RESUMEN
Fc-glycosite-specific antibody-drug conjugation represents a promising direction for the preparation of site-specific antibody-drug conjugates (ADCs). In the present research, we conducted a systemic evaluation of two endoglycosidase-catalyzed chemoenzymatic glycoengineering technologies to prepare glycosite-specific ADCs. In the first two-step approach, the antibody was deglycosylated and then reglycosylated with a modified intact N-glycan oxazoline. In the second one-pot approach, antibodies were deglycosylated and simultaneously glycosylated with a functionalized disaccharide oxazoline. For the comprehensive evaluation, we first optimized and scaled-up the preparation of azido glycan oxazolines. Afterwards, we proved that the one-pot glycan-remodeling approach was efficient for all IgG subclasses. Subsequently, we assembled respective ADCS using two technology routes, with two different linker-payloads combinations, and performed systemic in vitro and in vivo evaluations. All the prepared ADCs achieved high homogeneity and illustrated excellent stability in buffers with minimum aggregates, and exceptional stability in rat serum. All ADCs displayed a potent killing of BT-474 breast cancer cells. Moving to the mouse study, the ADCs prepared from two technology routes displayed potent and similar efficacy in a BT-474 xenograft model, which was comparable to an FDA-approved ADC generated from random conjugation. These ADCs also demonstrated excellent safety and did not cause body weight loss at the tested dosages.
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Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer where no effective therapy has been developed. Here, we report that the natural product ER translocon inhibitor ipomoeassin F is a selective inhibitor of TNBC cell growth. A proteomic analysis of TNBC cells revealed that ipomoeassin F significantly reduced the levels of ER molecular chaperones, including PDIA6 and PDIA4, and induced ER stress, unfolded protein response (UPR) and autophagy in TNBC cells. Mechanistically, ipomoeassin F, as an inhibitor of Sec61α-containing ER translocon, blocks ER translocation of PDIA6, inducing its proteasomal degradation. Silencing of PDIA6 or PDIA4 by RNA interferences or treatment with a small molecule inhibitor of the protein disulfide isomerases in TNBC cells successfully recapitulated the ipomoeassin F phenotypes, including the induction of ER stress, UPR and autophagy, suggesting that the reduction of PDIAs is the key mediator of the pharmacological effects of ipomoeassin F. Moreover, ipomoeassin F significantly suppressed TNBC growth in a mouse tumor xenograft model, with a marked reduction in PDIA6 and PDIA4 levels in the tumor samples. Our study demonstrates that Sec61α-containing ER translocon and PDIAs are potential drug targets for TNBC and suggests that ipomoeassin F could serve as a lead for developing ER translocon-targeted therapy for TNBC.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Humanos , Animales , Ratones , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Proteómica , Glicoconjugados , Modelos Animales de Enfermedad , Chaperonas MolecularesRESUMEN
Targeted degradation using cell-specific lysosome targeting receptors is emerging as a new therapeutic strategy for the elimination of disease-associated proteins. The liver-specific human asialoglycoprotein receptor (ASGPR) is a particularly attractive lysosome targeting receptor leveraged for targeted protein degradation (TPD). However, the efficiency of different glycan ligands for ASGPR-mediated lysosomal delivery remains to be further characterized. In this study, we applied a chemoenzymatic Fc glycan remodeling method to construct an array of site-specific antibody-ligand conjugates carrying natural bi- and tri-antennary N-glycans as well as synthetic tri-GalNAc ligands. Alirocumab, an anti-PCSK9 (proprotein convertase subtilisin/kexin type 9) antibody, and cetuximab (an anti-EGFR antibody) were chosen to demonstrate the ASGPR-mediated degradation of extracellular and membrane-associated proteins, respectively. It was found that the nature of the glycan ligands and the length of the spacer in the conjugates are critical for the receptor binding and the receptor-mediated degradation of PCSK9, which blocks low-density lipoprotein receptor (LDLR) function and adversely affects clearance of low-density lipoprotein cholesterol. Interestingly, the antibody-tri-GalNAc conjugates showed a clear hook effect for its binding to ASGPR, while antibody conjugates carrying the natural N-glycans did not. Both the antibody-tri-antennary N-glycan conjugate and the antibody-tri-GalNAc conjugate could significantly decrease extracellular PCSK9, as shown in the cell-based assays. However, the tri-GalNAc conjugate showed a clear hook effect in the receptor-mediated degradation of PCSK9, while the antibody conjugate carrying the natural N-glycans did not. The cetuximab-tri-GalNAc conjugates also showed a similar hook effect on degradation of the membrane-associated protein, epidermal growth factor receptor (EGFR). These results suggest that the two types of ligands may involve a distinct mode of interactions in the receptor binding and target-degradation processes. Interestingly, the alirocumab-tri-GalNAc conjugate was also found to upregulate LDLR levels in comparison with the antibody alone. This study showcases the potential of the targeted degradation strategy against PCSK9 for reducing low-density lipoprotein cholesterol, a risk factor for heart disease and stroke.
