RESUMEN
The immune system produces a diverse collection of antiglycan antibodies that are critical for host defense. At present, however, we know very little about the binding properties, origins, and sequences of these antibodies because of a lack of access to a variety of defined individual antibodies. To address this challenge, we used a glycan microarray with over 800 different components to screen a panel of 516 human monoclonal antibodies that had been randomly cloned from different B-cell subsets originating from healthy human subjects. We obtained 26 antiglycan antibodies, most of which bound microbial carbohydrates. The majority of the antiglycan antibodies identified in the screen displayed selective binding for specific glycan motifs on our array and lacked polyreactivity. We found that antiglycan antibodies were about twice as likely than expected to originate from IgG+ memory B cells, whereas none were isolated from naïve, early emigrant, or immature B cells. Therefore, our results indicate that certain B-cell subsets in our panel are enriched in antiglycan antibodies, and IgG+ memory B cells may be a promising source of such antibodies. Furthermore, some of the newly identified antibodies bound glycans for which there are no reported monoclonal antibodies available, and these may be useful as research tools, diagnostics, or therapeutic agents. Overall, the results provide insight into the types and properties of antiglycan antibodies produced by the human immune system and a framework for the identification of novel antiglycan antibodies in the future.
Asunto(s)
Anticuerpos Monoclonales , Polisacáridos , Humanos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Carbohidratos , Inmunoglobulina G/inmunología , Análisis por Micromatrices , Polisacáridos/metabolismo , Células B de Memoria/inmunologíaRESUMEN
Germline antibodies, the initial set of antibodies produced by the immune system, are critical for host defense, and information about their binding properties can be useful for designing vaccines, understanding the origins of autoantibodies, and developing monoclonal antibodies. Numerous studies have found that germline antibodies are polyreactive with malleable, flexible binding pockets. While insightful, it remains unclear how broadly this model applies, as there are many families of antibodies that have not yet been studied. In addition, the methods used to obtain germline antibodies typically rely on assumptions and do not work well for many antibodies. Herein, we present a distinct approach for isolating germline antibodies that involves immunizing activation-induced cytidine deaminase (AID) knockout mice. This strategy amplifies antigen-specific B cells, but somatic hypermutation does not occur because AID is absent. Using synthetic haptens, glycoproteins, and whole cells, we obtained germline antibodies to an assortment of clinically important tumor-associated carbohydrate antigens, including Lewis Y, the Tn antigen, sialyl Lewis C, and Lewis X (CD15/SSEA-1). Through glycan microarray profiling and cell binding, we demonstrate that all but one of these germline antibodies had high selectivity for their glycan targets. Using molecular dynamics simulations, we provide insights into the structural basis of glycan recognition. The results have important implications for designing carbohydrate-based vaccines, developing anti-glycan monoclonal antibodies, and understanding antibody evolution within the immune system.
Asunto(s)
Anticuerpos Monoclonales , Antígenos de Carbohidratos Asociados a Tumores , Animales , Anticuerpos Monoclonales/química , Biomarcadores de Tumor , Carbohidratos , Células Germinativas , Ratones , Ratones Noqueados , Polisacáridos/químicaRESUMEN
Glycosylation is a vital post-translational modification involved in a range of biological processes including protein folding, signaling, and cell-cell interactions. In 2011, a new type of O-linked glycosylation was discovered, wherein the side-chain oxygen of tyrosine is modified with a GalNAc residue (GalNAc-Tyr). At present, very little is known about GalNAc-Tyr prevalence, function, or biosynthesis. Herein, we describe the design and synthesis of a GalNAc-Tyr-derived hapten and its use in generating a GalNAc-Tyr selective monoclonal antibody. The antibody, G10C, has an unusually high affinity (app KD = 100 pM) and excellent selectivity for GalNAc-Tyr. We also obtained a crystal structure of the G10C Fab region in complex with 4-nitrophenyl-N-acetyl-α-d-galactosaminide (a small molecule mimic of GalNAc-Tyr) providing insights into the structural basis for high affinity and selectivity. Using this antibody, we discovered that GalNAc-Tyr is widely expressed in most human tissues, indicating that it is a ubiquitous and underappreciated post-translational modification. Localization to specific cell types and organ substructures within those tissues indicates that GalNAc-Tyr is likely regulated in a cell-specific manner. GalNAc-Tyr was also observed in a variety of cell lines and primary cells but was only present on the external cell surface in certain cancer cell lines, suggesting that GalNAc-Tyr localization may be altered in cancer cells. Collectively, the results shed new light on this under-studied form of glycosylation and provide access to new tools that will enable expanded biochemical and clinical investigations.
