Publications by authors named "James C Paulson"

202 Publications

Site-specific O-glycosylation analysis of SARS-CoV-2 spike protein produced in insect and human cells.

bioRxiv 2021 Feb 4. Epub 2021 Feb 4.

Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S - insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complimentary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.
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http://dx.doi.org/10.1101/2021.02.03.429627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872350PMC
February 2021

Glycoengineering of NK Cells with Glycan Ligands of CD22 and Selectins for B-Cell Lymphoma Therapy.

Angew Chem Int Ed Engl 2021 Feb 14;60(7):3603-3610. Epub 2020 Dec 14.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, 92037, USA.

CD22, a member of Siglec family of sialic acid binding proteins, has restricted expression on B cells. Antibody-based agents targeting CD22 or CD20 on B lymphoma and leukemia cells exhibit clinical efficacy for treating these malignancies, but also attack normal B cells leading to immune deficiency. Here, we report a chemoenzymatic glycocalyx editing strategy to introduce high-affinity and specific CD22 ligands onto NK-92MI and cytokine-induced natural killer cells to achieve tumor-specific CD22 targeting. These CD22-ligand modified cells exhibited significantly enhanced tumor cell binding and killing in vitro without harming healthy B cells. For effective lymphoma cell killing in vivo, we further functionalized CD22 ligand-modified NK-92MI cells with the E-selectin ligand sialyl Lewis X to promote trafficking to bone marrow. The dual-functionalized cells resulted in the efficient suppression of B lymphoma in a xenograft model. Our results suggest that natural killer cells modified with glycan ligands to CD22 and selectins promote both targeted killing of B lymphoma cells and improved trafficking to sites where the cancer cells reside, respectively.
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http://dx.doi.org/10.1002/anie.202005934DOI Listing
February 2021

Adaptation of Influenza Viruses to Human Airway Receptors.

J Biol Chem 2020 11 3. Epub 2020 Nov 3.

Molecular Medicine, The Scripps Research Institute, United States.

Through annual epidemics and global pandemics, influenza A viruses (IAVs) remain a significant threat to human health as the leading cause of severe respiratory disease. Within the last century, four global pandemics have resulted from the introduction of novel IAVs into humans, with components of each originating from avian viruses. IAVs infect many avian species wherein they maintain a diverse natural reservoir, posing a risk to humans through the occasional emergence of novel strains with enhanced zoonotic potential. One natural barrier for transmission of avian IAVs into humans is the specificity of the receptor-binding protein, hemagglutinin (HA), that recognizes sialic acid-containing glycans on host cells. HAs from human IAVs exhibit "human-type" receptor specificity, binding exclusively to glycans on cells lining the human airway where terminal sialic acids are attached in the α2-6 configuration (NeuAcα2-6Gal). In contrast, HAs from avian viruses exhibit specificity for "avian-type" α2-3-linked (NeuAcα2-3Gal) receptors, and thus require adaptive mutations to bind human-type receptors. Since all human IAV pandemics can be traced to avian origins, there remains ever-present concern over emerging IAVs with human-adaptive potential that might lead to the next pandemic. This concern has been brought into focus through emergence of SARS-CoV-2, aligning both scientific and public attention to the threat of novel respiratory viruses from animal sources. In this review we summarize receptor-binding adaptations underlying the emergence of all prior IAV pandemics in humans, maintenance and evolution of human-type receptor specificity in subsequent seasonal IAVs, and potential for future human-type receptor adaptation in novel avian HAs.
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http://dx.doi.org/10.1074/jbc.REV120.013309DOI Listing
November 2020

Visualization of the HIV-1 Env glycan shield across scales.

Proc Natl Acad Sci U S A 2020 11 22;117(45):28014-28025. Epub 2020 Oct 22.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037;

The dense array of N-linked glycans on the HIV-1 envelope glycoprotein (Env), known as the "glycan shield," is a key determinant of immunogenicity, yet intrinsic heterogeneity confounds typical structure-function analysis. Here, we present an integrated approach of single-particle electron cryomicroscopy (cryo-EM), computational modeling, and site-specific mass spectrometry (MS) to probe glycan shield structure and behavior at multiple levels. We found that dynamics lead to an extensive network of interglycan interactions that drive the formation of higher-order structure within the glycan shield. This structure defines diffuse boundaries between buried and exposed protein surface and creates a mapping of potentially immunogenic sites on Env. Analysis of Env expressed in different cell lines revealed how cryo-EM can detect subtle changes in glycan occupancy, composition, and dynamics that impact glycan shield structure and epitope accessibility. Importantly, this identified unforeseen changes in the glycan shield of Env obtained from expression in the same cell line used for vaccine production. Finally, by capturing the enzymatic deglycosylation of Env in a time-resolved manner, we found that highly connected glycan clusters are resistant to digestion and help stabilize the prefusion trimer, suggesting the glycan shield may function beyond immune evasion.
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http://dx.doi.org/10.1073/pnas.2000260117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668054PMC
November 2020

Structural analysis of full-length SARS-CoV-2 spike protein from an advanced vaccine candidate.