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Proproteína Convertasas , Serina Endopeptidasas , Humanos , Receptor de Asialoglicoproteína , Ligandos , Serina Endopeptidasas/metabolismo , Proproteína Convertasas/metabolismo , Asialoglicoproteínas , Cetuximab , LDL-Colesterol/metabolismoRESUMEN
Mass spectrometry (MS) can unlock crucial insights into the intricate world of glycosylation analysis. Despite its immense potential, the qualitative and quantitative analysis of isobaric glycopeptide structures remains one of the most daunting hurdles in the field of glycoproteomics. The ability to distinguish between these complex glycan structures poses a significant challenge, hindering our ability to accurately measure and understand the role of glycoproteins in biological systems. A few recent publications described the use of collision energy (CE) modulation to improve structural elucidation, especially for qualitative purposes. Different linkages of glycan units usually demonstrate different stabilities under CID/HCD fragmentation conditions. Fragmentation of the glycan moiety produces low molecular weight ions (oxonium ions) that can serve as a structure-specific signature for specific glycan moieties; however, the specificity of these fragments has never been examined closely. Here, we particularly focused on N-glycoproteomics analysis and investigated fragmentation specificity using synthetic stable isotope-labeled N-glycopeptide standards. These standards were isotopically labeled at the reducing terminal GlcNAc, which allowed us to resolve fragments produced by the oligomannose core moiety and fragments generated from outer antennary structures. Our research identified the potential for false-positive structure assignments due to the occurrence of "Ghost" fragments resulting from single glyco unit rearrangement or mannose core fragmentation within the collision cell. To mitigate this issue, we have established a minimal intensity threshold for these fragments to prevent misidentification of structure-specific fragments in glycoproteomics analysis. Our findings provide a crucial step forward in the quest for more accurate and reliable glycoproteomics measurements.
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Glicoproteínas , Espectrometría de Masas en Tándem , Espectrometría de Masas en Tándem/métodos , Glicoproteínas/química , Polisacáridos/química , Glicopéptidos/análisis , Iones/químicaRESUMEN
Mass spectrometry (MS) can unlock crucial insights into the intricate world of glycosylation analysis. Despite its immense potential, the qualitative and quantitative analysis of isobaric glycopeptide structures remains one of the most daunting hurdles in the field of glycoproteomics. The ability to distinguish between these complex glycan structures poses a significant challenge, hindering our ability to accurately measure and understand the role of glycoproteins in biological systems. A few recent publications described the use of collision energy (CE) modulation to improve structural elucidation, especially for qualitative purposes. Different linkages of glycan units usually demonstrate different stabilities under CID/HCD fragmentation conditions. Fragmentation of the glycan moiety produces low molecular weight ions (oxonium ions) that can serve as a structure-specific signature for specific glycan moieties, however, specificity of these fragments has never been examined closely. Here, we investigated fragmentation specificity using synthetic stable isotope-labelled glycopeptide standards. These standards were isotopically labelled at the reducing terminal GlcNAc, which allowed us to resolve fragments produced by oligomannose core moiety and fragments generated from outer antennary structures. Our research identified the potential for false positive structure assignments due to the occurrence of "Ghost" fragments resulting from single glyco unit rearrangement or mannose core fragmentation within the collision cell. To mitigate this issue, we have established a minimal intensity threshold for these fragments to prevent the misidentification of structure-specific fragments in glycoproteomics analysis. Our findings provide a crucial step forward in the quest for more accurate and reliable glycoproteomics measurements.
