The minimum information required for a glycomics experiment (MIRAGE) project: improving the standards for reporting glycan microarray-based data

MIRAGE (Minimum Information Required for A Glycomics Experiment) is an initiative that was created by experts in the fields of glycobiology, glycoanalytics and glycoinformatics to produce guidelines for reporting results from the diverse types of experiments and analyses used in structural and functional studies of glycans in the scientific literature. As a sequel to the guidelines for sample preparation (Struwe et al. 2016, Glycobiology, 26:907–910) and mass spectrometry  data (Kolarich et al. 2013, Mol. Cell Proteomics, 12:991–995), here we present the first version of guidelines intended to improve the standards for reporting data from glycan microarray analyses. For each of eight areas in the workflow of a glycan microarray experiment, we provide guidelines for the minimal information that should be provided in reporting results. We hope that the MIRAGE glycan microarray guidelines proposed here will gain broad acceptance by the community, and will facilitate interpretation and reproducibility of the glycan microarray results with implications in comparison of data from different laboratories and eventual deposition of glycan microarray data in international databases

Harnessing glycomics technologies: Integrating structure with function for glycan characterization

Glycans, or complex carbohydrates, are a ubiquitous class of biological molecule which impinge on a variety of physiological processes ranging from signal transduction to tissue development and microbial pathogenesis. In comparison to DNA and proteins, glycans present unique challenges to the study of their structure and function owing to their complex and heterogeneous structures and the dominant role played by multivalency in their sequence-specific biological interactions. Arising from these challenges, there is a need to integrate information from multiple complementary methods to decode structure–function relationships. Focusing on acidic glycans, we describe here key glycomics technologies for characterizing their structural attributes, including linkage, modifications, and topology, as well as for elucidating their role in biological processes. Two cases studies, one involving sialylated branched glycans and the other sulfated glycosaminoglycans, are used to highlight how integration of orthogonal information from diverse datasets enables rapid convergence of glycan characterization for development of robust structure–function relationships.National Institutes of Health (U.S.) (GM R37 GM057073-13)Singapore-MIT Alliance for Research and Technolog

Ontogeny-based immunogens for the induction of V2-directed HIV broadly neutralizing antibodies.

CAPRISA, 2017.Abstract available in pdf

Integrating glycomics, proteomics and glycoproteomics to understand the structural basis for influenza a virus evolution and glycan mediated immune interactions

Glycosylation modulates the range and specificity of interactions among glycoproteins and their binding partners. This is important in influenza A virus (IAV) biology because binding of host immune molecules depends on glycosylation of viral surface proteins such as hemagglutinin (HA). Circulating viruses mutate rapidly in response to pressure from the host immune system. As proteins mutate, the virus glycosylation patterns change. The consequence is that viruses evolve to evade host immune responses, which renders vaccines ineffective. Glycan biosynthesis is a non-template driven process, governed by stoichiometric and steric relationships between the enzymatic machinery for glycosylation and the protein being glycosylated. Consequently, protein glycosylation is heterogeneous, thereby making structural analysis and elucidation of precise biological functions extremely challenging. The lack of structural information has been a limiting factor in understanding the exact mechanisms of glycan-mediated interactions of the IAV with host immune-lectins. Genetic sequencing methods allow prediction of glycosylation sites along the protein backbone but are unable to provide exact phenotypic information regarding site occupancy. Crystallography methods are also unable to determine the glycan structures beyond the core residues due to the flexible nature of carbohydrates. This dissertation centers on the development of chromatography and mass spectrometry methods for characterization of site-specific glycosylation in complex glycoproteins and application of these methods to IAV glycomics and glycoproteomics. We combined the site-specific glycosylation information generated using mass spectrometry with information from biochemical assays and structural modeling studies to identify key glycosylation sites mediating interactions of HA with immune lectin surfactant protein-D (SP-D). We also identified the structural features that control glycan processing at these sites, particularly those involving glycan maturation from high-mannose to complex-type, which, in turn, regulate interactions with SP-D. The work presented in this dissertation contributes significantly to the improvement of analytical and bioinformatics methods in glycan and glycoprotein analysis using mass spectrometry and greatly advances the understanding of the structural features regulating glycan microheterogeneity on HA and its interactions with host immune lectins