Science 2020 11 20;370(6520):1089-1094. Epub 2020 Oct 20.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Vaccine efforts to combat the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the current coronavirus disease 2019 (COVID-19) pandemic, are focused on SARS-CoV-2 spike glycoprotein, the primary target for neutralizing antibodies. We performed cryo-election microscopy and site-specific glycan analysis of one of the leading subunit vaccine candidates from Novavax, which is based on a full-length spike protein formulated in polysorbate 80 detergent. Our studies reveal a stable prefusion conformation of the spike immunogen with slight differences in the S1 subunit compared with published spike ectodomain structures. We also observed interactions between the spike trimers, allowing formation of higher-order spike complexes. This study confirms the structural integrity of the full-length spike protein immunogen and provides a basis for interpreting immune responses to this multivalent nanoparticle immunogen.
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http://dx.doi.org/10.1126/science.abe1502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857404PMC
November 2020

Hemagglutinin Traits Determine Transmission of Avian A/H10N7 Influenza Virus between Mammals.

Cell Host Microbe 2020 10;28(4):602-613.e7

Department of Viroscience, Postgraduate School of Molecular Medicine, Erasmus MC University Medical Center, 3015GE, Rotterdam, the Netherlands. Electronic address:

In 2014, an outbreak of avian A/H10N7 influenza virus occurred among seals along North-European coastal waters, significantly impacting seal populations. Here, we examine the cross-species transmission and mammalian adaptation of this influenza A virus, revealing changes in the hemagglutinin surface protein that increase stability and receptor binding. The seal A/H10N7 virus was aerosol or respiratory droplet transmissible between ferrets. Compared with avian H10 hemagglutinin, seal H10 hemagglutinin showed stronger binding to the human-type sialic acid receptor, with preferential binding to α2,6-linked sialic acids on long extended branches. In X-ray structures, changes in the 220-loop of the receptor-binding pocket caused similar interactions with human receptor as seen for pandemic strains. Two substitutions made seal H10 hemagglutinin more stable than avian H10 hemagglutinin and similar to human hemagglutinin. Consequently, identification of avian-origin influenza viruses across mammals appears critical to detect influenza A viruses posing a major threat to humans and other mammals.
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http://dx.doi.org/10.1016/j.chom.2020.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556738PMC
October 2020

Structural analysis of full-length SARS-CoV-2 spike protein from an advanced vaccine candidate.

bioRxiv 2020 Aug 6. Epub 2020 Aug 6.

Dept. of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA.

Vaccine efforts against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the current COVID-19 pandemic are focused on SARS-CoV-2 spike glycoprotein, the primary target for neutralizing antibodies. Here, we performed cryo-EM and site-specific glycan analysis of one of the leading subunit vaccine candidates from Novavax based on a full-length spike protein formulated in polysorbate 80 (PS 80) detergent. Our studies reveal a stable prefusion conformation of the spike immunogen with slight differences in the S1 subunit compared to published spike ectodomain structures. Interestingly, we also observed novel interactions between the spike trimers allowing formation of higher order spike complexes. This study confirms the structural integrity of the full-length spike protein immunogen and provides a basis for interpreting immune responses to this multivalent nanoparticle immunogen.
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http://dx.doi.org/10.1101/2020.08.06.234674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418715PMC
August 2020

Efficient Chemoenzymatic Synthesis of N-Glycans with a β1,4-Galactosylated Bisecting GlcNAc Motif.

Chembiochem 2020 Nov 19;21(22):3212-3215. Epub 2020 Aug 19.

University of Bayreuth, Bioorganic Chemistry, Universitätsstraße 30, 95447, Bayreuth, Germany.

In human serum immunoglobulin G (IgG), a rare modification of biantennary complex N-glycans lead to a β1,4-galactosylated bisecting GlcNAc branch. We found that the bisecting GlcNAc on a biantennary core-fucosylated N-glycan was enzymatically galactosylated under stringent reaction conditions. Further optimizations led to an efficient enzymatic approach to this particular modification for biantennary substrates. Notably, tri- and tetra-antennary complex N-glycans were not converted by bovine galactosyltransferase. An N-glycan with a galactosylated bisecting GlcNAc was linked to a lanthanide binding tag. The pseudo-contact shifts (PCS) obtained from the corresponding Dy-complex were used to calculate the conformational preferences of the rare N-glycan. Besides two extended conformations only a single folded conformation was found.
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http://dx.doi.org/10.1002/cbic.202000268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723014PMC
November 2020

Salmonella Typhoid Toxin PltB Subunit and Its Non-typhoidal Salmonella Ortholog Confer Differential Host Adaptation and Virulence.