RESUMEN
Targeted quantification of proteins is a standard methodology with broad utility, but targeted quantification of glycoproteins has not reached its full potential. The lack of optimized workflows and isotopically labeled standards limits the acceptance of glycoproteomics quantification. In this work, we introduce an efficient and streamlined chemoenzymatic synthesis of a library of isotopically labeled glycopeptides of IgG1 which we use for quantification in an energy optimized LC-MS/MS-PRM workflow. Incorporation of the stable isotope labeled N-acetylglucosamine enables an efficient monitoring of all major fragment ions of the glycopeptides generated under the soft higher-energy C-trap dissociation (HCD) conditions, which reduces the coefficients of variability (CVs) of the quantification to 0.7-2.8%. Our results document, for the first time, that the workflow using a combination of stable isotope labeled standards with intrascan normalization enables quantification of the glycopeptides by an electron transfer dissociation (ETD) workflow, as well as the HCD workflow, with the highest sensitivity compared to traditional workflows. This was exemplified by a rapid quantification (13 min) of IgG1 Fc glycoforms from COVID-19 patients.
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COVID-19 , Inmunoglobulina G , Humanos , Espectrometría de Masas en Tándem/métodos , Glicopéptidos , Cromatografía Liquida/métodosRESUMEN
Multivalent interactions are a key characteristic of protein-carbohydrate recognition. Phospholipid-based liposomes have been explored as a popular platform for multivalent presentation of glycans, but this platform has been plagued by the instability of typical liposomal formulations in biological media. We report here the exploitation of catanionic vesicles as a stable lipid-based nanoparticle scaffold for displaying large natural N-glycans as multivalent ligands. Hydrophobic insertion of lipidated N-glycans into the catanionic vesicle bilayer was optimized to allow for high-density display of structurally diverse N-glycans on the outer membrane leaflet. In an enzyme-linked competitive lectin-binding assay, the N-glycan-coated vesicles demonstrated a clear clustering glycoside effect, with significantly enhanced affinity for the corresponding lectins including Sambucus nigra agglutinin (SNA), concanavalin A (ConA), and human galectin-3, in comparison with their respective natural N-glycan ligands. Our results showed that relatively low density of high-mannose and sialylated complex type N-glycans gave the maximal clustering effect for binding to ConA and SNA, respectively, while relatively high-density display of the asialylated complex type N-glycan provided maximal clustering effects for binding to human galectin 3. Moreover, we also observed a macromolecular crowding effect on the binding of ConA to high-mannose N-glycans when catanionic vesicles bearing mixed high-mannose and complex-type N-glycans were used. The N-glycan-coated catanionic vesicles are stable and easy to formulate with varied density of ligands, which could serve as a feasible vehicle for drug delivery and as potent inhibitors for intervening protein-carbohydrate interactions implicated in disease.
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Carbohidratos , Manosa , Humanos , Ligandos , Carbohidratos/química , Polisacáridos/química , ProteínasRESUMEN
Protein glycosylation is a crucial mediator of biological functions and is tightly regulated in health and disease. However, interrogating complex protein glycoforms is challenging, as current lectin tools are limited by cross-reactivity while mass spectrometry typically requires biochemical purification and isolation of the target protein. Here, we describe a method to identify and characterize a class of nanobodies that can distinguish glycoforms without reactivity to off-target glycoproteins or glycans. We apply this technology to immunoglobulin G (IgG) Fc glycoforms and define nanobodies that specifically recognize either IgG lacking its core-fucose or IgG bearing terminal sialic acid residues. By adapting these tools to standard biochemical methods, we can clinically stratify dengue virus and SARS-CoV-2 infected individuals based on their IgG glycan profile, selectively disrupt IgG-Fcγ receptor binding both in vitro and in vivo, and interrogate the B cell receptor (BCR) glycan structure on living cells. Ultimately, we provide a strategy for the development of reagents to identify and manipulate IgG Fc glycoforms.
