Association of copeptin levels with patient prognosis and survival in sepsis syndromes: a meta-analysis.

BACKGROUND: Sepsis syndromes are a major burden in the ICU with very high mortality. Vasopressin and copeptin are released in response to hypovolemia and have shown potential significance in diagnosing sepsis. OBJECTIVE: To investigate the levels of copeptin in patients with sepsis syndromes and evaluate its relation with patient prognosis and mortality. METHODS: Four databases were searched for literature published from inception to the 8th of November 2022. Original research articles where copeptin was measured in sepsis patients and compared with controls were included. Data extraction and synthesis: study characteristics, levels of copeptin in the participants, and copeptin assay description were extracted. Levels of copeptin in patients were pooled and compared with controls in terms of the standard mean difference (SMD) generated using a random-effects model. RESULTS: Fifteen studies met the selection criteria. Copeptin levels were significantly higher in patients with sepsis, severe sepsis, and septic shock as compared to controls [(SMD: 1.49, 95% CI: 0.81-2.16, P<0.0001), (SMD: 1.94, 95% CI: 0.34-3.54, P=0.02), and (SMD: 2.17, 95% CI: 0.68-3.66, P=0.004), respectively]. The highest copeptin levels were noted in septic shock patients. The admission copeptin levels were significantly lower in survivors as compared to nonsurvivors (SMD: -1.73; 95% CI: -2.41 to -1.06, P<0.001). CONCLUSION AND RELEVANCE: Copeptin was significantly elevated in sepsis, severe sepsis, and septic shock. Survivors had a significantly lower copeptin during admission. Copeptin offered an excellent predictability to predict 1-month mortality. Measuring the copeptin in sepsis patients can aid treating physicians to foresee patients' prognosis.

Uncovering the roles of Mycobacterium tuberculosis melH in redox and bioenergetic homeostasis: implications for antitubercular therapy.

Mycobacterium tuberculosis (Mtb), the pathogenic bacterium that causes tuberculosis, has evolved sophisticated defense mechanisms to counteract the cytotoxicity of reactive oxygen species (ROS) generated within host macrophages during infection. The melH gene in Mtb and Mycobacterium marinum (Mm) plays a crucial role in defense mechanisms against ROS generated during infection. We demonstrate that melH encodes an epoxide hydrolase and contributes to ROS detoxification. Deletion of melH in Mm resulted in a mutant with increased sensitivity to oxidative stress, increased accumulation of aldehyde species, and decreased production of mycothiol and ergothioneine. This heightened vulnerability is attributed to the increased expression of whiB3, a universal stress sensor. The absence of melH also resulted in reduced intracellular levels of NAD+, NADH, and ATP. Bacterial growth was impaired, even in the absence of external stressors, and the impairment was carbon source dependent. Initial MelH substrate specificity studies demonstrate a preference for epoxides with a single aromatic substituent. Taken together, these results highlight the role of melH in mycobacterial bioenergetic metabolism and provide new insights into the complex interplay between redox homeostasis and generation of reactive aldehyde species in mycobacteria. IMPORTANCE: This study unveils the pivotal role played by the melH gene in Mycobacterium tuberculosis and in Mycobacterium marinum in combatting the detrimental impact of oxidative conditions during infection. This investigation revealed notable alterations in the level of cytokinin-associated aldehyde, para-hydroxybenzaldehyde, as well as the redox buffer ergothioneine, upon deletion of melH. Moreover, changes in crucial cofactors responsible for electron transfer highlighted melH's crucial function in maintaining a delicate equilibrium of redox and bioenergetic processes. MelH prefers epoxide small substrates with a phenyl substituted substrate. These findings collectively emphasize the potential of melH as an attractive target for the development of novel antitubercular therapies that sensitize mycobacteria to host stress, offering new avenues for combating tuberculosis.

Development and validation of a model and nomogram for breast cancer diagnosis based on quantitative analysis of serum disease-specific haptoglobin N-glycosylation.

