Naked Peptide Research Articles

Abstract 4111: Multi-peptide cancer vaccines targeting KRAS induce significant anti-tumor efficacy

Abstract Background and objective: Kirsten rat sarcoma viral oncogene homologue (KRAS) is frequently mutated in human cancers. KRAS mutants offer the potential for the development of cancer-specific immunotherapy, as epitopes harbouring mutations are strong neoantigens to which the immune system generates cancer-specific anti-tumor effects. However, previous clinical trials of KRAS-targeted vaccines failed to generate satisfactory anti-cancer efficacy. Evidence suggested that CD4 T cell activity plays a critical role in augmenting the cytotoxic effect of CD8 T cells. Here, we design long peptides based on the MHC II hotspots on the entire KRAS protein that activate both CD4 and CD8 T cells to kill KRAS-driven cancer cells. Long peptides also cover a wider range of HLA alleles to increase their utility in a broader spectrum of patients. Our objective is to create a multi-peptide vaccine simultaneously targeting the mutant and wild-type (WT) regions of human KRAS protein. Methods: By selecting MHC II hotspots via multiple epitope prediction algorithms, we designed and synthesized 8 long peptides with lengths between 25 to 30mers. One of them harboured a G12D mutation, while the others covered the WT regions of KRAS. In addition, the corresponding keyhole limpet hemocyanin (KLH) conjugated peptides were also generated. We first demonstrated their immunogenicity in mouse in vivo models. Different combinations of peptides adjuvanted with either CpG oligodeoxynucleotide + aluminum (CpG+alum) or complete/incomplete Freud’s adjuvant (CFA/IFA) were immunized into Balb/c mice before determining the immune response by mouse interferon γ (mIFNγ) ELISPOT assay. Subsequently, an in vivo prophylactic anti-tumor study was conducted by immunizing this multi-peptide vaccine in Balb/c mice and observing the growth inhibition of the CT26-derived tumors. Results: T cell responses were observed in two peptide mixes with or without G12D mutation, suggesting that both mutant-harbouring peptide and WT peptides were immunogenic. We showed that KLH-conjugated peptides and CpG+alum elicited stronger immune responses than the naked peptides and CFA/IFA respectively. Furthermore, peptide mapping showed that the G12D mutant-harbouring peptide and two other WT peptides may contribute to the overall T cell response in the Balb/c mouse. In the prophylactic anti-tumor study (n = 10), immunization of KRAS naked peptide and KLH-conjugated peptide vaccines significantly inhibited tumor growth by 51.2% (****p < 0.0001) and 44.7% (***p < 0.001) respectively, compared to control. Subsequent mIFNγ ELISPOT assay showed that the tumor inhibitory effect was attributed to the activation of antigen-specific T cell response of the vaccines. Conclusion: Our multi-peptide vaccines targeting both the mutant and WT regions of KRAS could generate KRAS-specific T cell responses and elicit significant anti-tumor effect in mouse models. Citation Format: Chi Han Samson Li, Melvin Toh. Multi-peptide cancer vaccines targeting KRAS induce significant anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4111.

Combined ROS Sensitive PEG-PPS-PEG with Peptide Agonist for Effective Target Therapy in Mouse Model.

Growth hormone-releasing hormone (GHRH) agonist, a 29-amino acid peptide, shows significant potential in treating myocardial infarction (MI) by aiding the repair of injured heart tissue. The challenge lies in the effective on-site delivery of GHRH agonist. This study explores the use of a targetable delivery system employing ROS-responsive PEG-PPS-PEG polymers to encapsulate and deliver GHRH agonist MR409 for enhanced therapeutic efficacy. We synthesized a self-assembling poly (ethylene glycol)-poly (propylene sulfide)-poly (ethylene glycol) polymer (PEG-PPS-PEG) amphiphilic polymer responsive to reactive oxygen species (ROS). The hydrophilic peptide GHRH agonist MR409 was encapsulated within these polymers to form nano PEG-PPS-PEG@MR409 vesicles (NPs). Cardiomyocyte apoptosis was induced under hypoxia and serum-free culture condition for 24 hours, and their production of ROS was detected by fluorescence dye staining. The cellular uptake of PEG-PPS-PEG@MR409 NPs was observed using fluorescence-labeled MR409. Targeting ability and therapeutic efficacy were evaluated using a mouse MI model. PEG-PPS-PEG@MR409 NPs were efficiently internalized by cardiomyocytes, reducing ROS levels and apoptosis. These NPs exhibited superior targeting to the infarcted heart compared to naked MR409 peptide. With a reduced injection frequency (once every three days), PEG-PPS-PEG@MR409 NPs significantly promoted cardiac function recovery post-MI, matching the efficacy of daily MR409 injections. ROS-responsive PEG-PPS-PEG polymers provide a novel and effective platform for the targeted delivery of GHRH agonist peptides, improving cardiac function and offering a new approach for peptide therapy in MI treatment.