Asunto(s)
COVID-19 , Anticuerpos de Dominio Único , Humanos , Inmunoglobulina G/metabolismo , SARS-CoV-2 , Fragmentos Fc de Inmunoglobulinas/metabolismo , Polisacáridos/metabolismoRESUMEN
Targeted quantification of proteins is a standard methodology with broad utility, but targeted quantification of glycoproteins has not reached its full potential. The lack of optimized workflows and isotopically labeled standards limits the acceptance of glycoproteomics quantification. In this paper, we introduce an efficient and streamlined chemoenzymatic synthesis of a library of isotopically labeled glycopeptides of IgG1 which we use for quantification in an energy optimized LC-MS/MS-PRM workflow. Incorporation of the stable isotope labeled N-acetylglucosamine enables an efficient monitoring of all major fragment ions of the glycopeptides generated under the soft collision induced dissociation (CID) conditions which reduces the CVs of the quantification to 0.7-2.8%. Our results document, for the first time, that the workflow using a combination of stable isotope labeled standards with intra-scan normalization enables quantification of the glycopeptides by an electron transfer dissociation (ETD) workflow as well as the CID workflow with the highest sensitivity compared to traditional workflows., This was exemplified by a rapid quantification (13-minute) of IgG1 Fc glycoforms from COVID-19 patients.
RESUMEN
Sulfation is a common modification of glycans and glycoproteins. Sulfated N-glycans have been identified in various glycoproteins and implicated for biological functions, but in vitro synthesis of structurally well-defined full length sulfated N-glycans remains to be described. We report here the first in vitro enzymatic sulfation of biantennary complex type N-glycans by recombinant human CHST2 (GlcNAc-6-O-sulfotransferase 1, GlcNAc6ST-1). We found that the sulfotransferase showed high antennary preference and could selectively sulfate the GlcNAc moiety located on the Manα1,3Man arm of the biantennary N-glycan. The glycan chain was further elongated by bacterial ß1,4 galactosyltransferase from Neiserria meningitidis and human ß1,4 galactosyltransferase IVï¼B4GALT4ï¼, which led to the formation of different sulfated N-glycans. Using rituximab as a model IgG antibody, we further demonstrated that the sulfated N-glycans could be efficiently transferred to an intact antibody by using a chemoenzymatic Fc glycan remodeling method, providing homogeneous sulfated glycoforms of antibodies. Preliminary binding analysis indicated that sulfation did not affect the apparent affinity of the antibody for FcγIIIa receptor.
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Sulfatos , Sulfotransferasas , Galactosiltransferasas , Glicoproteínas , Humanos , Inmunoglobulina G , Polisacáridos/metabolismo , Sulfotransferasas/metabolismo , Carbohidrato SulfotransferasasRESUMEN
The plant-derived macrocyclic resin glycoside ipomoeassin F (Ipom-F) binds to Sec61α and significantly disrupts multiple aspects of Sec61-mediated protein biogenesis at the endoplasmic reticulum, ultimately leading to cell death. However, extensive assessment of Ipom-F as a molecular tool and a therapeutic lead is hampered by its limited production scale, largely caused by intramolecular assembly of the macrocyclic ring. Here, using in vitro and/or in cellula biological assays to explore the first series of ring-opened analogues for the ipomoeassins, and indeed all resin glycosides, we provide clear evidence that macrocyclic integrity is not required for the cytotoxic inhibition of Sec61-dependent protein translocation by Ipom-F. Furthermore, our modeling suggests that open-chain analogues of Ipom-F can interact with multiple sites on the Sec61α subunit, most likely located at a previously identified binding site for mycolactone and/or the so-called lateral gate. Subsequent in silico-aided design led to the discovery of the stereochemically simplified analogue 3 as a potent, alternative lead compound that could be synthesized much more efficiently than Ipom-F and will accelerate future ipomoeassin research in chemical biology and drug discovery. Our work may also inspire further exploration of ring-opened analogues of other resin glycosides.