BACKGROUND: A better diagnostic marker is in need to distinguish breast cancer from suspicious breast lesions. The abnormal glycosylation of haptoglobin has been documented to assist cancer diagnosis. This study aims to evaluate disease-specific haptoglobin (DSHp)-ß N-glycosylation as a potential biomarker for breast cancer diagnosis. METHODS: DSHp-ß chains of 497 patients with suspicious breast lesions who underwent breast surgery were separated from serum immunoinflammatory-related protein complexes. DSHp-ß N-glycosylation was quantified by mass spectrometric analysis. After missing data imputation and propensity score matching, patients were randomly assigned to the training set (n = 269) and validation set (n = 113). Logistic regression analysis was employed in model and nomogram construction. The diagnostic performance was analyzed with receiver operating characteristic and calibration curves. RESULTS: 95 N-glycopeptides at glycosylation sites N207/N211, N241, and N184 were identified in 235 patients with benign breast diseases and 262 patients with breast cancer. DSHp-ß N-tetrafucosyl and hexafucosyl were significantly increased in breast cancer compared with benign diseases (p < 0.001 and p = 0.001, respectively). The new diagnostic model and nomogram included GN2F2, G6N3F6, GN2FS at N184, G-N&G2S2, G2&G3NFS, G2N3F, GN3 at N207/N211, CEA, CA153, and could reliably distinguish breast cancer from benign diseases. For the training set, validation set, and training and validation sets, the area under the curves (AUCs) were 0.80 (95% CI: 0.75-0.86, specificity: 87%, sensitivity: 62%), 0.77 (95% CI:0.69-0.86, specificity: 75%, sensitivity: 69%), and 0.80 (95% CI:0.76-0.84, specificity: 77%, sensitivity: 68%), respectively. CEA, CA153, and their combination yielded AUCs of 0.62 (95% CI: 0.56-0.67, specificity: 29%, sensitivity: 90%), 0.65 (95% CI: 0.60-0.71, specificity: 74%, sensitivity: 51%), and 0.67 (95% CI: 0.62-0.73, specificity: 60%, sensitivity: 68%), respectively. CONCLUSIONS: The combination of DSHp-ß N-glycopeptides, CEA, and CA153 might be a better serologic marker to differentiate between breast cancer and benign breast diseases. The dysregulated N-glycosylation of serum DSHp-ß could provide insights into breast tumorigenesis.

Global O-glycoproteome enrichment and analysis enabled by a combinatorial enzymatic workflow.

A comprehensive analysis of site-specific protein O-glycosylation is hindered by the absence of a consensus O-glycosylation motif, the diversity of O-glycan structures, and the lack of a universal enzyme that cleaves attached O-glycans. Here, we report the development of a robust O-glycoproteomic workflow for analyzing complex biological samples by combining four different strategies: removal of N-glycans, complementary digestion using O-glycoprotease (IMPa) with/without another protease, glycopeptide enrichment, and mass spectrometry with fragmentation of glycopeptides using stepped collision energy. Using this workflow, we cataloged 474 O-glycopeptides on 189 O-glycosites derived from 79 O-glycoproteins from human plasma. These data revealed O-glycosylation of several abundant proteins that have not been previously reported. Because many of the proteins that contained unannotated O-glycosylation sites have been extensively studied, we wished to confirm glycosylation at these sites in a targeted fashion. Thus, we analyzed selected purified proteins (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) in independent experiments and validated the previously unknown O-glycosites.

A complement C4-derived glycopeptide is a biomarker for PMM2-CDG.