In vitro efficacy of different PEGylation designs on cathelicidin-like peptide with high antibacterial and antifungal activity

Recent reports on antibiotic resistance have highlighted the need to reduce the impact of this global health issue through urgent prevention and control. The World Health Organization currently considers antibiotic resistance as one of the most dangerous threats to global health. Therefore, Antimicrobial peptides (AMPs) are promising for the development of novel antibiotic molecules due to their high antimicrobial effects, non-inducing antimicrobial resistance (AMR) properties, and broad spectrum. Hence, in this study, we developed novel antimicrobial peptide/polymer conjugates to reduce the adverse effects of TN6 (RLLRLLLRLLR) peptide. We demonstrate how our constructs function in vitro in terms of antimicrobial activity, hemolytic activity, cytotoxicity, and protease resistance. Our findings show that our molecules are effective against different types of microorganisms such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant S. aureus, vancomycin-resistant Enteroccus faecium, and Candida albicans, which are known to be pathogenic and antibiotic-resistant. Our constructs generally showed low cytotoxicity relative to the peptide in HaCaT and 3T3 cells. Especially these structures are very successful in terms of hemotoxicity. In the bacteremia model with S. aureus, the naked peptide (TN6) was hemotoxic even at 1 µg/mL, while the hemotoxicity of the conjugates was considerably lower than the peptide. Remarkably in this model, the hemolytic activity of PepC-PEG-pepC conjugate decreased 15-fold from 2.36 to 31.12 µg/mL compared to the bacteria-free 60-min treatment. This is proof that in the case of bacteremia and sepsis, the conjugates specifically direct to bacterial cell membranes rather than red blood cells. In addition, the PepC-PEG-pepC conjugate is resistant to plasma proteases. Moreover, morphological and intracellular damage of the peptide/conjugates to Escherichia coli are demonstrated in SEM and TEM images. These results suggest our molecules can be considered potential next-generation broad-spectrum antibiotic molecule/drug candidates that might be used in clinical cases such as bacteremia and sepsis.

Phospholipid Encapsulation of an Anti-Fibrotic Endopeptide to Enhance Cellular Uptake and Myocardial Retention.

Cardioprotective effects of N-acetyl-ser-asp-lys-pro (Ac-SDKP) have been reported in preclinical models of myocardial remodeling. However, the rapid degradation of this endogenous peptide in vivo limits its clinical use. To prolong its bioavailability, Ac-SDKP was encapsulated by phosphocholine lipid bilayers (liposomes) similar to mammalian cell membranes. The physical properties of the liposome structures were assessed by dynamic light scattering and scanning electron microscopy. The uptake of Ac-SDKP by RAW 264.7 macrophages and human and murine primary cardiac fibroblasts was confirmed by fluorescence microscopy and flow cytometry. Spectrum computerized tomography and competitive enzyme-linked immunoassays were performed to measure the ex vivo cardiac biodistribution of Ac-SDKP. The biological effects of this novel synthetic compound were examined in cultured macrophages and cardiac fibroblasts and in a murine model of acute myocardial infarction induced by permanent coronary artery ligation. A liposome formulation resulted in the greater uptake of Ac-SDKP than the naked peptide by cultured RAW 264.7 macrophages and cardiac fibroblasts. Liposome-delivered Ac-SDKP decreased fibroinflammatory genes in cultured cardiac fibroblasts co-treated with TGF-β1 and macrophages stimulated with LPS. Serial tissue and serum immunoassays showed the high bioavailability of Ac-SDKP in mouse myocardium and in circulation. Liposome-delivered Ac-SDKP improved cardiac function and reduced myocardial fibroinflammatory responses in mice with acute myocardial infarction. Encapsulation of Ac-SDKP in a cell membrane-like phospholipid bilayer enhances its plasma and tissue bioavailability and offers cardioprotection against ischemic myocardial injury. Future clinical trials can use this novel approach to test small protective endogenous peptides in myocardial remodeling.