Effects of galectins and antibodies in HIV infection; Novel Assays

The high variability of the HIV envelope glycoproteins (Env), and their heavy glycan coating, contributes to the limited host immune control. Still, broadly neutralizing antibodies (bnAbs) are found in some chronically infected HIV-infected individuals, which has spurred the research on antibody-based vaccines. An important tool in detecting and studying bnAbs, are neutralization assays. Here we developed an image-based, high-content automated version of a plaque reduction (PR) assay, which uses green fluorescent protein expression as a reporter of HIV infection. This permitted simultaneous detection of antibodies mediating neutralization and inhibition of virus induced cell-cell fusion. In a multicenter study, Neutnet II, the assay compared well with other neutralization assays and was suggested to be an alternative to the traditional peripheral blood monocyte (PBMC)-based assay and the TZMbl assay. The glycans of Env can also take part in HIV host cell adhesion and infection, via host glycan-binding protein, such as galectins. To explore this, we examined the interaction of gp120 (the surface Env protein) with a panel of galectins, by adapting the fluorescent anisotropy (FA) assay to microscale. Galectin-8, a galectin with two carbohydrate recognition domains (CRDs), had high affinity for gp120 as well as the HIV receptor CD4. The N-terminal CRD mediated the strongest interaction with gp120. The results of the FAassay correlated well with binding of whole virions screened in another assay against the same panel of galectins, now immobilized on beads. In the PR assay described above, here used as an infectivity assay, added intact galectin-8 enhanced infectivity of some HIV-1- strains, while this was not seen with the N-CRD, demonstrating that both CRDs of galectin-8 are required for the effect on infectivity. The enhancement effect mediated by galectin-8 was most pronounced with HIV-1 isolates obtained during the relative immune competent chronic phase, as compared to viruses isolated after AIDS onset. Hence, galectin-8 binding carbohydrate motifs on Env appear to be altered at severe immunodeficiency, adding to the knowledge on the evolution of Env glycosylation patterns related to HIV pathogenesis. These results add to the basic knowledge of virus-host interactions, which hopefully could be used for identification of antibodies and galectin-inhibitors effective in HIV prophylactic interventions

Identification and Analysis of the Heparan Sulfate-Binding Domain and Cellular Factors Involved in the Entry of Human Endogenous Retrovirus K HERV-K (HML-2)