BACKGROUNDDiagnosis of PMM2-CDG, the most common congenital disorder of glycosylation (CDG), relies on measuring carbohydrate-deficient transferrin (CDT) and genetic testing. CDT tests have false negatives and may normalize with age. Site-specific changes in protein N-glycosylation have not been reported in sera in PMM2-CDG.METHODSUsing multistep mass spectrometry-based N-glycoproteomics, we analyzed sera from 72 individuals to discover and validate glycopeptide alterations. We performed comprehensive tandem mass tag-based discovery experiments in well-characterized patients and controls. Next, we developed a method for rapid profiling of additional samples. Finally, targeted mass spectrometry was used for validation in an independent set of samples in a blinded fashion.RESULTSOf the 3,342 N-glycopeptides identified, patients exhibited decrease in complex-type N-glycans and increase in truncated, mannose-rich, and hybrid species. We identified a glycopeptide from complement C4 carrying the glycan Man5GlcNAc2, which was not detected in controls, in 5 patients with normal CDT results, including 1 after liver transplant and 2 with a known genetic variant associated with mild disease, indicating greater sensitivity than CDT. It was detected by targeted analysis in 2 individuals with variants of uncertain significance in PMM2.CONCLUSIONComplement C4-derived Man5GlcNAc2 glycopeptide could be a biomarker for accurate diagnosis and therapeutic monitoring of patients with PMM2-CDG and other CDGs.FUNDINGU54NS115198 (Frontiers in Congenital Disorders of Glycosylation: NINDS; NCATS; Eunice Kennedy Shriver NICHD; Rare Disorders Consortium Disease Network); K08NS118119 (NINDS); Minnesota Partnership for Biotechnology and Medical Genomics; Rocket Fund; R01DK099551 (NIDDK); Mayo Clinic DERIVE Office; Mayo Clinic Center for Biomedical Discovery; IA/CRC/20/1/600002 (Center for Rare Disease Diagnosis, Research and Training; DBT/Wellcome Trust India Alliance).

Enrichment methods of N-linked glycopeptides from human serum or plasma: A mini-review.

Human diseases often correlate with changes in protein glycosylation, which can be observed in serum or plasma samples. N-glycosylation, the most common form, can provide potential biomarkers for disease prognosis and diagnosis. However, glycoproteins constitute a relatively small proportion of the total proteins in human serum and plasma compared to the non-glycosylated protein albumin, which constitutes the majority. The detection of microheterogeneity and low glycan abundance presents a challenge. Mass spectrometry facilitates glycoproteomics research, yet it faces challenges due to interference from abundant plasma proteins. Therefore, methods have emerged to enrich N-glycans and N-linked glycopeptides using glycan affinity, chemical properties, stationary phase chemical coupling, bioorthogonal techniques, and other alternatives. This review focuses on N-glycans and N-glycopeptides enrichment in human serum or plasma, emphasizing methods and applications. Although not exhaustive, it aims to elucidate principles and showcase the utility and limitations of glycoproteome characterization.

Beyond the model organism-Mechanistic analysis of protein post-translational modifications is ready for all challengers.

A historic challenge for shotgun proteomics has been the requirement for high quality, simple, and nonredundant curated protein sequences in small .fasta text files. Due to the intrinsic informatic challenges and time required to assemble these files, proteomics has struggled to expand beyond the confines of a few model organisms. When considering post-translational modifications that may or may not be present on a specific peptide sequence, these factors inevitably compound. A study on how mangos continue to ripen on the shelf may not be the first thing you'd think of as proof of a scientific discipline shedding historic limitations. However, Bautiste-Valle et al., may be just that. These authors present a quantitative comparison of both peptide and glycopeptide alterations through the complexity of the fruit ripening process and in this we see the present state of a field that no longer needs to wait on genomics to obtain deep mechanistic insights.

Targeted and Self-Adjuvated Nanoglycovaccine Candidate for Cancer Immunotherapy.

Advanced-stage solid primary tumors and metastases often express mucin 16 (MUC16), carrying immature glycans such as the Tn antigen, resulting in specific glycoproteoforms not found in healthy human tissues. This presents a valuable approach for designing targeted therapeutics, including cancer glycovaccines, which could potentially promote antigen recognition and foster the immune response to control disease spread and prevent relapse. In this study, we describe an adjuvant-free poly(lactic-co-glycolic acid) (PLGA)-based nanoglycoantigen delivery approach that outperforms conventional methods by eliminating the need for protein carriers while exhibiting targeted and adjuvant properties. To achieve this, we synthesized a library of MUC16-Tn glycoepitopes through single-pot enzymatic glycosylation, which were then stably engrafted onto the surface of PLGA nanoparticles, generating multivalent constructs that better represent cancer molecular heterogeneity. These glycoconstructs demonstrated affinity for Macrophage Galactose-type Lectin (MGL) receptor, known to be highly expressed by immature antigen-presenting cells, enabling precise targeting of immune cells. Moreover, the glycopeptide-grafted nanovaccine candidate displayed minimal cytotoxicity and induced the activation of dendritic cells in vitro, even in the absence of an adjuvant. In vivo, the formulated nanovaccine candidate was also nontoxic and elicited the production of IgG specifically targeting MUC16 and MUC16-Tn glycoproteoforms in cancer cells and tumors, offering potential for precise cancer targeting, including targeted immunotherapies.