Cell Host Microbe 2020 06 11;27(6):937-949.e6. Epub 2020 May 11.

Department of Microbiology and Immunology, Cornell University College of Veterinary Medicine, Ithaca, NY 14853, USA. Electronic address:

Typhoidal and non-typhoidal Salmonelleae (NTS) cause typhoid fever and gastroenteritis, respectively, in humans. Salmonella typhoid toxin contributes to typhoid disease progression and chronic infection, but little is known about the role of its NTS ortholog. We found that typhoid toxin and its NTS ortholog induce different clinical presentations. The PltB subunit of each toxin exhibits different glycan-binding preferences that correlate with glycan expression profiles of host cells targeted by each bacterium at the primary infection or intoxication sites. Through co-crystal structures of PltB subunits bound to specific glycan receptor moieties, we show that they induce markedly different glycan-binding preferences and virulence outcomes. Furthermore, immunization with the NTS S. Javiana or its toxin offers cross-reactive protection against lethal-dose typhoid toxin challenge. Cumulatively, these results offer insights into the evolution of host adaptations in Salmonella AB toxins, their cell and tissue tropisms, and the design for improved typhoid vaccines and therapeutics.
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http://dx.doi.org/10.1016/j.chom.2020.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7292776PMC
June 2020

Phenotypic Effects of Substitutions within the Receptor Binding Site of Highly Pathogenic Avian Influenza H5N1 Virus Observed during Human Infection.

J Virol 2020 06 16;94(13). Epub 2020 Jun 16.

Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands.

Highly pathogenic avian influenza (HPAI) viruses are enzootic in wild birds and poultry and continue to cause human infections with high mortality. To date, more than 850 confirmed human cases of H5N1 virus infection have been reported, of which ∼60% were fatal. Global concern persists that these or similar avian influenza viruses will evolve into viruses that can transmit efficiently between humans, causing a severe influenza pandemic. It was shown previously that a change in receptor specificity is a hallmark for adaptation to humans and evolution toward a transmittable virus. Substantial genetic diversity was detected within the receptor binding site of hemagglutinin of HPAI A/H5N1 viruses, evolved during human infection, as detected by next-generation sequencing. Here, we investigated the functional impact of substitutions that were detected during these human infections. Upon rescue of 21 mutant viruses, most substitutions in the receptor binding site (RBS) resulted in viable virus, but virus replication, entry, and stability were often impeded. None of the tested substitutions individually resulted in a clear switch in receptor preference as measured with modified red blood cells and glycan arrays. Although several combinations of the substitutions can lead to human-type receptor specificity, accumulation of multiple amino acid substitutions within a single hemagglutinin during human infection is rare, thus reducing the risk of virus adaptation to humans. H5 viruses continue to be a threat for public health. Because these viruses are immunologically novel to humans, they could spark a pandemic when adapted to transmit between humans. Avian influenza viruses need several adaptive mutations to bind to human-type receptors, increase hemagglutinin (HA) stability, and replicate in human cells. However, knowledge on adaptive mutations during human infections is limited. A previous study showed substantial diversity within the receptor binding site of H5N1 during human infection. We therefore analyzed the observed amino acid changes phenotypically in a diverse set of assays, including virus replication, stability, and receptor specificity. None of the tested substitutions resulted in a clear step toward a human-adapted virus capable of aerosol transmission. It is notable that acquiring human-type receptor specificity needs multiple amino acid mutations, and that variability at key position 226 is not tolerated, reducing the risk of them being acquired naturally.
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http://dx.doi.org/10.1128/JVI.00195-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307160PMC
June 2020

Human Influenza Virus Hemagglutinins Contain Conserved Oligomannose N-Linked Glycans Allowing Potent Neutralization by Lectins.

Cell Host Microbe 2020 05 15;27(5):725-735.e5. Epub 2020 Apr 15.