Design of a New Coating Agent Based on Graphene Oxide and Antimicrobial/Spermicidal Peptide (Sarcotoxin Pd) for Condom Coating: New Strategy for Prevention of Unplanned Pregnancy and Sexually Transmitted Infections

In this study, sarcotoxin Pd-functionalized graphene oxide (GO-Pd) was synthesized as a new condom-coating agent. Antimicrobial activity was evaluated by radial diffusion assay (RDA) and absorbance-based methods. Sperm motility and morphology were assessed in different concentrations of designed nanostructures. Peptide stability on the GO structures was assessed by the CD technique. GO-Pd showed the highest contact angle. The results approved that GO-Pd had broad-spectrum antimicrobial activities against examined pathogens, especially vaginal infections such as Candida vulvovaginitis. This antimicrobial activity was more than pristine peptides, vancomycin, and fluconazole. GO-Pd also had a higher inhibitory activity on the sperm motility and viability than pristine peptides. GO-Pd had high stability and activity in all examined conditions. But, naked peptides had low stability and activity after incubation in acidic pH and high temperatures (>38°C). In all tests, GO-Pd showed a significant difference compared to naked peptide. Based on the results, GO-Pd can be used as a condom coating to prevent unplanned pregnancy and sexually transmitted infections.

PpGalNAc-T4-catalyzed O-Glycosylation of TGF-β type Ⅱ receptor regulates breast cancer cells metastasis potential

GalNAc-type O-glycosylation, initially catalyzed by polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), is one of the most abundant and complex posttranslational modifications of proteins. Emerging evidence has proven that aberrant ppGalNAc-Ts are involved in malignant tumor transformation. However, the exact molecular functions of ppGalNAc-Ts are still unclear. Here, the role of one isoform, ppGalNAc-T4, in breast cancer cell lines was investigated. The expression of ppGalNAc-T4 was found to be negatively associated with migration of breast cancer cells. Loss-of-function studies revealed that ppGalNAc-T4 attenuated the migration and invasion of breast cancer cells by inhibiting the epithelial–mesenchymal transition (EMT) process. Correspondingly, transforming growth factor beta (TGF-β) signaling, which is the upstream pathway of EMT, was impaired by ppGalNAc-T4 expression. ppGalNAc-T4 knockout decreased O-GalNAc modification of TGF-β type Ⅰ and Ⅱ receptor (TβR Ⅰ and Ⅱ) and led to the elevation of TGF-β receptor dimerization and activity. Importantly, a peptide from TβR Ⅱ was identified as a naked peptide substrate of ppGalNAc-T4 with a higher affinity than ppGalNAc-T2. Further, Ser31, corresponding to the extracellular domain of TβR Ⅱ, was identified as the O-GalNAcylation site upon in vitro glycosylation by ppGalNAc-T4. The O-GalNAc-deficient S31 A mutation enhanced TGF-β signaling activity and EMT in breast cancer cells. Together, these results identified a novel mechanism of ppGalNAc-T4-catalyzed TGF-β receptors O-GalNAcylation that suppresses breast cancer cell migration and invasion via the EMT process. Targeting ppGalNAc-T4 may be a potential therapeutic strategy for breast cancer treatment.

Report on biopharmaceutical profile of recent biotherapeutics and insilco docking studies on target bindings of known aptamer biotherapeutics

ABSTRACT Accumulated Toxicity, disease recurrence and drug resistivity problems have been observed due to the synthetic and semisynthetic therapeutic practices, which alternatively led to focus on Bio-therapeutics production than xenobiotics. Quick plasma clearance and high potency are the reasons for trending research with huge pharma market of numerous Bio-therapeutics than ever before. Researchers proved that most of the nano and micro Bio-therapeutics have multiple beneficial therapeutic effects. We have analyzed the past, and present scenario of some notable clinically approved Bio-therapeutics to identify the future formulation needs with advanced techniques. Protein-related drugs are the foremost Bio-therapeutics such as antibodies, enzymes, and short, fragmented polypeptides show aggregation properties during storage, naked peptide moieties are resisted by the polar cell membrane, and also the antidrug antibodies were reported. Even though Nucleic acid nano-bodies are excellent target binders than proteins, they had only a few minutes of half-life. Maintaining homogeneousness upon storage of Bio-therapeutics is still a significant challenge in industrial-scale formulation. Notably, plant systems are identified as most useful cost-effective hosts to produce human enzymes than animal systems without any possible viral loads. Irrespective of numerous advancements in routes of administration and additives, subcutaneous is still a golden one to achieve better dynamics. Additionally, the interactions and effective bonds made by each class of well-known aptamer biotherapeutics which are considered as future drugs were studied.