Mass Spectrometry Analysis of Glycopeptides Enriched by Anion Exchange-Mediated Methods Reveals PolyLacNAc-Extended N-Glycans in Integrins and Tetraspanins in Melanoma Cells.

Glycosylation is a key modulator of the functional state of proteins. Recent developments in large-scale analysis of intact glycopeptides have enabled the identification of numerous glycan structures that are relevant in pathophysiological processes. However, one motif found in N-glycans, poly-N-acetyllactosamine (polyLacNAc), still poses a substantial challenge to mass spectrometry-based glycoproteomic analysis due to its relatively low abundance and large size. In this work, we developed approaches for the systematic mapping of polyLacNAc-elongated N-glycans in melanoma cells. We first evaluated five anion exchange-based matrices for enriching intact glycopeptides and selected two materials that provided better overall enrichment efficiency. We then tested the robustness of the methodology by quantifying polyLacNAc-containing glycopeptides as well as changes in protein fucosylation and sialylation. Finally, we applied the optimal enrichment methods to discover glycopeptides containing polyLacNAc motifs in melanoma cells and found that integrins and tetraspanins are substantially modified with these structures. This study demonstrates the feasibility of glycoproteomic approaches for identification of glycoproteins with polyLacNAc motifs.

Comparison of N-Glycopeptide to Released N-Glycan Abundances and the Influence of Glycopeptide Mass and Charge States on N-Linked Glycosylation of IgG Antibodies.

We report the comparison of mass-spectral-based abundances of tryptic glycopeptides to fluorescence abundances of released labeled glycans and the effects of mass and charge state and in-source fragmentation on glycopeptide abundances. The primary glycoforms derived from Rituximab, NISTmAb, Evolocumab, and Infliximab were high-mannose and biantennary complex galactosylated and fucosylated N-glycans. Except for Evolocumab, in-source ions derived from the loss of HexNAc or HexNAc-Hex sugars are prominent for other therapeutic IgGs. After excluding in-source fragmentation of glycopeptide ions from the results, a linear correlation was observed between fluorescently labeled N-glycan and glycopeptide abundances over a dynamic range of 500. Different charge states of human IgG-derived glycopeptides containing a wider variety of abundant attached glycans were also investigated to examine the effects of the charge state on ion abundances. These revealed a linear dependence of glycopeptide abundance on the mass of the glycan with higher charge states favoring higher-mass glycans. Findings indicate that the mass spectrometry-based bottom-up approach can provide results as accurate as those of glycan release studies while revealing the origin of each attached glycan. These site-specific relative abundances are conveniently displayed and compared using previously described glycopeptide abundance distribution spectra "GADS" representations. Mass spectrometry data are available from the MAssIVE repository (MSV000093562).

Comprehensive Review on Chiral Stationary Phases in Single-Column Simultaneous Chiral-Achiral HPLC Separation Methods.

This comprehensive review explores the utilization of chiral stationary phases (CSPs) in the context of single-column simultaneous chiral-achiral high-performance liquid chromatography (HPLC) separation methods. While CSPs have traditionally been pivotal for enantioselective drug analysis, contemporary CSPs often exhibit notable chemoselective properties. Consequently, there is a discernible trend towards the development of methodologies that enable simultaneous enantio- and chemoselective separations utilizing a single CSP-based chromatographic column. This review provides an exhaustive overview of reported HPLC methods in this domain, with a focus on four major CSP types: cyclodextrin-, glycopeptide antibiotic-, protein-, and polysaccharide-based CSPs. This article delves into the diverse applications of CSPs, encompassing various chromatographic modes such as normal phase (NP), reverse phase (RP), and polar organic (PO). This review critically discusses method development, emphasizing the additional chemoselective separation mechanisms of CSPs. It also explores possibilities for method optimization and development, concluding with future perspectives on this evolving field. Despite the inherent challenges in understanding the retention mechanisms involved in chemoselective separations, this review highlights promising trends and anticipates a growing number of simultaneous enantio- and chemoselective methods in pharmaceutical analyses, pharmacokinetic studies, and environmental sample determinations.