The human endogenous retroviruses (HERVs) are remnant elements of ancient retroviruses that infected the human ancestors' germline and integrated into their genome, and thereby passed down to their descendants in a mendelian fashion. The HERV-K (HML-2) family includes the most intact and complete sequences of human-specific elements with conserved ORF for most of their proteins and the ability to produce viral particles. The viral envelope protein (Env) promoted the initial endogenization activities and was shown to have a broad tropism. It mediates the viral entry through interaction with the cell surface heparan sulfates (HS). In this study, we demonstrated first that the specified receptor binding site (RBS) of HML-2 Env is not involved in binding HS. Further, to identify the involved domain in binding HS, we aligned the protein sequence of HML-2 Env to that of the mouse mammary tumor virus (MMTV). This way, we identified an HS-binding domain (HBD) at the N-terminus of the HML-2 Env between residues 216 and 236, corresponding to MMTV HBD. Generated mutations in all positively charged residues of this domain impaired binding to heparin-coated beads and blocked viral entry. Moreover, mutations in the residues R216, K219, and K223, were influential in HS binding and the viral entry. In the second and third parts of the study, we aimed to identify the cellular factors or requirements for HML-2 Env in its early entry pathway utilizing two approaches. In the first approach, we successfully generated a trimer fusion protein for HML-2 consisting of the SU subunit fused with fibritin, a trimerization domain derived from the bacteriophage T4. The trimer fusion protein bound HS similarly to the native Env. However, it did not block HML-2 Env viral entry. Using this trimer fusion protein, we specifically co-purified the Golgi membrane protein 73 (GP73) as a potential attachment factor for HML-2 Env. The second approach included using HML-2 Env pseudotyped viral particles in conducting a functional screening of a cDNA library for transmembrane proteins in a non-permissive cell line. Screening results identified six transmembrane proteins (TMEM9, C12ORF59, IL1RAP, PSCA, LETMD1, and MPEG1) involved in the entry pathway of HML-2 that confer the susceptibility to it. We also found that only IL1RAP and MPEG1 proteins confer susceptibility in a different non-permissive cell line. Our results affirmed the role of HS in the HML-2 Env attachments and identified the specific HBD involved. They also affirmed the contribution of another molecule(s) that serves as the receptor(s). In those lines seven transmembrane proteins involved in the entry pathway of HML-2 Env were identified.Die Humanen Endogenen Retroviren (HERVs) sind Restbestandteile alter Retroviren, die die Keimbahn der menschlichen Vorfahren infizierten und in ihr Genom integriert wurden. Sie werden seither nach den Mendelschen Regeln an ihre Nachkommen weitergegeben. Die HERV-K (HML-2)-Familie umfasst die intaktesten und vollständigsten Sequenzen mit konservierten Leserahmen für die meisten ihrer Proteine und die Fähigkeit, virale Partikel zu produzieren. Das virale Hüllprotein (Env) förderte die anfängliche Endogenisierung und zeigt einen breiten Tropismus. Es vermittelt die Infektion durch erste Wechselwirkung mit Heparansulfaten (HS) auf der Zelloberfläche. In dieser Studie zeigten wir, dass die Rezeptorbindungsstelle (RBS) von HML-2 Env nicht an der Bindung von HS beteiligt ist. Um die daran beteiligte Domäne zu identifizieren, haben wir die Proteinsequenz von HML-2 Env mit der des Maus-Mammatumorvirus (MMTV) verglichen. Am N-Terminus des HML-2 Env wurde so eine potentielle homologe HS-bindende Domäne (HBD) zwischen den Positionen 216 und 236 identifiziert. Mutationen in allen positiv geladenen Resten dieser Domäne beeinträchtigten die Bindung an Heparin-beschichtete Beads und blockierten die Infektion. Darüber hinaus waren Mutationen an den Positionen R216, K219 und K223 für die HS-Bindung und den viralen Zelleintritt maßgeblich. Im zweiten und dritten Teil der Studie wollten wir die zellulären Faktoren für den HML-2 Env vermittelten Zelleintritt mit zwei Ansätzen identifizieren. Im ersten Ansatz ist es gelungen, ein Trimer-Fusionsprotein für HML-2 zu erzeugen, das aus der SU-Untereinheit besteht, die mit der Trimerisierungs-domäne fusioniert ist. Sie ist vom Fibritin des Bakteriophagen T4 abgeleitet. Das Trimer-Fusionsprotein bindet HS ähnlich wie das native Env. Es blockierte jedoch nicht den Eintritt von HML-2 in die Zellen. Mit diesem Fusionsprotein wurde das Golgi-Membranprotein 37 (GP73) als potenzieller Bindungsfaktor für HML-2 Env gefunden. Der zweite Ansatz umfasste die Verwendung von HML-2 Env-Pseudoviren bei der Durchführung eines funktionellen Screenings einer cDNA-Bibliothek mit einer nicht-permissiven Zelllinie. Damit wurden 6 Transmembranproteine (TMEM9, C12ORF59, IL1RAP, PSCA, LETMD1 und MPEG1) identifiziert, die am Infektionsprozess von HML-2 beteiligt sind. Nur IL1RAP und MPEG1 konnten allerding den Eintritt in eine andere nicht-permissiven Zelllinie vermitteln. Die Arbeiten bestätigen die Rolle von HS bei der Zelladhärenz von HML-2 Env und identifizierten die spezifische HBD. Sie bestätigten auch die Beteiligung eines oder mehrerer anderer Moleküle, die als Rezeptor(en) dienen. In diesem Zusammenhang wurden 7 Transmembranproteine identifiziert, die am Eintrittsweg von HML-2 beteiligt sind