Department of Molecular Medicine, Scripps Research, La Jolla, CA 92037, USA; Department of Immunology & Microbiology, Scripps Research, La Jolla, CA 92037, USA. Electronic address:

Hemagglutinins (HAs) from human influenza viruses adapt to bind α2-6-linked sialosides, overcoming a receptor-defined species barrier distinct from the α2-3 specificity of avian virus progenitors. Additionally, human-adapted HAs gain glycosylation sites over time, although their biological function is poorly defined. Using quantitative glycomic analysis, we show that HAs from human pandemic viruses exhibit significant proportions of high-mannose type N-linked glycans throughout the head domain. By contrast, poorly adapted avian-origin HAs contain predominately complex-type glycans, which have greater structural diversity. Although oligomannose levels vary, they are present in all tested recombinant HAs and whole viruses and can be specifically targeted for universal detection. The positions of high-mannose glycosites on the HA of human H1N1 and H3N2 strains are conserved. Additionally, high-mannose-binding lectins possess a broad capacity to neutralize and prevent infection with contemporary H3N2 strains. These findings reveal the biological significance of HA glycosylation and therapeutic potential of targeting these structures.
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http://dx.doi.org/10.1016/j.chom.2020.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7158820PMC
May 2020

Siglecs as Immune Cell Checkpoints in Disease.

Annu Rev Immunol 2020 04 27;38:365-395. Epub 2020 Jan 27.

Departments of Molecular Medicine, and Immunology and Microbiology, Scripps Research, La Jolla, California 92037, USA; email:

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are expressed on the majority of white blood cells of the immune system and play critical roles in immune cell signaling. Through recognition of sialic acid-containing glycans as ligands, they help the immune system distinguish between self and nonself. Because of their restricted cell type expression and roles as checkpoints in immune cell responses in human diseases such as cancer, asthma, allergy, neurodegeneration, and autoimmune diseases they have gained attention as targets for therapeutic interventions. In this review we describe the Siglec family, its roles in regulation of immune cell signaling, current efforts to define its roles in disease processes, and approaches to target Siglecs for treatment of human disease.
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http://dx.doi.org/10.1146/annurev-immunol-102419-035900DOI Listing
April 2020

Repression of phagocytosis by human CD33 is not conserved with mouse CD33.

Commun Biol 2019 3;2:450. Epub 2019 Dec 3.

1Department of Chemistry, University of Alberta, Alberta, Canada.

CD33 is an immunomodulatory receptor linked to Alzheimer's disease (AD) susceptibility via regulation of phagocytosis in microglia. Divergent features between human CD33 (hCD33) and murine CD33 (mCD33) include a unique transmembrane lysine in mCD33 and cytoplasmic tyrosine in hCD33. The functional consequences of these differences in restraining phagocytosis remains poorly understood. Using a new αmCD33 monoclonal antibody, we show that mCD33 is expressed at high levels on neutrophils and low levels on microglia. Notably, cell surface expression of mCD33 is entirely dependent on Dap12 due to an interaction with the transmembrane lysine in mCD33. In RAW264.7 cultured macrophages, BV-2 cultured microglia, primary neonatal and adult microglia, uptake of cargo - including aggregated Aβ - is not altered upon genetic ablation of mCD33. Alternatively, deletion of hCD33 in monocytic cell lines increased cargo uptake. Moreover, transgenic mice expressing hCD33 in the microglial cell lineage showed repressed cargo uptake in primary microglia. Therefore, mCD33 and hCD33 have divergent roles in regulating phagocytosis, highlighting the importance of studying hCD33 in AD susceptibility.
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http://dx.doi.org/10.1038/s42003-019-0698-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890642PMC
July 2020

Structural Basis of Protection against H7N9 Influenza Virus by Human Anti-N9 Neuraminidase Antibodies.

Cell Host Microbe 2019 12 19;26(6):729-738.e4. Epub 2019 Nov 19.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA; Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA. Electronic address:

Influenza virus neuraminidase (NA) is a major target for small-molecule antiviral drugs. Antibodies targeting the NA surface antigen could also inhibit virus entry and egress to provide host protection. However, our understanding of the nature and range of target epitopes is limited because of a lack of human antibody structures with influenza neuraminidase. Here, we describe crystal and cryogenic electron microscopy (cryo-EM) structures of NAs from human-infecting avian H7N9 viruses in complex with five human anti-N9 antibodies, systematically defining several antigenic sites and antibody epitope footprints. These antibodies either fully or partially block the NA active site or bind to epitopes distant from the active site while still showing neuraminidase inhibition. The inhibition of antibodies to NAs was further analyzed by glycan array and solution-based NA activity assays. Together, these structural studies provide insights into protection by anti-NA antibodies and templates for the development of NA-based influenza virus vaccines and therapeutics.
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http://dx.doi.org/10.1016/j.chom.2019.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908751PMC
December 2019

A Sulfonamide Sialoside Analogue for Targeting Siglec-8 and -F on Immune Cells.

J Am Chem Soc 2019 09 30;141(36):14032-14037. Epub 2019 Aug 30.

Department of Molecular Medicine , The Scripps Research Institute , La Jolla , California 92037 , United States.