In Vitro and In Silico Explorations of the Protein Conformational Changes of Corynebacterium glutamicum MshA, a Model Retaining GT-B Glycosyltransferase.

MshA is a GT-B glycosyltransferase catalyzing the first step in the biosynthesis of mycothiol. While many GT-B enzymes undergo an open-to-closed transition, MshA is unique because its 97° rotation is beyond the usual range of 10-25°. Molecular dynamics (MD) simulations were carried out for MshA in both ligand bound and unbound states to investigate the effect of ligand binding on localized protein dynamics and its conformational free energy landscape. Simulations showed that both the unliganded "opened" and liganded "closed" forms of the enzyme sample a wide degree of dihedral angles and interdomain distances with relatively low overlapping populations. Calculation of the free energy surface using replica exchange MD for the apo "opened" and an artificial generated apo "closed" structure revealed overlaps in the geometries sampled, allowing calculation of a barrier of 2 kcal/mol for the open-to-closed transition in the absence of ligands. MD simulations of fully liganded MshA revealed a smaller sampling of the dihedral angles. The localized protein fluctuation changes suggest that UDP-GlcNAc binding activates the motions of loops in the 1-l-myo-inositol-1-phosphate (I1P)-binding site despite little change in the interactions with UDP-GlcNAc. Circular dichroism, intrinsic fluorescence spectroscopy, and mutagenesis studies were used to confirm the ligand-induced structural changes in MshA. The results support a proposed mechanism where UDP-GlcNAc binds with rigid interactions to the C-terminal domain of MshA and activates flexible loops in the N-terminal domain for binding and positioning of I1P. This model can be used for future structure-based drug development of inhibitors of the mycothiol biosynthetic pathway.

Mass Spectrometry-Compatible Elution Technique Enables an Improved Mucin-Selective Enrichment Strategy to Probe the Mucinome.

Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of healthy and disease-driven biological functions. Previously, we developed a mucin-selective enrichment strategy by employing a catalytically inactive mucinase (StcE) conjugated to a solid support. While this method was effective, it suffered from low throughput and high sample requirements. Further, the elution step required boiling in SDS, thus necessitating an in-gel digest with trypsin. Here, we introduce innovative elution conditions amenable to mucinase digestion and downstream analysis using mass spectrometry. This increased throughput and lowered sample input while maintaining mucin selectivity and enhancing the glycopeptide signal. We then benchmarked this technique against different O-glycan binding moieties for their ability to enrich mucins from various cell lines and human serum. Overall, the new method outperformed our previous procedure and all of the other enrichment techniques tested. This allowed for the effective isolation of more mucin-domain glycoproteins, resulting in a high number of O-glycopeptides, thus enhancing our ability to analyze the mucinome.

The potential of polyaniline-coated stationary phase in hydrophilic interaction liquid chromatography-based solid-phase extraction for glycopeptide enrichment.

Glycopeptide enrichment is a crucial step in glycoproteomic analysis, often achieved through solid-phase extraction (SPE) on polar stationary phases in hydrophilic interaction liquid chromatography (HILIC). This study explores the potential of polyaniline (PANI)-coated silica gel for enriching human immunoglobulin G (IgG). Experimental conditions were varied to assess their impact on glycopeptide enrichment efficiency, comparing PANI-cotton wool SPE with conventional cotton wool as SPE sorbents. Two formic acid concentrations (0.1% and 1%) in elution solvent were tested, revealing that higher concentrations led to earlier elution of studied glycopeptides, especially for sialylated glycopeptides. Substituting formic acid with acetic acid increased the interaction of neutral glycopeptides with the PANI-modified sorbent, while sialylated glycopeptides showed no significant change in enrichment efficiency. Acetonitrile concentration in the elution solvent (5%, 10%, and 20%) affected the enrichment efficiency with most glycopeptides eluting at the lowest acetonitrile concentration. The acetonitrile concentration in conditioning and washing solutions (65%, 75%, and 85%) played a crucial role; at 65% acetonitrile, glycopeptides were least retained on the stationary phase, and neutral glycopeptides were even detected in the flow-through fraction. This study shows the potential of in-house-prepared PANI-modified sorbents for SPE-HILIC glycopeptide enrichment, highlighting the crucial role of tuning experimental conditions in sample preparation to enhance enrichment efficiency and selectivity.