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Antineoplásicos , Glicoconjugados , Antineoplásicos/química , Glicoconjugados/química , Glicósidos/farmacología , Canales de Translocación SEC/metabolismoRESUMEN
Recent observations that abiotic materials can engage in redox-based interactive communication motivates the search for new redox-active materials. Here we fabricated a hydrogel from a four-armed thiolated polyethylene glycol (PEG-SH) and the bacterial metabolite, pyocyanin (PYO). We show that: (i) the PYO-PEG hydrogel is reversibly redox-active; (ii) the molecular-switching and directed electron flow within this PYO-PEG hydrogel requires both a thermodynamic driving force (i.e., potential difference) and diffusible electron carriers that serve as nodes in a redox network; (iii) this redox-switching and electron flow is controlled by the redox network's topology; and (iv) the ability of the PYO-PEG hydrogel to "transmit" electrons to a second insoluble redox-active material (i.e., a catechol-PEG hydrogel) is context-dependent (i.e., dependent on thermodynamic driving forces and appropriate redox shuttles). These studies provide an experimental demonstration of important features of redox-communication and also suggest technological opportunities for the fabrication of interactive materials.
RESUMEN
N-Glycosylation plays an important role in many biological recognition processes. However, very few N-glycan-specific antibodies are available for functional studies and potentially for therapeutic development. In this study, we sought to synthesize bacteriophage Qß conjugates with representative N-glycans and investigate their immunogenicity for raising N-glycan-specific antibodies. An array of Qß glycoconjugates bearing five different human N-glycans and two different chemical linkers were synthesized, and the immunization of the N-glycan-Qß conjugates was performed in mice. We found that the N-glycan-Qß conjugates raised significant IgG antibodies that recognize N-glycans, but, surprisingly, most of the glycan-dependent antibodies were directed to the shared chitobiose core and were nonspecific for respective N-glycan structures. The linker chemistry was found to affect antibody specificity with adipic acid-linked N-glycan-Qß immunogens raising antibodies capable of recognizing both the N-acetylglucosamine (GlcNAc) moieties of the chitobiose core. In contrast, antibodies raised by N-glycan-Qß immunogens with a triazole linker preferentially recognized the innermost N-acetylglucosamine moiety at the reducing end. We also found that sialylation of the N-glycans significantly suppressed the immune response. Furthermore, the N-glycan-Qß immunogens with an adipic acid linker elicited higher glycan-specific antibody titers than the N-glycan-triazole-Qß immunogens. These findings delineate several challenges in eliciting mammalian N-glycan-specific antibodies through the conventional glycoconjugate vaccine design and immunization.
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Acetilglucosamina , Formación de Anticuerpos , Allolevivirus/química , Animales , Antígenos , Disacáridos , Glicoconjugados , Humanos , Mamíferos , Ratones , Polisacáridos/química , TriazolesRESUMEN
Monoclonal antibodies (mAbs) are one of the most rapidly growing drug classes used for the treatment of cancer, infectious and autoimmune diseases. Complement-dependent cytotoxicity (CDC) is one of the effector functions for antibodies to deplete target cells. We report here an efficient chemoenzymatic synthesis of structurally well-defined conjugates of a monoclonal antibody with a rhamnose- and an αGal trisaccharide-cluster to recruit natural anti-rhamnose and anti-αGal antibodies, respectively, to enhance the CDC-dependent targeted cell killing. The synthesis was achieved by using a modular antibody Fc-glycan remodeling method that includes site-specific chemoenzymatic Fc-glycan functionalization and subsequent click conjugation of synthetic rhamnose- and αGal trisaccharide-cluster to provide the respective homogeneous antibody conjugates. Cell-based assays indicated that the antibody-rhamnose cluster conjugates could mediate potent CDC activity for targeted cancer cell killing and showed much more potent efficacy than the antibody-αGal trisaccharide cluster conjugates for CDC effects.
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Inmunoconjugados , Ramnosa , Anticuerpos Monoclonales , Apoptosis , Fragmentos Fc de InmunoglobulinasRESUMEN
The broadly neutralizing antibody PG9 recognizes a unique glycopeptide epitope in the V1V2 domain of HIV-1 gp120 envelope glycoprotein. The present study describes the design, synthesis, and antibody-binding analysis of HIV-1 V1V2 glycopeptide-Qß conjugates as a mimic of the proposed neutralizing epitope of PG9. The glycopeptides were synthesized using a highly efficient chemoenzymatic method. The alkyne-tagged glycopeptides were then conjugated to the recombinant bacteriophage (Qß), a virus-like nanoparticle, through a click reaction. Antibody-binding analysis indicated that the synthetic glycoconjugates showed significantly enhanced affinity for antibody PG9 compared with the monomeric glycopeptides. It was also shown that the affinity of the Qß-conjugates for antibody PG9 was dependent on the density of the glycopeptide antigen display. The glycopeptide-Qß conjugates synthesized represent a promising candidate of HIV-1 vaccine.
