Quantifying Protein-Specific N-Glycome Profiles by Focused Protein and Immunoprecipitation Glycomics.

Serum N-glycans have been reported to be potential diagnostic and therapeutic biomarkers for many diseases and conditions, such as inflammation, fibrosis, and cancer progression. We previously described the focused protein glycomic analysis (FPG) from gel-separated serum proteins. With this methodology, we sought novel glycan biomarkers for nonalcoholic steatohepatitis (NASH) and successfully identified some N-glycans that were significantly elevated in NASH patients compared to nonalcoholic fatty liver patients. Among them, trisialylated monofucosylated triantennary glycan (A3F) of alpha-1 antitrypsin showed the most dynamic change. For rapid identification of N-glycans on the focused proteins, we constructed a simplified method called immunoprecipitation glycomics (IPG), where the target proteins were immunoprecipitated with affinity beads and subsequently subjected to glycomic analysis by MALDI-TOF MS. Focusing on alpha-1 antitrypsin and ceruloplasmin as the target proteins, we compared the values of N-glycans determined by FPG and IPG. The quantified values of each N-glycan by these two methods showed a statistically significant correlation, indicating that high throughput and quantitative N-glycomics of targeted proteins can be achieved by the simplified IPG method. Thus, an analytical strategy combining FPG and IPG can be adapted to general biomarker discovery and validation in appropriate disease areas.

Discovery of novel differentiation markers in the early stage of chondrogenesis by glycoform-focused reverse proteomics and genomics.

BACKGROUND: Osteoarthritis (OA) is one of the most common chronic diseases among adults, especially the elderly, which is characterized by destruction of the articular cartilage. Despite affecting more than 100 million individuals all over the world, therapy is currently limited to treating pain, which is a principal symptom of OA. New approaches to the treatment of OA that induce regeneration and repair of cartilage are strongly needed. METHODS: To discover potent markers for chondrogenic differentiation, glycoform-focused reverse proteomics and genomics were performed on the basis of glycoblotting-based comprehensive approach. RESULTS: Expression levels of high-mannose type N-glycans were up-regulated significantly at the late stage of differentiation of the mouse chondroprogenitor cells. Among 246 glycoproteins carrying this glycotype identified by ConA affinity chromatography and LC/MS, it was demonstrated that 52% are classified as cell surface glycoproteins. Gene expression levels indicated that mRNAs for 15 glycoproteins increased distinctly in the earlier stages during differentiation compared with Type II collagen. The feasibility of mouse chondrocyte markers in human chondrogenesis model was demonstrated by testing gene expression levels of these 15 glycoproteins during differentiation in human mesenchymal stem cells. CONCLUSION: The results showed clearly an evidence of up-regulation of 5 genes, ectonucleotide pyrophosphatase/phosphodiesterase family member 1, collagen alpha-1(III) chain, collagen alpha-1(XI) chain, aquaporin-1, and netrin receptor UNC5B, in the early stages of differentiation. GENERAL SIGNIFICANCE: These cell surface 5 glycoproteins become highly sensitive differentiation markers of human chondrocytes that contribute to regenerative therapies, and development of novel therapeutic reagents.

Induction of endoplasmic reticulum-endosome fusion for antigen cross-presentation induced by poly (γ-glutamic acid) nanoparticles.

We previously reported that poly (γ-glutamic acid)-based nanoparticles (γ-PGA NPs) are excellent vaccine carriers for inducing efficient cross-presentation in dendritic cells, thereby producing strong antitumor immunity in vivo. Analyzing the mechanism of cross-presentation induced by γ-PGA NPs will be useful toward designing novel vaccine carriers. In this study, we show an intracellular mechanism of efficient cross-presentation induced by OVA-loaded γ-PGA NPs. Cross-presentation induced by γ-PGA NPs depended on cytoplasmic proteasomes and TAP, similar to the classical MHC class I presentation pathway for endogenous Ags. Intracellular behavior analyzed by confocal laser scanning microscopy revealed that encapsulated OVA and γ-PGA accumulated in both the endoplasmic reticulum (ER) and endosome compartments within 2 h. At the same time, electron microscopy analysis clearly showed that intracellular γ-PGA NPs and encapsulated Au NPs were enveloped in endosome-like vesicles, not in the ER. These findings strongly suggest that γ-PGA NPs enhance ER-endosome fusion for cross-presentation. Moreover, inhibition of ER translocon sec61 significantly decreased the γ-PGA NP/OVA-mediated cross-presentation efficiency, indicating that sec61 is important for transporting Ags from the fused ER-endosome to the cytoplasm. These findings imply that the ER-endosome complex is key for the efficient cross-presentation of Ags encapsulated in γ-PGA NPs.

Distinct features of SARS-CoV-2 humoral immunity against Omicron breakthrough infection.