Prediction of glycopeptide fragment mass spectra by deep learning.

Deep learning has achieved a notable success in mass spectrometry-based proteomics and is now emerging in glycoproteomics. While various deep learning models can predict fragment mass spectra of peptides with good accuracy, they cannot cope with the non-linear glycan structure in an intact glycopeptide. Herein, we present DeepGlyco, a deep learning-based approach for the prediction of fragment spectra of intact glycopeptides. Our model adopts tree-structured long-short term memory networks to process the glycan moiety and a graph neural network architecture to incorporate potential fragmentation pathways of a specific glycan structure. This feature is beneficial to model explainability and differentiation ability of glycan structural isomers. We further demonstrate that predicted spectral libraries can be used for data-independent acquisition glycoproteomics as a supplement for library completeness. We expect that this work will provide a valuable deep learning resource for glycoproteomics.

Hydrophilic Interaction Liquid Chromatography (HILIC) Enrichment of Glycopeptides Using PolyHYDROXYETHYL A.

Glycosylation of proteins is an important post-translational modification that plays a role in a wide range of biological processes, including immune response, intercellular signaling, inflammation, and host-pathogen interaction. Abnormal protein glycosylation has been correlated with various diseases. However, the study of protein glycosylation remains challenging due to its low abundance, microheterogeneity of glycosylation sites, and low ionization efficiency. During the past decade, several methods for enrichment and for isolation of glycopeptides from biological samples have been developed and successfully employed in glycoproteomics research. In this chapter, we discuss the sample preparation protocol and the strategies for effectively isolating and enriching glycopeptides from biological samples, using PolyHYDROXYETHYL A as a hydrophilic interaction liquid chromatography (HILIC) enrichment technique.

O-Glycoproteomics Sample Preparation and Analysis Using NanoHPLC and Tandem MS.

Glycosylation refers to the biological processes that covalently attach carbohydrates to the peptide backbone after the synthesis of proteins. As one of the most common post-translational modifications (PTMs), glycosylation can greatly affect proteins' features and functions. Moreover, aberrant glycosylation has been linked to various diseases. There are two major types of glycosylation, known as N-linked and O-linked glycosylation. Here, we focus on O-linked glycosylation and thoroughly describe a bottom-up strategy to perform O-linked glycoproteomics studies. The experimental section involves enzymatic digestions using trypsin and O-glycoprotease at 37 °C. The prepared samples containing O-glycopeptides are analyzed using nanoHPLC coupled with tandem mass spectrometry (MS) for accurate identification and quantification.

Copeptin for the differentiation of type 1 versus type 2 myocardial infarction or myocardial injury.