Abstract B123: Local treatment with PeptiCRAd-1, a novel cancer immunotherapy approach, mediates a systemic antitumour CD8+ T-cell response and infiltration of CD8+ and CD4+ T-cells into distant untreated tumors in a clinically relevant humanized mouse melanoma model

Abstract Multiple oncolytic viruses (OV) are currently in clinical development as cancer immunotherapy candidates, due to their high immunogenicity and immune activation capacity. However, only anecdotal clinical evidence for OV-mediated tumor-specific immune responses exists. Peptide vaccines are highly tumor-specific, but the low immunogenicity and lack of CD8+ T-cell responses limit the clinical utility of this class of cancer immunotherapy. We have developed a unique cancer immunotherapy platform, PeptiCRAd™, which utilizes a highly immunogenic genetically engineered oncolytic adenovirus to deliver tumor-specific peptides to the immune system. This creates robust Th1 biased cellular immune responses against multiple tumor-specific targets simultaneously. This study compares the immune activation characteristics of intratumorally administered PeptiCRAd-1 to intratumoral OV treatment and standard intradermal peptide vaccination in a humanized mouse melanoma model. PeptiCRAd™ is an immunotherapy platform where multiple tumor-specific peptides are adsorbed onto the negatively charged capsid of genetically engineered oncolytic adenovirus via electrostatic interactions. Our lead clinical candidate PeptiCRAd-1 is based on a state-of-the-art oncolytic adenovirus coding for human CD40L and OX40L transgenes, coated with poly-lysine extended NY-ESO-1 and MAGE-A3 peptides. NOD/Shi-scid/IL-2Rγnull immunodeficient mice were humanized using hematopoietic stem cells (CD34+, HLA-B35+) isolated from human cord blood. A375 melanoma tumors were implanted subcutaneously and treated either with PeptiCRAd-1 or the naked virus. Peptide vaccines were given intradermally with Poly-IC as an adjuvant. Secondary tumors were implanted into the contralateral flank two days after the treatments were stopped. No treatments for secondary tumors were given. Peripheral blood mononuclear cells (PBMCs) and tumor infiltrating CD8+ lymphocytes (TILs) were analyzed for NY-ESO-1 and MAGE-A3 specific CD8+ T-cells by flow cytometry with dextramer analysis. Different immune cell subsets among PBMCs and TILs were assessed. All active treatments increased the number of immune cells in primary tumors in comparison to mock treated animals. Both OV and PeptiCRAd-1 treated animals showed more T-cells (CD3, CD4, CD8) in primary tumors in comparison to peptide vaccine or mock treated animals post treatment. Furthermore, the number of T regulatory cells (CD3+/CD4+/FoxP3+) was smaller in OV and PeptiCRAd-1 treated primary tumors in comparison to primary tumors from peptide vaccine or mock treated animals. This suggests that intratumorally administered immunogenic adenovirus (either naked virus or PeptiCRAd-1) modulates the tumor microenvironment by reducing local immune-suppression. Unlike OV treated animals, PeptiCRAd-1 treated animals showed more CD4+ and CD8+ T-cells in untreated secondary tumors than in treated primary tumors, suggesting that tumor-targeting via peptide-coating of the virus was critically important for the induction of an effect in distant untreated tumors. Furthermore, PeptiCRAd-1 treated animals had more NYESO-specific CD8+ T-cells in blood post priming (mean= 4.3% of total CD8+ cells) in comparison to OV treated (mean= 0.6%) or peptide vaccine treated (mean= 0.6%) animals. PeptiCRAd™ is superior to naked oncolytic adenovirus or standard peptide vaccination in triggering systemic tumor-targeted CD8+ T-cell responses and infiltration of CD8+ TILs into untreated distant tumors. The data suggest that PeptiCRAd improves the tumor targeting specificity of a standard OV. This easily adaptable technology could potentially be used to improve the tumor-focused immune responses generated by any oncolytic adenovirus currently in development. Importantly, PeptiCRAd™ could potentially be used for personalized tumor vaccination by delivering patient-specific neoantigens. A phase I clinical trial with PeptiCRAd-1 in several solid tumor indications is under preparation. Citation Format: Erkko Ylosmaki, Tuuli Ranki, Petri Priha, Charlotta Backman, Matthew Vaughn, Vincenzo Cerullo, Sari Pesonen. Local treatment with PeptiCRAd-1, a novel cancer immunotherapy approach, mediates a systemic antitumour CD8+ T-cell response and infiltration of CD8+ and CD4+ T-cells into distant untreated tumors in a clinically relevant humanized mouse melanoma model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B123.

Dendrons and Multiarm Polymers with Thiol-Exchangeable Cores: A Reversible Conjugation Platform for Delivery.