The Siglec family of cell surface receptors have emerged as attractive targets for cell-directed therapies due to their restricted expression on immune cells, endocytic properties, and ability to modulate receptor signaling. Human Siglec-8, for instance, has been identified as a therapeutic target for the treatment of eosinophil and mast cell disorders. A promising strategy to target Siglecs involves the use of liposomal nanoparticles with a multivalent display of Siglec ligands. A key challenge for this approach is the identification of a high affinity ligand for the target Siglec. Here, we report the development of a ligand of Siglec-8 and its closest murine functional orthologue Siglec-F that is capable of targeting liposomes to cells expressing Siglec-8 or -F. A glycan microarray library of synthetic 9--sulfonyl sialoside analogues was screened to identify potential lead compounds. The best ligand, 9--(2-naphthyl-sulfonyl)-Neu5Acα2-3-[6--sulfo]-Galβ1-4GlcNAc (6'--sulfo Neu5Ac) combined the lead 2-naphthyl sulfonyl C-9 substituent with the preferred sulfated scaffold. The ligand 6'--sulfo Neu5Ac was conjugated to lipids for display on liposomes to evaluate targeted delivery to cells. Targeted liposomes showed strong in vitro binding/uptake and selectivity to cells expressing Siglec-8 or -F and, when administered to mice, exhibit in vivo targeting to Siglec-F eosinophils.
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http://dx.doi.org/10.1021/jacs.9b05769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6861165PMC
September 2019

An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells.

Mol Cell 2019 07 18;75(2):394-407.e5. Epub 2019 Jun 18.

Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and Odontology, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, Copenhagen, Denmark. Electronic address:

The structural diversity of glycans on cells-the glycome-is vast and complex to decipher. Glycan arrays display oligosaccharides and are used to report glycan hapten binding epitopes. Glycan arrays are limited resources and present saccharides without the context of other glycans and glycoconjugates. We used maps of glycosylation pathways to generate a library of isogenic HEK293 cells with combinatorially engineered glycosylation capacities designed to display and dissect the genetic, biosynthetic, and structural basis for glycan binding in a natural context. The cell-based glycan array is self-renewable and reports glycosyltransferase genes required (or blocking) for interactions through logical sequential biosynthetic steps, which is predictive of structural glycan features involved and provides instructions for synthesis, recombinant production, and genetic dissection strategies. Broad utility of the cell-based glycan array is demonstrated, and we uncover higher order binding of microbial adhesins to clustered patches of O-glycans organized by their presentation on proteins.
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http://dx.doi.org/10.1016/j.molcel.2019.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6660356PMC
July 2019

Author Correction: In vivo tropism of Salmonella Typhi toxin to cells expressing a multiantennal glycan receptor.

Nat Microbiol 2019 07;4(7):1242

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41564-019-0505-7DOI Listing
July 2019

Preventing an Antigenically Disruptive Mutation in Egg-Based H3N2 Seasonal Influenza Vaccines by Mutational Incompatibility.

Cell Host Microbe 2019 Jun 28;25(6):836-844.e5. Epub 2019 May 28.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA; The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

Egg-based seasonal influenza vaccines are the major preventive countermeasure against influenza virus. However, their effectiveness can be compromised when antigenic changes arise from egg-adaptive mutations on influenza hemagglutinin (HA). The L194P mutation is commonly observed in egg-based H3N2 vaccine seed strains and significantly alters HA antigenicity. An approach to prevent L194P would therefore be beneficial. We show that emergence of L194P during egg passaging can be impeded by preexistence of a G186V mutation, revealing strong incompatibility between these mutations. X-ray structures illustrate that individual G186V and L194P mutations have opposing effects on the HA receptor-binding site (RBS), and when both G186V and L194P are present, the RBS is severely disrupted. Importantly, wild-type HA antigenicity is maintained with G186V, but not L194P. Our results demonstrate that these epistatic interactions can be used to prevent the emergence of mutations that adversely alter antigenicity during egg adaptation.
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http://dx.doi.org/10.1016/j.chom.2019.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6579542PMC
June 2019

Virus recognition of glycan receptors.

Curr Opin Virol 2019 02 5;34:117-129. Epub 2019 Mar 5.

Departments of Molecular Medicine, Immunology & Microbiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address:

Attachment of viruses to cell-surface receptors is the initial step in infection. Many mammalian viruses have evolved to recognize receptors that are glycans on cell-surface glycoproteins or glycolipids. Although glycans are a ubiquitous component of mammalian cells, the types of terminal structures expressed vary among different cell-types and tissues, and even between comparable cells and tissues from different species, frequently leading to specific tissue and species tropisms as a direct consequence of glycan receptor recognition. Covering the majority of known virus families, this review provides an overview of mammalian viruses that use glycans as receptors, and their roles in determining in host recognition and tropism.
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http://dx.doi.org/10.1016/j.coviro.2019.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6476673PMC
February 2019

Exploiting CD22 To Selectively Tolerize Autoantibody Producing B-Cells in Rheumatoid Arthritis.

