company Synpeptide Co. Ltd. (Shanghai, China) chemically synthesized the peptides with a purity of >90%.…”

“…The neurodegeneration in this model was elicited by axotomy of the optic nerve from the retina resulting in reproducible and significant loss of RGCs during 24 h of cultivation ex vivo. It provided excellent requirements to mimic glaucomatous-like conditions ex vivo and was frequently applied to deliver first hints for neuroprotection of potential glaucoma medications, particularly immunotherapeutics [8,10,11]. Accordingly, the retinal explants were either treated with 50 µg/mL and 100 µg/mL of synthetic CDR1 peptide or remained untreated, serving as the control group (CTRL) (see Figure 1 and Table S1).…”

“…To elucidate the neuroprotective mode of action of the synthetic CDR1 peptide (SCTGTSSDVGGYNYVSWYQ), we screened for potential interaction partners in the retina proteome using CDR1-specific immunoprecipitation experiments. This state-of-the-art MSbased technology enabled the identification of direct protein interaction partners of largely unexplored biological compounds and was already successfully applied by our group for target structure (biomarker) identification [8,11]. For this reason, we immobilized the synthetic CDR1 peptide on magnetic beads by biotin-streptavidin conjugation and performed a protein enrichment from retinal protein homogenate.…”

Neuroretinal Cell Culture Model as a Tool for the Development of New Therapeutic Approaches for Oxidative Stress-Induced Ocular Diseases, with a Focus on Glaucoma

“…The tryptic digestion of proteins was performed as described in our previous studies [31,32]. In brief, protein fractions were dissolved in 20 µL of 10 mM ammonium bicarbonate (ABC) and sonicated for 10 min on ice.…”