Asunto(s)
Anticuerpos Monoclonales , N-Acetilgalactosaminiltransferasas , Anticuerpos Monoclonales/metabolismo , Línea Celular , Glicosilación , Humanos , N-Acetilgalactosaminiltransferasas/metabolismo , Tirosina/metabolismoRESUMEN
Carbohydrate-binding antibodies play diverse and critical roles in human health. Endogenous carbohydrate-binding antibodies that recognize bacterial, fungal, and other microbial carbohydrates prevent systemic infections and help maintain microbiome homeostasis. Anti-glycan antibodies can have both beneficial and detrimental effects. For example, alloantibodies to ABO blood group carbohydrates can help reduce the spread of some infectious diseases, but they also impose limitations for blood transfusions. Antibodies that recognize self-glycans can contribute to autoimmune diseases, such as Guillain-Barre syndrome. In addition to endogenous antibodies that arise through natural processes, a variety of vaccines induce anti-glycan antibodies as a primary mechanism of protection. Some examples of approved carbohydrate-based vaccines that have had a major impact on human health are against pneumococcus, Haemophilus influeanza type b, and Neisseria meningitidis. Monoclonal antibodies specifically targeting pathogen associated or tumor associated carbohydrate antigens (TACAs) are used clinically for both diagnostic and therapeutic purposes. This review aims to highlight some of the well-studied and critically important applications of anti-carbohydrate antibodies.
Asunto(s)
Síndrome de Guillain-Barré/inmunología , Infecciones por Haemophilus/inmunología , Meningitis Meningocócica/inmunología , Neumonía Neumocócica/inmunología , Polisacáridos/inmunología , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/uso terapéutico , Autoanticuerpos/biosíntesis , Autoanticuerpos/sangre , Vacunas Bacterianas/biosíntesis , Vacunas Bacterianas/uso terapéutico , Secuencia de Carbohidratos , Síndrome de Guillain-Barré/patología , Infecciones por Haemophilus/microbiología , Infecciones por Haemophilus/prevención & control , Vacunas contra Haemophilus/biosíntesis , Vacunas contra Haemophilus/uso terapéutico , Haemophilus influenzae/inmunología , Humanos , Meningitis Meningocócica/microbiología , Meningitis Meningocócica/prevención & control , Neisseria meningitidis/inmunología , Vacunas Neumococicas/biosíntesis , Vacunas Neumococicas/uso terapéutico , Neumonía Neumocócica/microbiología , Neumonía Neumocócica/prevención & control , Polisacáridos/antagonistas & inhibidores , Polisacáridos/química , Streptococcus pneumoniae/inmunologíaRESUMEN
Protein-carbohydrate interactions play significant roles in a wide variety of biological systems. Glycan microarrays are commonly utilized to interrogate the selectivity, sensitivity, and breadth of these complex protein-carbohydrate interactions. During the past two decades, numerous distinct glycan microarray platforms have been developed, each assembled from a variety of slide-surface chemistries, glycan-attachment chemistries, glycan presentations, linkers, and glycan densities. Comparative analyses of glycan microarray data have shown that while many protein-carbohydrate interactions behave predictably across microarrays, there are instances when various array formats produce different results. For optimal construction and use of this technology, it is important to understand sources of variances across array platforms. In this study, we performed a systematic comparison of microarray data from 8 lectins across a range of concentrations on the CFG and neoglycoprotein array platforms. While there was good general agreement on the binding specificity of the lectins on the two arrays, there were some cases of large discrepancies. Differences in glycan density and linker composition contributed significantly to variability. The results provide insights for interpreting microarray data and designing future glycan microarrays.