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Allolevivirus/inmunología , Glicopéptidos/inmunología , Infecciones por VIH/prevención & control , VIH-1/inmunología , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Vacunas contra el SIDA/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Antígenos/inmunología , Epítopos/genética , Epítopos/inmunología , Glicopéptidos/genética , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/patogenicidad , Humanos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fagocitosis/inmunologíaRESUMEN
Functionalization of therapeutic lysosomal enzymes with mannose-6-phosphate (M6P) glycan ligands represents a major strategy for enhancing the cation-independent M6P receptor (CI-MPR)-mediated cellular uptake, thus improving the overall therapeutic efficacy of the enzymes. However, the minimal high-affinity M6P-containing N-glycan ligands remain to be identified and their efficient and site-selective conjugation to therapeutic lysosomal enzymes is a challenging task. We report here the chemical synthesis of truncated M6P-glycan oxazolines and their use for enzymatic glycan remodeling of recombinant human acid α-glucosidase (rhGAA), an enzyme used for treatment of Pompe disease which is a disorder caused by a deficiency of the glycogen-degrading lysosomal enzyme. Structure-activity relationship studies identified M6P tetrasaccharide oxazoline as the minimal substrate for enzymatic transglycosylation yielding high-affinity M6P glycan ligands for the CI-MPR. Taking advantage of the substrate specificity of endoglycosidases Endo-A and Endo-F3, we found that Endo-A and Endo-F3 could efficiently deglycosylate the respective high-mannose and complex type N-glycans in rhGAA and site-selectively transfer the synthetic M6P N-glycan to the deglycosylated rhGAA without product hydrolysis. This discovery enabled a highly efficient one-pot deglycosylation/transglycosylation strategy for site-selective M6P-glycan remodeling of rhGAA to obtain a more homogeneous product. The Endo-A and Endo-F3 remodeled rhGAAs maintained full enzyme activity and demonstrated 6- and 20-fold enhanced binding affinities for CI-MPR receptor, respectively. Using an in vitro cell model system for Pompe disease, we demonstrated that the M6P-glycan remodeled rhGAA greatly outperformed the commercial rhGAA (Lumizyme) and resulted in the reversal of cellular pathology. This study provides a general and efficient method for site-selective M6P-glycan remodeling of recombinant lysosomal enzymes to achieve enhanced M6P receptor binding and cellular uptake, which could lead to improved overall therapeutic efficacy of enzyme replacement therapy.
RESUMEN
Antibody-drug conjugates (ADCs) are an important class of therapeutic agents that harness the highly specific antigen targeting property of antibodies to deliver toxic drugs for targeted cell killing. Site-specific conjugation methods are highly desirable for constructing homogeneous ADCs that possess a well-defined antibody-to-drug ratio, stability, ideal pharmacological profile, and optimal therapeutic index. We report here a facile synthesis of functionalized glycan oxazolines from free sialoglycans that are key donor substrates for enzymatic Fc glycan remodeling and the application of an efficient endoglycosidase mutant (Endo-S2 D184M) for site-specific glycan transfer to construct homogeneous ADCs. We found that by a sequential use of two coupling reagents under optimized conditions, free sialoglycans could be efficiently converted to selectively functionalized glycan oxazolines carrying azide-, cyclopropene-, and norbornene-tags, respectively, in excellent yield and in a simple one-pot manner. We further demonstrated that the recently reported Endo-S2 D184 M mutant was highly efficient for Fc glycan remodeling with the selectively modified glycan oxazolines to introduce tags into an antibody, which required a significantly smaller amount of glycan oxazolines and a much shorter reaction time than that of the Endo-S D233Q-catalyzed reaction, thus minimizing the side reactions. Finally homogeneous ADCs were constructed with three different click reactions. The resulting ADCs showed excellent serum stability, and in vitro cytotoxicity assays indicated that all the three ADCs generated from the distinct click reactions possessed potent and comparable cytotoxicity for targeted cancer cell killing.