Disulfide exchange reaction has emerged as a powerful tool for reversible conjugation of proteins, peptides and thiol containing molecules to polymeric supports. In particular, the pyridyl disulfide group provides an efficient handle for the site-specific conjugation of therapeutic peptides and proteins bearing cysteine moieties. In this study, novel biodegradable dendritic platforms containing a pyridyl disulfide unit at their focal point were designed. Presence of hydroxyl groups at the periphery of these dendrons allows their elaboration to multivalent initiators that yield poly(ethylene glycol) based multiarm star polymers via controlled radical polymerization. The pyridyl disulfide unit at the core of these star polymers undergoes efficient reaction with thiol functional group containing molecules such as a hydrophobic dye, namely, Bodipy-SH, glutathione, and KLAK sequence containing peptide. While conjugation of the hydrophobic fluorescent dye to the PEG-based multiarm polymer renders it water-soluble, it can be cleaved off the construct through thiol-disulfide exchange in the presence of an external thiol such as dithiothreitol. The multiarm polymer was conjugated with a thiol group containing apoptotic peptide to increase its solubility and cellular transport. In vitro cytotoxicity and apoptosis assays demonstrated that the resultant peptide-polymer conjugate had almost five times more apoptotic potential primarily through triggering apoptosis by disrupting mitochondrial membranes of human breast cancer cell line (MDA-MB-231) compared to naked peptide. The novel dendritic platform disclosed here offers an attractive template that can be modified to multiarm polymeric constructs bearing a "tag and release" characteristic.

Glycosylation of MUC1 influences the binding of a therapeutic antibody by altering the conformational equilibrium of the antigen.

In cancer cells, the glycoprotein Mucin 1 (MUC1) undergoes abnormal, truncated glycosylation. The truncated glycosylation exposes cryptic peptide epitopes that can be recognized by antibodies. Since these immunogenic regions are cancer specific, they represent ideal targets for therapeutic antibodies. We investigated the role of tumor-specific glycosylation on antigen recognition by the therapeutic antibody AR20.5. We explored the affinity of AR20.5 to a synthetic cancer-specific MUC1 glycopeptide and peptide. The antibody bound to the glycopeptide with an order of magnitude stronger affinity than the naked peptide. Given these results, we postulated that AR20.5 must specifically bind the carbohydrate as well as the peptide. Using X-ray crystallography, we examined this hypothesis by determining the structure of AR20.5 in complex with both peptide and glycopeptide. Surprisingly, the structure revealed that the carbohydrate did not form any specific polar contacts with the antibody. The high affinity of AR20.5 for the glycopeptide and the lack of specific binding contacts support a hypothesis that glycosylation of MUC1 stabilizes an extended bioactive conformation of the peptide recognized by the antibody. Since high affinity binding of AR20.5 to the MUC1 glycopeptide may not driven by specific antibody-antigen contacts, but rather evidence suggests that glycosylation alters the conformational equilibrium of the antigen, which allows the antibody to select the correct conformation. This study suggests a novel mechanism of antibody-antigen interaction and also suggests that glycosylation of MUC1 is important for the generation of high affinity therapeutic antibodies.

Solid-phase synthesis of C-mannosylated glycopeptide on WSXWS motif of human erythropoietin receptor

Protein C-mannosylation is a widely conserved posttranslational modification in multicellular organisms although the function of this modification is yet to be clarified. To evaluate the effect of this modification, a C-mannosyltryptophan containing glycopeptide of human erythropoietin receptor (EPOR), which includes WSXWS motif conserved in cytokine receptor superfamily, was synthesized by microwave assisted solid-phase synthesis using per-O-benzyl protected amino acids and C-mannosyltryptophan on Sieber amide resin. Comparative NMR analysis of the C-mannosyl glycopeptide and a corresponding naked peptide revealed that the C-mannosylation enhances the NOE signal between the peptide main chain and the aromatic side chain to suggest enhancement of conformational stability of the WSXWS motif and its surrounding regions.

Abstract 2443: Optides (optimized peptides), a new knottin-based cancer drug discovery platform

Abstract Some of the highest value targets in cancer therapeutics involve protein:protein interactions that are difficult to inhibit with small molecule therapeutics. Peptide drug candidates offer an alternative, but many peptide drugs have poor pharmacokinetic properties and some are immunogenic. Knottin peptides have long been promoted as scaffolds for human drug discovery efforts because the exquisitely tight knot formed by disulfide bridges creates resistance to proteolytic and other forms of degradation. Furthermore, some knottins cross the blood brain barrier, the gut wall, or cell membranes which is ideal for certain targets. Unfortunately, creating knottins synthetically or in bacteria results in a large number of disulfide isomers, which has hampered research. We have created a mammalian expression system that enables production of most knottin scaffolds and variants of native knottins encoded in plant and animal genomes. The platform routinely produces approximately 10 mg/liter of near pure naked peptide, which is sufficient to conduct in vivo studies without major scale-up. The system is endotoxin free. In addition to building the platform, we identified over 200,000 new potential knottin peptides in various genomes, creating a rich collection of scaffolds from which to establish diversity libraries of native and optimized variants. These candidates have potential for targeting therapeutics to cancer cells as a solid tumor penetrating alternative to antibody drug conjugates. Optides also have potential to be developed as anti-cancer drugs themselves. The fact that knottin peptides are not substrates for resistance mechanisms such as p-glycoprotein pumps underscores the potential importance of this class of drugs for those with previously treated cancer. Citation Format: James M. Olson, Roland Strong, Christopher Mehlin, Colin Correnti. Optides (optimized peptides), a new knottin-based cancer drug discovery platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2443. doi:10.1158/1538-7445.AM2015-2443