The role of α2,3- and α2,6-sialylation

RESUMO: Os glicoconjugados que decoram a superfície celular e os lípidos e proteínas secretados ocupam o ponto de encontro onde normalmente ocorrem interacções críticas homólogas (hospedeiro-hospedeiro) e heterólogas (hospedeiro-patogénio). Apesar de ser largamente aceite que os glicanos são parte integrante do processo de imunidade, continua a não ser claro qual o papel que os glicanos, em toda a sua diversidade, tomam no quadro geral da imunidade. Os glicanos, que são frequentemente terminados por resíduos de ácido siálico, podem ser alterados por factores externos, tais como patogénios, ou por acontecimentos fisiológicos celulares específicos. Normalmente em posição terminal, as glico-estruturas que contêm ácido siálico assumem um papel fundamental numa quantidade substancial de receptores imunes envolvidos na adesividade e tráfico celular, tal como as Selectinas e as Siglecs, das quais se sabe apresentarem uma relevante função imune. À altura do início desta tese, era sabido que os ácidos siálicos expressos à superfície das células poderiam modular mecanismos importantes nas respostas imunes adaptativas. Considerando a posição de charneira que as células dendríticas (DCs) ocupam na transição da resposta imune inata para a adaptativa, antecipámos que os ácidos siálicos poderiam também modular mecanismos relevantes nas DCs humanas. As DCs têm uma função muito relevante na verificação e captura antigénica, migração para os gânglios linfáticos e apresentação antigénica aos linfócitos, uma sequência de funções que conduz, em ultima instância, à indução da resposta inata adaptativa. Considerando estas premissas, a nossa hipótese principal foi que os ácidos siálicos podem influenciar funções relevantes das DCs, tais como captura de antigénios, maturação, migração para os gânglios linfáticos e apresentação antigénica às células Para testar esta hipótese, dividimos o trabalho em quatro partes: 1) Analisámos os glicanos sialilados de superfície, expressos durante a diferenciação de monócitos humanos em DCs (moDCs). Os nossos dados mostraram que a expressão dos glicanos com ligações em O (O-glicanos) e sialilados em α2,3, assim como glicanos com ligações em N (N-glicanos) sialilados em α2,6 e α2,3 aumentou durante o processo de diferenciação das moDCs. Contribuindo para esta nova configuração glicosídica, três sialiltransferases (STs) poderão estar envolvidas: a ST6Gal-1 correlaciona-se com a expressão aumentada de N-glicanos sialilados em α2,6; a ST3Gal-1 contribui para a sialilação em α2,3 de O-glicanos, em especial de antigénios T; e a ST3Gal-4 poderá ser responsável pelo aumento de N-glicanos sialilados em α2,3. Após estímulo e consequente maturação das moDCs, ambos os níveis de expressão génica de ST6Gal-1 e ST3Gal-4 são negativamente modificados sendo, também, que a expressão de ST3Gal-1 varia consoante o estímulo. 2) Estudámos posteriormente as consequências da modulação dos ácidos siálicos de superfície nas funções das DCs. Observámos que a remoção dos ácidos siálicos de superfície diminui significativamente a capacidade de macropinocitose e endocitose mediada por receptores nas moDCs. Em contrapartida, o tratamento com sialidase aumentou significativamente a capacidade das moDCs para fagocitar Escherichia coli. Determinou-se também que este mecanismo requer a existência de ácido siálico presente nas E. coli indicando um mecanismo de interacção hospedeiro-patogénio dependente de ácido siálico em ambas as partes envolvidas. As moDCs tratadas com sialidase também apresentam um nível superior de expressão de moléculas de MHC e moléculas co-estimulatórias, sugerindo um fenótipo celular mais maduro. Recorrendo ao modelo de ratinho, utilizaram-se DCs derivadas de células da medula (BMDCs) de ratinhos deficientes em ST3Gal-1 e ST6Gal-1. Estes ensaios revelaram que quer a endocitose quer a maturação são influenciadas por modificações 37 nos glicanos sialilados em α2,3 ou α2,6. A detecção e quantificação de proteínas Nglicosiladas e sialiladas em α2,6 apontou para um potencial envolvimento de integrinas β2 nestes mecanismos. 