ACS Chem Biol 2019 04 20;14(4):644-654. Epub 2019 Mar 20.

Department of Molecular Medicine , The Scripps Research Institute , North Torrey Pines Road , La Jolla , California 92037 , United States.

Rheumatoid arthritis (RA) is an autoimmune disease that primarily affects the synovial joints and can lead to bone erosion and cartilage damage. One hallmark of RA is anticitrullinated protein autoantibodies (ACPA) and memory citrulline-specific B-cells, which have been implicated in RA pathogenesis. While depletion of B-cells with Rituximab improves clinical responses in RA patients, this treatment strategy leaves patients susceptible to infections. Here we use of Siglec-engaging Tolerance-inducing Antigenic Liposomes (STALs) to selectively target the citrulline-specific B-cells. ACPA production from purified human RA patients' B-cells in vitro was achieved through a set of stimulation conditions, which includes the following: BAFF, anti-CD40, IL-21, and LPS. In vivo generation of citrulline specific B-cells and ACPA production was accomplished by antigenic liposomes consisting of monophosphoryl lipid A (MPLA) and a cyclic citrullinated peptide (CCP) administered to SJL/J mice. We show that STALs that codisplay a high affinity CD22 glycan ligand and synthetic citrullinated antigen (CCP STALs) can prevent ACPA production from RA patients' memory B-cells in vitro. These CCP STALs were also effective in inducing tolerance to citrullinated antigens in SJL/J mice. The results demonstrate that tolerization of the B-cells responsible for ACPA can be achieved by exploiting the inhibitory receptor CD22 with high-affinity glycan ligands. Such a treatment strategy could be beneficial in the treatment of RA.
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http://dx.doi.org/10.1021/acschembio.8b01018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941425PMC
April 2019

CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen.

J Clin Invest 2019 03 18;129(3):1387-1401. Epub 2019 Feb 18.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, USA.

Allergen immunotherapy for patients with allergies begins with weekly escalating doses of allergen under medical supervision to monitor and treat IgE mast cell-mediated anaphylaxis. There is currently no treatment to safely desensitize mast cells to enable robust allergen immunotherapy with therapeutic levels of allergen. Here, we demonstrated that liposomal nanoparticles bearing an allergen and a high-affinity glycan ligand of the inhibitory receptor CD33 profoundly suppressed IgE-mediated activation of mast cells, prevented anaphylaxis in Tg mice with mast cells expressing human CD33, and desensitized mice to subsequent allergen challenge for several days. We showed that high levels of CD33 were consistently expressed on human skin mast cells and that the antigenic liposomes with CD33 ligand prevented IgE-mediated bronchoconstriction in slices of human lung. The results demonstrated the potential of exploiting CD33 to desensitize mast cells to provide a therapeutic window for administering allergen immunotherapy without triggering anaphylaxis.
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http://dx.doi.org/10.1172/JCI125456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391081PMC
March 2019

Fluorescent Trimeric Hemagglutinins Reveal Multivalent Receptor Binding Properties.

J Mol Biol 2019 02 29;431(4):842-856. Epub 2018 Dec 29.

Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584, CG, Utrecht, the Netherlands. Electronic address:

Influenza A virus carries hundreds of trimeric hemagglutinin (HA) proteins on its viral envelope that interact with various sialylated glycans on a host cell. This interaction represents a multivalent binding event that is present in all the current receptor binding assays, including those employing viruses or precomplexed HA trimers. To study the nature of such multivalent binding events, we fused a superfolder green fluorescent protein (sfGFP) to the C-terminus of trimeric HA to allow for direct visualization of HA-receptor interactions without the need for additional fluorescent antibodies. The multivalent binding of the HA-sfGFP proteins was studied using glycan arrays and tissue staining. The HA-sfGFP with human-type receptor specificity was able to bind to a glycan array as the free trimer. In contrast, the HA-sfGFP with avian-type receptor specificity required multimerization by antibodies before binding to glycans on the glycan array could be observed. Interestingly, multimerization was not required for binding to tissues. The array data may be explained by the possible bivalent binding mode of a single human-specific HA trimer to complex branched N-glycans, which is not possible for the avian-specific HA due to geometrical constrains of the binding sites. The fact that this specificity pattern changes upon interaction with a cell surface probably represents the enhanced amount of glycan orientations and variable densities versus those on the glycan array.
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http://dx.doi.org/10.1016/j.jmb.2018.12.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397626PMC
February 2019

Flexibility of Amino Acid 226 in the Receptor-Binding Site of an H9 Subtype Influenza A Virus and Its Effect on Virus Replication, Tropism, and Transmission.