Asunto(s)
Lectinas/metabolismo , Análisis por Micromatrices/métodos , Polisacáridos/metabolismo , Animales , Modelos Moleculares , Polisacáridos/química , Unión ProteicaRESUMEN
Carboxylic acid-functionalized Pd and Pt PNNNP pincer complexes were used for the assembly of two porous Zr metal-organic frameworks (MOFs), 2-PdX and 2-PtX. Powder X-ray diffraction analysis shows that the new MOFs adopt cubic framework structures similar to the previously reported Zr6O4(OH)4[(POCOP)PdX]3, [POCOP = 2,6-(OPAr2)2C6H3); Ar = p-C6H4CO2-, X = Cl-, I-] (1-PdX). Elemental analysis and spectroscopic characterization indicate the presence of missing linker defects, and 2-PdX and 2-PtX were formulated as Zr6O4(OH)4(OAc)2.4[M(PNNNP)X]2.4 [M = Pd, Pt; PNNNP = 2,6-(HNPAr2)2C5H3N; Ar = p-C6H4CO2-; X = Cl-, I-]. Postsynthetic halide ligand exchange reactions were carried out by treating 2-PdX with Ag(O3SCF3) or NaI followed by PhI(O2CCF3)2. The latter strategy proved to be more effective at activating the MOF for the catalytic intramolecular hydroamination of an o-substituted alkynyl aniline, underscoring the advantage of using halide exchange reagents that produce soluble byproducts.
RESUMEN
SELMA (SELection with Modified Aptamers) is a directed evolution method which can be used to develop DNA-supported clusters of carbohydrates in which the geometry of clustering is optimized for strong recognition by a lectin of interest. Herein, we report a modification of SELMA which results in glycoclusters which achieve dramatically stronger target recognition (100-fold) with dramatically fewer glycans (2-3-fold). Our first applications of SELMA yielded clusters of 5-10 oligomannose glycans which were recognized by broadly neutralizing HIV antibody 2G12 with moderate affinities (150-500 nM Kd's). In the present manuscript, we report glycoclusters containing just 3-4 glycans, which are recognized by 2G12 with Kd's as low as 1.7 nM. These glycoclusters are recognized by 2G12 as tightly as is the HIV envelope protein gp120, and they are the first constructs to achieve this tight recognition with the minimal number of Man9 units (3-4) necessary to occupy the binding sites on 2G12. They are thus of great interest as immunogens which might elicit broadly neutralizing antibodies against HIV.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , ADN/química , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Manosa/análogos & derivados , Anticuerpos Monoclonales/química , Anticuerpos Neutralizantes/química , Secuencia de Bases , Sitios de Unión de Anticuerpos , Anticuerpos ampliamente neutralizantes , Anticuerpos Anti-VIH/química , VIH-1/inmunología , Calor , Humanos , Manosa/inmunología , Imitación Molecular , Datos de Secuencia MolecularRESUMEN
Herein, we report a method for in vitro selection of multivalent glycopeptides, combining mRNA display with incorporation of unnatural amino acids and "click" chemistry. We have demonstrated the use of this method to design potential glycopeptide vaccines against HIV. From libraries of ~10(13) glycopeptides containing multiple Man9 glycan(s), we selected variants that bind to HIV broadly neutralizing antibody 2G12 with picomolar to low nanomolar affinity. This is comparable to the strength of the natural 2G12-gp120 interaction, and is the strongest affinity achieved to date with constructs containing 3-5 glycans. These glycopeptides are therefore of great interest in HIV vaccine design.
Asunto(s)
Glicopéptidos/química , Anticuerpos Anti-VIH/química , Vacunas contra el SIDA/química , Vacunas contra el SIDA/inmunología , Química Clic , Glicopéptidos/síntesis química , Glicopéptidos/inmunología , Anticuerpos Anti-VIH/inmunología , Estructura MolecularRESUMEN
Anti-glycan monoclonal antibodies have important applications in human health and basic research. Therapeutic antibodies that recognize cancer- or pathogen-associated glycans have been investigated in numerous clinical trials, resulting in two FDA-approved biopharmaceuticals. Anti-glycan antibodies are also utilized to diagnose, prognosticate, and monitor disease progression, as well as to study the biological roles and expression of glycans. High-quality anti-glycan mAbs are still in limited supply, highlighting the need for new technologies for anti-glycan antibody discovery. This review discusses anti-glycan monoclonal antibodies with applications to basic research, diagnostics, and therapeutics, focusing on recent advances in mAbs targeting cancer- and infectious disease-associated glycans.