Incorporation of Naked Peptide Nucleic Acids into Liposomes Leads to Fast and Efficient Delivery.

The delivery of peptide nucleic acids (PNAs) to cells is a very challenging task. We report here that a liposomal formulation composed of egg PC/cholesterol/DSPE-PEG2000 can be loaded, according to different encapsulation techniques, with PNA or fluorescent PNA oligomers. PNA loaded liposomes efficiently and quickly promote the uptake of a PNA targeting the microRNA miR-210 in human erythroleukemic K562 cells. By using this innovative delivery system for PNA, down-regulation of miR-210 is achieved at a low PNA concentration.

MAGIC: an automated N-linked glycoprotein identification tool using a Y1-ion pattern matching algorithm and in silico MS² approach.

Glycosylation is a highly complex modification influencing the functions and activities of proteins. Interpretation of intact glycopeptide spectra is crucial but challenging. In this paper, we present a mass spectrometry-based automated glycopeptide identification platform (MAGIC) to identify peptide sequences and glycan compositions directly from intact N-linked glycopeptide collision-induced-dissociation spectra. The identification of the Y1 (peptideY0 + GlcNAc) ion is critical for the correct analysis of unknown glycoproteins, especially without prior knowledge of the proteins and glycans present in the sample. To ensure accurate Y1-ion assignment, we propose a novel algorithm called Trident that detects a triplet pattern corresponding to [Y0, Y1, Y2] or [Y0-NH3, Y0, Y1] from the fragmentation of the common trimannosyl core of N-linked glycopeptides. To facilitate the subsequent peptide sequence identification by common database search engines, MAGIC generates in silico spectra by overwriting the original precursor with the naked peptide m/z and removing all of the glycan-related ions. Finally, MAGIC computes the glycan compositions and ranks them. For the model glycoprotein horseradish peroxidase (HRP) and a 5-glycoprotein mixture, a 2- to 31-fold increase in the relative intensities of the peptide fragments was achieved, which led to the identification of 7 tryptic glycopeptides from HRP and 16 glycopeptides from the mixture via Mascot. In the HeLa cell proteome data set, MAGIC processed over a thousand MS(2) spectra in 3 min on a PC and reported 36 glycopeptides from 26 glycoproteins. Finally, a remarkable false discovery rate of 0 was achieved on the N-glycosylation-free Escherichia coli data set. MAGIC is available at http://ms.iis.sinica.edu.tw/COmics/Software_MAGIC.html .

GM-CSF-neuroantigen fusion proteins reverse experimental autoimmune encephalomyelitis and mediate tolerogenic activity in adjuvant-primed environments: association with inflammation-dependent, inhibitory antigen presentation.

Single-chain fusion proteins comprised of GM-CSF and neuroantigen (NAg) are potent, NAg-specific inhibitors of experimental autoimmune encephalomyelitis (EAE). An important question was whether GMCSF-NAg tolerogenic vaccines retained inhibitory activity within inflammatory environments or were contingent upon steady-state conditions. GM-CSF fused to the myelin oligodendrocyte glycoprotein MOG35-55 peptide (GMCSF-MOG) reversed established paralytic disease in both passive and active models of EAE in C57BL/6 mice. The fusion protein also reversed EAE in CD4-deficient and B cell-deficient mice. Notably, GMCSF-MOG inhibited EAE when coinjected adjacent to the MOG35-55/CFA emulsion. GMCSF-MOG also retained dominant inhibitory activity when directly emulsified with MOG35-55 in the CFA emulsion in both C57BL/6 or B cell-deficient models of EAE. Likewise, when combined with proteolipid protein 139-151 in CFA, GM-CSF fused to proteolipid protein 139-151 peptide inhibited EAE in SJL mice. When deliberately emulsified in CFA with the NAg, GMCSF-NAg inhibited EAE even though NAg was present at >30-fold molar excess. In vitro studies revealed that the GM-CSF domain of GMCSF-MOG stimulated growth and differentiation of inflammatory dendritic cells (DC) and simultaneously targeted the MOG35-55 domain for enhanced presentation by these DC. These inflammatory DC presented MOG35-55 to MOG-specific T cells by an inhibitory mechanism that was mediated in part by IFN-γ signaling and NO production. In conclusion, GMCSF-NAg was tolerogenic in CFA-primed proinflammatory environments by a mechanism associated with targeted Ag presentation by inflammatory DC and an inhibitory IFN-γ/NO pathway. The inhibitory activity of GMCSF-NAg in CFA-primed lymphatics distinguishes GMCSF-NAg fusion proteins as a unique class of inflammation-dependent tolerogens that are mechanistically distinct from naked peptide or protein-based tolerogens.