3) O efeito da sialilação em α2,6 na migração das DCs para os gânglios linfáticos foi também analisado. Observámos que BMDCs deficientes para ST6Gal-1 apresentam uma redução de cerca de 50% nos níveis de migração das DCs para os gânglios linfáticos, tal como aferido em ensaios de inflamação in situ e estudos de transferência adoptiva de células. Uma redução dos níveis deste tipo de migração foi também observada quando BMDCs nativas foram transferidas para ratinhos receptores deficientes em ST6Gal-1. São, contudo, necessários mais ensaios de forma a identificar as moléculas envolvidas neste processo. 4) Por último, analisámos o impacto da sialilação na estimulação antigénica das DCs às células T. Assim, concluiu-se que moDCs tratadas com sialidase apresentam um nível de expressão superior de IL-12, TNF-ɑ, IL-6 e IL-10, e activação do factor de transcrição nuclear kappa B (NF-κB). As DCs tratadas com sialidase induziram uma maior proliferação nas células T, com expressão correspondente de interferão-γ. Este dado sugere que a remoção de ácidos siálicos de superfície contribui para o desenvolvimento de uma resposta pro-inflamatória do tipo 1 por células T auxiliares (resposta Th1). Considerando estes dados no seu todo, concluímos que o ácido siálico tem um papel marcante nas funções imunes das DCs. Alterações à concentração de ácido siálico à superfície das células podem alterar a endocitose/fagocitose, maturação, migração para os tecidos e gânglios linfáticos e capacidade estimulatória para com as células T. Complementando estes dados, as ligações glicosídicas de ácidos siálicos criados por ST6Gal-1 e ST3Gal-1 são funcionalmente relevantes. A modulação programada da sialilação do glicocálice, mediada por sialidases individuais ou sialiltransferases é uma possibilidade aceitável para a melhoria da fagocitose por DCs e da sua potência imunológica. Este facto tem um significado particular para imunoterapias baseadas em DCs, podendo provar-se decisivo para a sua eficiência e aplicabilidade num futuro muito próximo.-------------------------------ABSTRACT: Glycans decorating cell surface and secreted proteins and lipids occupy the junction where critical host–host and host-pathogen interactions occur. In spite of the wide acceptance that glycans are centrally implicated in immunity, exactly how glycans and their variety and variability contribute to the overall immune response remains poorly defined. Glycans, frequently terminated by sialic acid residues, may be modified by external factors such as pathogens or upon specific physiological cellular events. The terminal, privileged positions of sialic acid-modified structures makes them key, fundamental determinants for a number of immune receptors with known involvement in cellular adhesiveness and cell trafficking, such as Selectins and Siglecs, with known relevant immune functions. At the time this thesis was initiated, it was established that sialic acids expressed at cell surface could modulate important mechanisms of the adaptive immune responses. Given the key role of dendritic cells (DCs) in the transition from innate to the adaptive immune responses, we anticipated that sialic acids could also modulate important mechanisms of human DCs. DCs have a relevant role in antigen screening and uptake, migration to lymph nodes and antigen presentation to lymphocytes, ultimately triggering the adaptive immune response. Therefore, our primary hypothesis was that sialic acids may modulate DC functions, such as antigen uptake, maturation, homing to lymph nodes and antigen presentation to T cells. To test this hypothesis, we divided our work in four parts. 1) Surface sialylated glycans expressed during differentiation from human monocytes to DCs (moDCs) were analyzed. Our data showed that α2,3-sialylated O-glycans and α2,6- and α2,3-sialylated N-glycans expression increased during moDC differentiation. Three main sialyltransferases (STs) are committed with this new glycan configuration: ST6Gal- 1 correlates with the increased expression of α2,6-sialylated N-glycans; ST3Gal-1 32 contributes for the α2,3-sialylation of O-glycans, especially T antigens; and ST3Gal-4 may contribute for the increased α2,3-sialylated N-glycans. Upon moDC maturation, ST6Gal-1 and ST3Gal-4 are downregulated and ST3Gal-1 is altered in a stimulus dependent manner. 