Asunto(s)
Anticuerpos Monoclonales , Neoplasias , Humanos , Anticuerpos Monoclonales/uso terapéutico , Polisacáridos/metabolismoRESUMEN
Glycan microarrays provide a high-throughput technology for rapidly profiling interactions between carbohydrates and glycan-binding proteins (GBPs). Use of glycan microarrays involves several general steps, including construction of the microarray, carrying out the assay, detection of binding events, and analysis of the results. While multiple platforms have been developed to construct microarrays, most utilize fluorescence for detection of binding events. This chapter describes methods to acquire and process microarray images, including generating GAL files, imaging of the slide, aligning the grid, detecting problematic spots, and evaluating the quality of the data. The chapter focuses on processing our neoglycoprotein microarrays, but many of the lessons we have learned are applicable to other array formats.
Asunto(s)
Carbohidratos , Polisacáridos , Proteínas Portadoras/metabolismo , Análisis por Micromatrices/métodos , Polisacáridos/metabolismoRESUMEN
The high mannose patch (HMP) of the HIV envelope protein (Env) is the structure most frequently targeted by broadly neutralizing antibodies; therefore, many researchers have attempted to use mimics of this region as a vaccine immunogen. In our previous efforts, vaccinating rabbits with evolved HMP mimic glycopeptides containing Man9 resulted in an overall antibody response targeting the glycan core and linker rather than the full glycan or Manα1â2Man tips of Man9 glycans. A possible reason could be processing of our immunogen by host serum mannosidases. We sought to test whether more prolonged dosing could increase the antibody response to intact glycans, possibly by increasing the availability of intact Man9 to germinal centers. Here, we describe a study investigating the impact of immunization regimen on antibody response by testing immunogen delivery through bolus, an exponential series of mini doses, or a continuously infusing mini-osmotic pump. Our results indicate that, with our glycopeptide immunogens, standard bolus immunization elicited the strongest HIV Env-binding antibody response, even though higher overall titers to the glycopeptide were elicited by the exponential and pump regimens. Antibody selectivity for intact glycan was, if anything, slightly better in the bolus-immunized animals.
Asunto(s)
Vacunas contra el SIDA/metabolismo , Glicopéptidos/química , Anticuerpos Anti-VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/química , Oligosacáridos/química , Vacunas Conjugadas/metabolismo , Animales , Anticuerpos Neutralizantes , Formación de Anticuerpos , Sitios de Unión , Glicosilación , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/orina , Infecciones por VIH/prevención & control , Humanos , Inmunización , Manosidasas/metabolismo , Oligosacáridos/orina , Unión Proteica , Conformación Proteica , Conejos , Bibliotecas de Moléculas Pequeñas/química , VacunaciónRESUMEN
We have previously studied the generation of immune responses after vaccination with tumor-associated carbohydrate antigen (TACA)-containing glycopeptides from the tandem repeat (TR) sequence of MUC4, an aberrantly expressed mucin in pancreatic adenocarcinomas. A specific lead antigen from that study containing the Thomsen-Friedenreich TACA disaccharide facilitated the pursuit of a monoclonal antibody to this synthetic hapten. Initial evaluation of polyclonal antiserum resulting from immunization with a KLH conjugate of this glycopeptide into rabbits showed high titer antibodies by ELISA assays, and selective immunoreactivity with MUC4+ cells by western blot and flow cytometry techniques. Glycan microarray analysis showed an intriguing binding pattern where the antiserum showed near complete specificity for MUC4 TR glycopeptides and peptides, relative to all components on the array. Tissue staining also showed distinct tumor specificity to pancreatic tumor tissue in relation to normal pancreatic tissue, with a preference for more aggressive tumor foci. Based on this data, we produced a monoclonal antibody whose binding and reactivity profile was similar to that of the polyclonal serum, with the added benefit of being more specific for the N-terminal glycosylated peptide domain. This epitope represents a novel immunogen to potentially develop diagnostic antibodies or immunotherapies against various MUC4-positive cancers.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Glicopéptidos/inmunología , Mucina 4/inmunología , Neoplasias Pancreáticas/inmunología , Animales , Formación de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Células Cultivadas , Epítopos/inmunología , Inmunización/métodos , Inmunoterapia/métodos , Ratones , Ratones Endogámicos BALB C , Conejos , Vacunación/métodos , Neoplasias PancreáticasRESUMEN
Linalyl diphosphate (LPP) is the postulated intermediate in the enzymatic cyclization of monoterpenes catalyzed by terpene synthases. LPP is considered an obligate intermediate due to the conformationally restrictive trans-C2-C3 double bond of the substrate, geranyl diphosphate (GPP), which precludes the proper positioning of carbons C1 and C6 to enable cyclization. However, because of the complexity of potential carbocation-mediated rearrangements in these enzymatic reactions, it has proven difficult to directly demonstrate the formation of LPP despite significant efforts. Here we synthesized a fluorinated substrate analog, 8,9-difluorogeranyl diphosphate (DFGPP), which is designed to allow initial ionization/isomerization and form the fluorinated equivalent of LPP (DFLPP) while preventing the subsequent ionization/cyclization to produce the α-terpinyl cation. Steady-state kinetic studies with the model enzyme (+)-limonene synthase (LS) under catalytic conditions show that the cyclization of DFGPP is completely blocked and a single linear product, difluoromyrcene, is produced. When crystals of apo-LS are soaked with DFGPP under conditions limiting turnover of the enzyme, we show, using X-ray crystallography, that DFLPP is produced in the enzyme active site and trapped in the crystals. Clear electron density is observed in the active site of the enzyme, but it cannot be appropriately fit with a model for the DFGPP substrate analog, whereas it can accommodate an extended conformation of DFLPP. This result supports the current model for monoterpene cyclization by providing direct evidence of LPP as an intermediate.