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Inmunoconjugados/química , Inmunoconjugados/farmacología , Polisacáridos/química , Receptor ErbB-2/inmunología , Trastuzumab/química , Línea Celular Tumoral , Supervivencia Celular , Química Clic , Humanos , Estructura Molecular , Trastuzumab/metabolismoRESUMEN
Glycosylation plays important roles in SARS-CoV-2 infection. We describe here a facile chemoenzymatic synthesis of core-fucosylated N-glycopeptides derived from the SARS-CoV-2 Spike protein and their binding with glycan-dependent neutralizing antibody S309 and human lectin CLEC4G. The synthetic glycopeptides provide tools for further functional characterization of viral glycosylation.
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Glicopéptidos/síntesis química , Glicopéptidos/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Anticuerpos Neutralizantes/inmunología , Técnicas de Química Sintética , Glicopéptidos/química , Glicopéptidos/inmunología , Glicosilación , Polisacáridos/metabolismoRESUMEN
The heterotrimeric Sec61 complex is a major site for the biogenesis of transmembrane proteins (TMPs), accepting nascent TMP precursors that are targeted to the endoplasmic reticulum (ER) by the signal recognition particle (SRP). Unlike most single-spanning membrane proteins, the integration of type III TMPs is completely resistant to small molecule inhibitors of the Sec61 translocon. Using siRNA-mediated depletion of specific ER components, in combination with the potent Sec61 inhibitor ipomoeassin F (Ipom-F), we show that type III TMPs utilise a distinct pathway for membrane integration at the ER. Hence, following SRP-mediated delivery to the ER, type III TMPs can uniquely access the membrane insertase activity of the ER membrane complex (EMC) via a mechanism that is facilitated by the Sec61 translocon. This alternative EMC-mediated insertion pathway allows type III TMPs to bypass the Ipom-F-mediated blockade of membrane integration that is seen with obligate Sec61 clients.
Asunto(s)
Retículo Endoplásmico/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Biosíntesis de Proteínas , Canales de Translocación SEC/metabolismo , Animales , Retículo Endoplásmico/efectos de los fármacos , Glicoconjugados/farmacología , Células HeLa , Humanos , Immunoblotting , Membranas Intracelulares/efectos de los fármacos , Modelos Biológicos , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , Canales de Translocación SEC/genética , Partícula de Reconocimiento de Señal/metabolismoRESUMEN
Core fucosylation is the attachment of an α-1,6-fucose moiety to the innermost N-acetyl glucosamine (GlcNAc) in N-glycans in mammalian systems. It plays a pivotal role in modulating the structural and biological functions of glycoproteins including therapeutic antibodies. Yet, few α-l-fucosidases appear to be capable of removing core fucose from intact glycoproteins. This paper describes a comparative study of the substrate specificity and relative activity of the human α-l-fucosidase (FucA1) and two bacterial α-l-fucosidases, the AlfC from Lactobacillus casei and the BfFuc from Bacteroides fragilis. This study was enabled by the synthesis of an array of structurally well-defined core-fucosylated substrates, including core-fucosylated N-glycopeptides and a few antibody glycoforms. It was found that AlfC and BfFuc could not remove core fucose from intact full-length N-glycopeptides or N-glycoproteins but could hydrolyze only the truncated Fucα1,6GlcNAc-peptide substrates. In contrast, the human α-l-fucosidase (FucA1) showed low activity on truncated Fucα1,6GlcNAc substrates but was able to remove core fucose from intact and full-length core-fucosylated N-glycopeptides and N-glycoproteins. In addition, it was found that FucA1 was the only α-l-fucosidase that showed low but apparent activity to remove core fucose from intact IgG antibodies. The ability of FucA1 to defucosylate intact monoclonal antibodies reveals an opportunity to evolve the human α-l-fucosidase for direct enzymatic defucosylation of therapeutic antibodies to improve their antibody-dependent cellular cytotoxicity.