Cellular and animal models for high-throughput screening of therapeutic agents for the treatment of the diseases of the elderly in general and Alzheimer's disease in particular(†).

It is currently thought that the dementia of Alzheimer’s disease is due to the neurotoxicity of the deposits or aggregates of amyloid-β (Aβ) in the extracellular space of the cerebral cortex. This model has been widely criticized because there is a poor correlation between deposits and dementia. Others have questioned whether Aβ is truly neurotoxic. Yet, in spite of these concerns, the search for therapeutic agents has been based on the development of mouse models transfected with mutant genes associated in humans with early onset Alzheimer’s disease. A major limitation of these models is that although they exhibit many of the pathological and clinical manifestation of the human disease, the bulk of individuals who develop the dementia of Alzheimer’s disease have none of these mutant genes. Furthermore, nine clinical trials of drugs that were effective in transgenic mice failed to show any benefit in patients. Finally, a major unresolved issue with the Aβ model is that since Aβ is produced in everyone, why are deposits only seen in the elderly? This issue must be resolved if we are to understand the etiology of the disease and develop test systems for both diagnosis and drug discovery. Published studies from my laboratory demonstrate that in human cerebrospinal fluid immunoreactive Aβ is only present as a complex with two chaperones, ERp57 and calreticulin and is N-glycosylated. This complex formation is catalyzed by the posttranslational protein processing system of the endoplasmic reticulum (ER). Others have reported that in plaque Aβ is present only as the naked peptide. Together these results suggest that both plaque and dementia are secondary to an age related decline in the capacity of the ER to catalyze protein, posttranslational processing. Since the synaptic membrane proteins necessary for a functioning memory are also processed in the ER, these findings would suggest that the loss of cognition is due to a decline in the capacity of the neuron to produce and maintain functioning synapses. Work from my laboratory and from others further indicate that the components of the ER, posttranslational, protein processing pathway do dramatically decline with age. These data suggest that this decline may be found in all cells and could account not only for the dementia of Alzheimer’s disease, but also for many of the other manifestations of the aging process. These observations also suggest that declining ER function has a role in two well-recognized phenomena associated with aging: a loss of mitochondrial function and a decrease in myelin. Finally, based on this paradigm I propose new cellular and animals models for high-throughput screening for drug discovery.

An acetylation site in lectin domain modulates the biological activity of polypeptide GalNAc-transferase-2

Polypeptide GalNAc-transferases (ppGalNAc-Ts) are a family of enzymes that catalyze the initiation of mucin-type O-glycosylation. All ppGalNAc-T family members contain a common (QXW)3 motif, which is present in the R-type lectin group. The acetylation site K521 is part of the QKW motif of β-trefoil in the lectin domain of ppGalNAc-T2. We used a combination of acetylation and site-directed mutagenesis approaches to examine the functional role of K521 in ppGalNAc-T2. Binding assays of non-acetylated and acetylated forms of the mutant ppGalNAc-T2K521Q to various naked and αGalNAc-glycosylated mucin peptides indicated that the degree of interaction of lectin domain with αGalNAc depends on the peptide sequence of mucin. Studies of the inhibitory effect of various carbohydrates on the interactions of ppGalNAc-T2 with MUC1αGalNAc indicate that point K521Q mutation enhance the carbohydrate specificity of lectin domain for αGalNAc. K521Q mutation resulted in an enzyme activity lower than that of the wild-type ppGalNAc-T2, similar to the acetylation of ppGalNAc-T2. We conclude that an acetylation site in the QKW motif of the lectin domain modulates carbohydrate recognition specificity and catalytic activity of ppGalNAc-T2 for partially preglycosylated acceptors and a certain naked peptide. Posttranslational modifications of ppGalNAc-Ts, such as acetylation, may play key roles in modulating the functions of the R-type lectin domains in cellular homeostasis.