Asunto(s)
Monoterpenos Acíclicos/química , Difosfatos/química , Diterpenos/química , Inhibidores Enzimáticos/química , Liasas Intramoleculares/antagonistas & inhibidores , Fosfatos de Poliisoprenilo/química , Dominio Catalítico , Citrus sinensis/enzimología , Cristalografía por Rayos X , Difosfatos/síntesis química , Diterpenos/síntesis química , Pruebas de Enzimas , Inhibidores Enzimáticos/síntesis química , Liasas Intramoleculares/químicaRESUMEN
Up to â¼20% of HIV-infected individuals eventually develop broadly neutralizing antibodies (bnAbs), and many of these antibodies (â¼40%) target a region of dense high-mannose glycosylation on gp120 of the HIV envelope protein, known as the "high-mannose patch" (HMP). Thus, there have been numerous attempts to develop glycoconjugate vaccine immunogens that structurally mimic the HMP and might elicit bnAbs targeting this conserved neutralization epitope. Herein, we report on the immunogenicity of glycopeptides, designed by in vitro selection, that bind tightly to anti-HMP antibody 2G12. By analyzing the fine carbohydrate specificity of rabbit antibodies elicited by these immunogens, we found that they differ from some natural human bnAbs, such as 2G12 and PGT128, in that they bind primarily to the core structures within the glycan, rather than to the Manα1 â 2Man termini (2G12) or to the whole glycan (PGT128). Antibody specificity for the glycan core may result from extensive serum mannosidase trimming of the immunogen in the vaccinated animals. This finding has broad implications for vaccine design aiming to target glycan-dependent HIV neutralizing antibodies.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Evolución Molecular Dirigida , Manosa/química , Azidas/química , ADN/química , ADN/metabolismo , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Humanos , Polisacáridos/química , Polisacáridos/metabolismo , TermodinámicaAsunto(s)
ADN/química , Evolución Molecular Dirigida/métodos , Diseño de Fármacos , Glicoconjugados , Anticuerpos Monoclonales/química , Azidas/química , Secuencia de Bases , Sitios de Unión , Anticuerpos ampliamente neutralizantes , Biblioteca de Genes , Glicoconjugados/síntesis química , Glicoconjugados/química , Glicosilación , Anticuerpos Anti-VIH , Proteína gp120 de Envoltorio del VIH/química , Ligandos , Manósidos/química , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
In vitro selection of nucleic acid aptamers, coined SELEX, has led to the discovery of novel therapeutics and aided in the structural and mechanistic understanding of many ligand-biomolecule interactions. A related method, selection with modified aptamers (SELMA), enables selection of DNA aptamers containing bases with a large modification that cannot undergo PCR. A key application of this method is the evolution of aptamers containing carbohydrate modifications. Carbohydrate-binding proteins normally require several copies of the carbohydrate moiety for strong recognition. Whereas it may be difficult to rationally design synthetic scaffolds that cluster glycans in the optimal spacing and orientation for target recognition, SELMA furnishes glycoaptamers with highly optimized glycan clustering, achieving low-nanomolar recognition. Although numerous applications can be envisioned, the protocols and discussions in this article describe procedures involved in applying SELMA to the discovery glycoDNAs that bind to the HIV broadly neutralizing antibody 2G12.