J Virol 2019 03 5;93(6). Epub 2019 Mar 5.

Poultry Diagnostic and Research Center, Department of Population Health, University of Georgia, Athens, Georgia, USA

Influenza A viruses (IAVs) remain a significant public health threat, causing more than 300,000 hospitalizations in the United States during the 2015-2016 season alone. While only a few IAVs of avian origin have been associated with human infections, the ability of these viruses to cause zoonotic infections further increases the public health risk of influenza. Of these, H9N2 viruses in Asia are of particular importance as they have contributed internal gene segments to other emerging zoonotic IAVs. Notably, recent H9N2 viruses have acquired molecular markers that allow for a transition from avian-like to human-like terminal sialic acid (SA) receptor recognition via a single amino acid change at position 226 (H3 numbering), from glutamine (Q226) to leucine (L226), within the hemagglutinin (HA) receptor-binding site (RBS). We sought to determine the plasticity of amino acid 226 and the biological effects of alternative amino acids on variant viruses. We created a library of viruses with the potential of having any of the 20 amino acids at position 226 on a prototypic H9 HA subtype IAV. We isolated H9 viruses that carried naturally occurring amino acids, variants found in other subtypes, and variants not found in any subtype at position 226. Fitness studies in quails revealed that some natural amino acids conferred an replication advantage. This study shows the flexibility of position 226 of the HA of H9 influenza viruses and the resulting effect of single amino acid changes on the phenotype of variants and A single amino acid change at position 226 in the hemagglutinin (HA) from glutamine (Q) to leucine (L) has been shown to play a key role in receptor specificity switching in various influenza virus HA subtypes, including H9. We tested the flexibility of amino acid usage and determined the effects of such changes. The results reveal that amino acids other than L226 and Q226 are well tolerated and that some amino acids allow for the recognition of both avian and human influenza virus receptors in the absence of other changes. Our results can inform better avian influenza virus surveillance efforts as well as contribute to rational vaccine design and improve structural molecular dynamics algorithms.
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http://dx.doi.org/10.1128/JVI.02011-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401463PMC
March 2019

Targeted Delivery of Antigen to Activated CD169 Macrophages Induces Bias for Expansion of CD8 T Cells.

Cell Chem Biol 2019 01 1;26(1):131-136.e4. Epub 2018 Nov 1.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

Macrophages (MØs) expressing the endocytic sialic acid-binding immunoglobulin-like lectin 1 (siglec-1, CD169, sialoadhesin) are known to be adept at antigen capture-primarily due to their strategic location within lymphatic tissues. Antigen concentrated in these cells can be harnessed to induce potent anti-tumor/anti-pathogen cytotoxic (CD8) T cell responses. Here, we describe a chemical platform that exploits the CD169-mediated antigen capture pathway for biased priming of antigen-specific CD4 or CD8 T cells in vivo. In the absence of a toll-like receptor (TLR) agonist, antigen delivery through CD169 produced robust CD4 T cell priming only. However, simultaneous treatment with targeted antigen and a TLR7 agonist induced CD8 T cell priming, with concomitant suppression of the CD4 T cell response. We exploited these observations to manipulate the activation ratio of CD4/CD8 T cells in the same animal. These findings represent a unique chemical strategy for targeting CD169 macrophages to modulate antigen-specific T cell immunity.
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http://dx.doi.org/10.1016/j.chembiol.2018.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338492PMC
January 2019

Plasticity of Amino Acid Residue 145 Near the Receptor Binding Site of H3 Swine Influenza A Viruses and Its Impact on Receptor Binding and Antibody Recognition.

J Virol 2019 01 4;93(2). Epub 2019 Jan 4.

Department of Population Health, Poultry Diagnostic and Research Center, University of Georgia, Athens, Georgia, USA