BACKGROUND: The rapid and reliable differentiation of myocardial infarction (MI) due to atherothrombosis (T1MI) from MI due to supply-demand mismatch (T2MI) or acute myocardial injury is of major clinical relevance due to very different treatments, but still a major unmet clinical need. This study aimed to investigate whether copeptin, a stress hormone produced in the hypothalamus, helps to differentiate between T1MI versus T2MI or injury. METHODS: In a retrospective analysis, 1271 unselected consecutive patients presenting with symptoms suggestive of MI to the emergency department were evaluated. Patients diagnosed with ST-elevation MI were excluded. All patients with elevated cardiac troponin I (cTnI) concentration possibly indicating MI were classified into T1MI, T2MI, or acute myocardial injury using detailed clinical assessment and coronary imaging. Copeptin plasma concentration was measured in a blinded fashion. A multicenter diagnostic study with central adjudication of the final diagnosis served as external validation cohort (n = 1390). RESULTS: Among 1161 patients, 154 patients had increased cTnI concentration. Of these, 78 patients (51%) were classified as T1MI and 76 (49%) as T2MI or myocardial injury. Patients with T2MI or myocardial injury had significantly higher copeptin plasma concentration between patients versus T1MI (21,4 pmol/l versus 8,1 pmol/l, p = 0,001). A multivariable regression analysis revealed that higher concentrations of copeptin and C-reactive protein, higher heart rate at presentation and lower frequency of smoking remained significantly associated with T2MI and myocardial injury. Findings were largely confirmed in the external validation cohort. CONCLUSION: In patients without ST-segment elevation, copeptin concentration was higher in T2MI and myocardial Injury versus T1MI and may help in their differential diagnosis.

Preparation of glycopeptide-modified pH-sensitive liposomes for promoting antigen cross-presentation and induction of antigen-specific cellular immunity.

Cross-presentation, exogenous antigen presentation onto major histocompatibility complex class I molecules on antigen presenting cells, is crucially important for inducing antigen-specific cellular immune responses for cancer immunotherapy and for the treatment of infectious diseases. One strategy to induce cross-presentation is cytosolic delivery of an exogenous antigen using fusogenic or endosomolytic molecule-introduced nanocarriers. Earlier, we reported liposomes modified with pH-responsive polymers to achieve cytosolic delivery of an antigen. Polyglycidol-based or polysaccharide-based pH-responsive polymers can provide liposomes with delivery performance of antigenic proteins into cytosol via membrane fusion with endosomes responding to acidic pH, leading to induction of cross-presentation. Mannose residue was introduced to pH-responsive polysaccharides to increase uptake selectivity to antigen presenting cells and to improve cross-presentation efficiency. However, direct introduction of mannose residue into pH-responsive polysaccharides suppressed cytoplasmic delivery performance of liposomes. To avoid such interference, for this study, mannose-containing glycans were incorporated separately into pH-responsive polysaccharide-modified liposomes. Soybean agglutinin-derived glycopeptide was used as a ligand for lectins on antigen presenting cells. Incorporation of glycopeptide significantly increased the cellular uptake of liposomes by dendritic cell lines and increased cross-presentation efficiency. Liposomes incorporated both glycopeptide and pH-responsive polysaccharides exhibited strong adjuvant effects in vitro and induced the increase of dendritic cells, M1 macrophages, and effector T cells in the spleen. Subcutaneous administration of these liposomes induced antigen-specific cellular immunity, resulting in strong therapeutic effects in tumor-bearing mice. These results suggest that separate incorporation of glycopeptides and pH-responsive polysaccharides into antigen-loaded liposomes is an effective strategy to produce liposome-based nanovaccines to achieve antigen cross-presentation and induction of cellular immunity towards cancer immunotherapy.

Preparation of Mucin Glycopeptides by Organic Synthesis.

Mucins are sugar-rich glycoproteins. Glycoprotein sugar moieties are structurally diverse, making it difficult to obtain naturally pure glycoproteins. Chemical synthesis is a powerful tool for obtaining target or designed compounds. Automated peptide synthesizers are commercially available, and many use the solid-phase peptide synthesis (SPPS) method. In addition, some of these synthesizers apply microwave irradiation to obtain higher reaction yields, thereby enabling the synthesis of 40 to 50 amino acid residual glycopeptides. Theoretically, glycopeptides can be synthesized using methods similar to those used for peptide synthesis, but glycosylated amino acid synthons are less stable than amino acid synthons and are also very expensive. Therefore, they are not suitable for use in large excess amounts. Many of oligosaccharide-linked amino acid synthons are not commercially available, so they must be specially prepared, and they also require careful handling that demands specific organic synthesis experience and techniques. However, monosaccharide-linked amino acid synthons are commercially available and are relatively easy to handle. Here, as an entry into glycopeptide synthesis, we describe a typical glycopeptide synthesis procedure for a 27 amino acid residual MUC1 repeating unit with monosaccharides.