BACKGROUND: SARS-CoV-2 Omicron breakthrough infection (Omicron-BTI) after vaccination has been frequently observed. A more detailed understanding of the humoral immunity against Omicron-BTI is required. METHODS: We measured strain-specific live-virus based neutralizing activity, anti-spike IgG, and anti-receptor-binding domain (RBD) IgG titers in individuals with Omicron/BA.1-BTI and directly compared them with controls with diverse combinations of wild-type (WT) mRNA vaccination and infection history. RESULTS: Omicron-BTI individuals showed markedly higher neutralizing titers against all the WT, Delta, and Omicron strains in convalescent sera, compared with unvaccinated Omicron-infection individuals with only Omicron neutralizing activity. Similar tendencies were found in strain-specific anti-spike and anti-RBD IgG titers. The Omicron-specificity (BA.1/WT neutralizing ratio), Omicron-neutralizing efficiency per antibody unit, and anti-Omicron RBD-directivity of anti-spike antibodies in Omicron-BTI individuals were all significantly lower than those in unvaccinated Omicron-infection individuals, but they were equivalent to or higher than those in uninfected vaccinees. The induction of Omicron-specific neutralizing activity after Omicron-BTI was not weakened for eight months from the last vaccination. CONCLUSIONS: These findings suggest that cross-reactive vaccine-induced immunity was intensively stimulated following Omicron breakthrough infection, which contributed to Omicron neutralization. Measuring SARS-CoV-2 variant-specific antibody levels as well as neutralizing activity is useful for evaluating humoral immunity after breakthrough infection in the current situation of antigenic gaps between vaccinated and epidemic (Omicron sub-lineages) strains.

The utility of poly(γ-glutamic acid) nanoparticles as antigen delivery carriers in dendritic cell-based cancer immunotherapy.

Cytotoxic T-lymphocytes (CTLs) specific for tumor-associated antigens (TAAs) act in the immune surveillance system as major effector cells to eliminate malignant cells. Immunization with TAA-loaded dendritic cells (DCs) has great potential for treating cancer, because DCs are potent antigen-presenting cells capable of inducing antigen-specific CTLs by the primary activation of naive T-lymphocytes. The establishment of a non-cytotoxic and efficient antigen delivery method is required to improve the efficacy of DC-based cancer immunotherapy. We developed biodegradable poly(γ-glutamic acid) nanoparticles (γ-PGA NPs) that can efficiently entrap various proteins as antigen delivery carriers. γ-PGA NPs efficiently delivered entrapped antigenic proteins into DCs without cytotoxicity and presented antigens to DCs via major histocompatibility complex class I and II molecules. Immunization with TAA-loaded DCs using γ-PGA NPs inhibited tumor growth by inducing TAA-specific CTLs. These findings indicate that γ-PGA NPs can function as useful antigen delivery carriers in DC-based cancer immunotherapy.

AM251 Suppresses Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells.

Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the causative mechanisms of kidney fibrosis. In our study, we screened lipophilic compounds using a lipid library including approximately 200 lipids to identify those that suppressed EMT induced by a transforming growth factor (TGF)-ß1 stimulus. Initial screening was performed with the immortalized HK-2 renal tubule epithelial cell line. The most promising compounds were further tested in RPTEC primary renal tubule epithelial cells. We found that the synthetic lipid AM251 suppressed two hallmark events associated with EMT, the upregulation of collagen 1A1 (COL1A1) and downregulation of E-cadherin. Though AM251 is known to act as an antagonist for the cannabinoid receptor type 1 (CB1) and an agonist for the G protein-coupled receptor 55 (GRP55), the suppression of EMT by AM251 was not mediated through either receptor. Microarray analyses revealed that AM251 inhibited induction of several EMT transcription factors such as SNAIL1, which is the key inducer of EMT, and the AP-1 transcription factors FOSB and JUNB. Activation of SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) was inhibited by AM251, with greater inhibition of the latter, indicating that AM251 acted upstream of SMAD/p38 MAPK in the TGF-ß signaling pathway. Our findings regarding the effects of AM251 on the TGF-ß signaling pathway may inform development of a novel therapeutic agent suppressing EMT, thus preventing kidney fibrosis.

Nanoparticles built by self-assembly of amphiphilic gamma-PGA can deliver antigens to antigen-presenting cells with high efficiency: a new tumor-vaccine carrier for eliciting effector T cells.

Nanotechnology is a fundamental technology for designing and generating innovative carriers for biomacromolecular drugs. Biodegradable poly(gamma-glutamic acid)-based nanoparticles (gamma-PGA NPs) are excellent vaccine carriers capable of delivering antigenic proteins to antigen-presenting cells (APCs) and eliciting potent immune responses based on antigen-specific cytotoxic T lymphocytes. In mice, subcutaneous immunization with gamma-PGA NPs entrapping ovalbumin (OVA) more effectively inhibited the growth of OVA-transfected tumors than immunization with OVA emulsified using Freund's complete adjuvant. In addition, gamma-PGA NPs did not induce histopathologic changes after subcutaneous injection or acute toxicity through intravenous injection. Importantly, gamma-PGA NPs efficiently delivered entrapped antigenic proteins into APCs, and these antigen-capturing APCs migrated to regional lymph nodes. Our results demonstrate that a gamma-PGA NP system for antigen delivery will advance the clinical utility of vaccines against cancer.