Shift Reagents for Multidimensional Ion Mobility Spectrometry-Mass Spectrometry Analysis of Complex Peptide Mixtures: Evaluation of 18-Crown-6 Ether Complexes

18-Crown-6 ether (18C6) is evaluated as a shift reagent for multidimensional ion mobility spectrometry-mass spectrometry (IMS-IMS-MS) analyses of tryptic protein digests. In this approach, 18C6 is spiked into the solution-phase mixture and noncovalent peptide-crown ion complexes are formed by electrospraying the mixture into the gas phase. After an initial mobility separation in the first IMS drift region, complexes of similar mobility are selected and dissociated via collisional activation prior to entering the second drift region. These dissociation products (including smaller complexes, naked peptide ions, charge transfer products, and fragment ions) differ in mobility from their precursor ion complexes and (in favorable cases) from one another, allowing the mixture to resolve further in the second IMS region. We estimate an IMS-IMS peak capacity of ~2400 when shift reagents are employed. The approach is illustrated by examining a tryptic digest of cytochrome c and by identifying a peptide out of a complex mixture obtained by digestion of human plasma proteins. Disadvantages arising from increased complexity of data sets as well as other advantages of this approach are considered.

An Efficient Approach for the Characterization of Mucin‐Type Glycopeptides: The Effect of O‐Glycosylation on the Conformation of Synthetic Mucin Peptides

Despite the growing importance of mucin core O-glycosylation in many biological processes including the protection of epithelial cell surfaces, the immune response, cell adhesion, inflammation, and tumorigenesis/metastasis, the regulation mechanism and conformational significance of the multiple introduction of α-GalNAc residues by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) remains unclear. Here we report an efficient approach by combining MS and NMR spectroscopy that allows for the identification of O-glycosylation site(s) and the effect of O-glycosylation on the peptide backbone structures during enzymatic mucin domain assembly by using an isoform UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T2 (ppGalNAcT2) in vitro. An electron-capture dissociation device in a linear radio-frequency quadrupole ion trap (RFQ-ECD) combined with a time-of-flight (TOF) mass spectrometer was employed for the identification of Thr/Ser residues occupied by α-GalNAc branching among multiple and potential O-glycosylation sites in the tandem repeats of human mucin glycoproteins MUC4 (Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp) and MUC5AC (Pro-Thr-Thr-Val-Gly-Ser-Thr-Thr-Val-Gly). In the present study, O-glycosylation was initiated specifically at Thr10 in naked MUC4 peptide and additional introduction of α-GalNAc proceeded preferentially but randomly at three other Thr residues to afford densely glycosylated MUC4 containing six α-GalNAc residues at Thr1, Ser2, Ser5, Thr6, Thr10, and Thr15. On the contrary, O-glycosylation of naked MUC5AC peptide occurred predominantly at consecutive Thr residues and led to MUC5AC with four α-GalNAc residues at Thr2, Thr3, Thr7, and Thr8. The solution structures determined by NMR spectroscopic studies elicited that the preferential introduction of α-GalNAc at Thr10 of MUC4 stabilizes specifically a β-like extended backbone structure at this area, whereas other synthetic models with a single α-GalNAc residue at Thr1, Thr6, or Thr15 did not exhibit any converged three-dimensional structure at the proximal peptide moiety. Such conformational impact on the underlying peptides was proved to be remarkable in the glycosylation at the consecutive Thr residues of MUC5AC.

An Efficient Biodelivery System for Antisense Polyamide Nucleic Acid (PNA)

With the aim of developing a general and straightforward procedure for the intracellular delivery of naked peptide nucleic acids (PNAs), we designed an intracellularly biodegradable triphenylphosphonium (TPP) cation based transporter system. In this system, TPP is linked, via a biolabile disulfide bridge, to an activated mercaptoethoxycarbonyl moiety, allowing its direct coupling to the N-terminal extremity of a free PNA through a carbamate bond. We found that such TPP-PNA-carbamate conjugates were highly stable in a cell culture medium containing fetal calf serum. In a glutathione-containing medium mimicking the cytosol, the conjugates were rapidly degraded into an unstable intermediate, which spontaneously decomposed, releasing the free PNA. Using a fluorescence-labeled PNA-TPP conjugate, we demonstrated that conjugates were taken up by cells. Efficient cellular uptake and release of the PNA into the cytosol was further confirmed by the anti-HIV activity measured for the TPP-conjugate of a 16-mer PNA targeting the TAR region of the HIV-1 genome. This conjugate exhibited an IC(50) value of 1 microM, while the free 16-mer PNA did not inhibit replication of HIV in the same cellular test.