The hemagglutinin (HA), a glycoprotein on the surface of influenza A virus (IAV), initiates the virus life cycle by binding to terminal sialic acid (SA) residues on host cells. The HA gradually accumulates amino acid substitutions that allow IAV to escape immunity through a mechanism known as antigenic drift. We recently confirmed that a small set of amino acid residues are largely responsible for driving antigenic drift in swine-origin H3 IAV. All identified residues are located adjacent to the HA receptor binding site (RBS), suggesting that substitutions associated with antigenic drift may also influence receptor binding. Among those substitutions, residue 145 was shown to be a major determinant of antigenic evolution. To determine whether there are functional constraints to substitutions near the RBS and their impact on receptor binding and antigenic properties, we carried out site-directed mutagenesis experiments at the single-amino-acid level. We generated a panel of viruses carrying substitutions at residue 145 representing all 20 amino acids. Despite limited amino acid usage in nature, most substitutions at residue 145 were well tolerated without having a major impact on virus replication All substitution mutants retained receptor binding specificity, but the substitutions frequently led to decreased receptor binding. Glycan microarray analysis showed that substitutions at residue 145 modulate binding to a broad range of glycans. Furthermore, antigenic characterization identified specific substitutions at residue 145 that altered antibody recognition. This work provides a better understanding of the functional effects of amino acid substitutions near the RBS and the interplay between receptor binding and antigenic drift. The complex and continuous antigenic evolution of IAVs remains a major hurdle for vaccine selection and effective vaccination. On the hemagglutinin (HA) of the H3N2 IAVs, the amino acid substitution N 145 K causes significant antigenic changes. We show that amino acid 145 displays remarkable amino acid plasticity , tolerating multiple amino acid substitutions, many of which have not yet been observed in nature. Mutant viruses carrying substitutions at residue 145 showed no major impairment in virus replication in the presence of lower receptor binding avidity. However, their antigenic characterization confirmed the impact of the 145 K substitution in antibody immunodominance. We provide a better understanding of the functional effects of amino acid substitutions implicated in antigenic drift and its consequences for receptor binding and antigenicity. The mutation analyses presented in this report represent a significant data set to aid and test the ability of computational approaches to predict binding of glycans and in antigenic cartography analyses.
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http://dx.doi.org/10.1128/JVI.01413-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321904PMC
January 2019

Avenues to Characterize the Interactions of Extended N-Glycans with Proteins by NMR Spectroscopy: The Influenza Hemagglutinin Case.

Angew Chem Int Ed Engl 2018 11 17;57(46):15051-15055. Epub 2018 Oct 17.

Dpto Química Orgánica I, Fac. Ciencias Químicas, Universidad Complutense de Madrid, Avd. Complutense s/n, 28040, Madrid, Spain.

Long-chain multiantenna N-glycans are extremely complex molecules. Their inherent flexibility and the presence of repetitions of monosaccharide units in similar chemical environments hamper their full characterization by X-ray diffraction or standard NMR methods. Herein, the successful conformational and interaction analysis of a sialylated tetradecasaccharide N-glycan presenting two LacNAc repetitions at each arm is presented. This glycan has been identified as the receptor of the hemagglutinin protein of pathogenic influenza viruses. To accomplish this study, a N-glycan conjugated with a lanthanide binding tag has been synthesized, enabling analysis of the system by paramagnetic NMR. Under paramagnetic conditions, the NMR signals of each sugar unit in the glycan have been determined. Furthermore, a detailed binding epitope of the tetradecasaccharide N-glycan in the presence of HK/68 hemagglutinin is described.
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http://dx.doi.org/10.1002/anie.201807162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282704PMC
November 2018

Proximity Ligation-Based Fluorogenic Imaging Agents for Neuraminidases.

Angew Chem Int Ed Engl 2018 10 14;57(41):13538-13541. Epub 2018 Sep 14.

Department of Chemistry, University of British Columbia, Vancouver, British Columbia, V6T 1Z1, Canada.

Reagents to visualize and localize neuraminidase activity would be valuable probes to study the role of neuraminidases in normal cellular processes as well as during viral infections or cancer development. Herein, a new class of neuraminidase-imaging probes that function as proximity ligation reagents by releasing a highly reactive fluorophore that tags nearby cellular material is described. It is further demonstrated that it is possible to create an influenza virus-specific reagent, which can specifically detect influenza virus infections in mammalian cells. These reagents have potential use as specific histological probes independent of viral antigenicity and, therefore, offer some advantages over commonly used anti-neuraminidase antibodies.
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http://dx.doi.org/10.1002/anie.201808196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311531PMC
October 2018

Differential processing of HIV envelope glycans on the virus and soluble recombinant trimer.

Nat Commun 2018 09 12;9(1):3693. Epub 2018 Sep 12.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, 92037, USA.

As the sole target of broadly neutralizing antibodies (bnAbs) to HIV, the envelope glycoprotein (Env) trimer is the focus of vaccination strategies designed to elicit protective bnAbs in humans. Because HIV Env is densely glycosylated with 75-90 N-glycans per trimer, most bnAbs use or accommodate them in their binding epitope, making the glycosylation of recombinant Env a key aspect of HIV vaccine design. Upon analysis of three HIV strains, we here find that site-specific glycosylation of Env from infectious virus closely matches Envs from corresponding recombinant membrane-bound trimers. However, viral Envs differ significantly from recombinant soluble, cleaved (SOSIP) Env trimers, strongly impacting antigenicity. These results provide a benchmark for virus Env glycosylation needed for the design of soluble Env trimers as part of an overall HIV vaccine strategy.
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http://dx.doi.org/10.1038/s41467-018-06121-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135743PMC
September 2018