2) We subsequently analyzed the consequences of the modulation of cell surface sialic acids in DC functions. We observed that removing surface sialic acid by sialidase significantly decreased the capacity of moDCs to micropinocytose and receptormediated endocytose. In contrast, treatment with a sialidase significantly improved the capacity of moDCs to phagocytose Escherichia coli. The improved phagocytosis mechanism required E. coli sialic acids, indicating a mechanism of host–pathogen interaction dependent on sialic acid moieties. Sialidase-treated moDCs have increased expression of MHC and co-stimulatory molecules, suggesting a more mature phenotype. Experiments using mouse bone marrow-derived DCs (BMDCs) from ST3Gal-1-/- and ST6Gal-1-/- strains indicated that endocytosis and maturation are influenced by changes in either α2,3 or α2,6-sialylated glycans. The analysis of α2,6-sialylated, N-glycosylated proteins, strongly suggested the potential involvement of β2 integrins, underlying these mechanisms. 3) The effect of α2,6-sialylation in DC homing to lymph nodes was also analyzed. We observed that BMDCs deficient for ST6Gal-1 have an almost 50% reduction in DC homing, as assayed by in situ inflammation and adoptive transfer studies. A reduction in DC homing was also observed when wild type BMDCs were transferred into ST6Gal-1-/- recipient mice. Further investigations are necessary to identify the molecules involved in this process. 4) Finally, we also analyzed the impact of sialylation on DCs ability to prime T cells. Sialidase-treated moDCs show increased gene expression of IL-12, TNF-α, IL-6 and IL- 10 cytokines, and activation of the transcription factor nuclear factor-κB. Sialidase33 treated DCs induced a higher proliferative response of T cells with concomitant higher expression of interferon-γ, suggesting that the clearance of cell surface sialic acids contributes to the development of a T helper type 1 proinflammatory response. Together, our data strongly support sialic acid’s relevance in DC immune functions. Alterations of cell surface sialic acid content can alter the endocytosis/phagocytosis, maturation, migration/homing and the ability for T cell priming in human DCs. Moreover, sialic acid linkages created by ST6Gal-1 and ST3Gal-1 are functionally relevant. The engineering of cell surface sialylation, mediated by individual sialidases or sialyltransferases is a likely possibility to fine tune DC phagocytosis and immunological potency, with particular significance to DC-based therapies

Structural and functional insights into species D adenovirus receptor usage: Implications for oncolytic virotherapy

Adenoviruses are a diverse virus family, infecting a range of animal hosts. The human adenoviruses comprise over 100 types, divided into seven species, A-G. Adenoviruses are clinically important as both engineered therapeutic vectors and pathogens. Adenoviruses are non-membrane bound viruses with double stranded DNA genomes, making them amenable to genetic manipulation and manufacturing at scale. This makes them attractive candidates as therapeutic vectors, currently under development as gene therapy vectors, viral vaccines, and oncolytic viruses. Adenovirus based vectors are in clinical trials for the prevention and treatment of diseases as diverse as Ebola infection and cancer, but are hindered by pre-existing antiviral immunity, leading to neutralisation of the virotherapy before it can have its therapeutic effects, and off-target tissue infections resulting in reduced delivery of therapeutic vector to the site of need. Many natural adenovirus infections do not require medical intervention as they are easily neutralised by the hosts immune system, though they can result in serious disease. Predominantly, this is a concern for immunocompromised patients. However, some types can cause symptoms including gastroenteritis, epidemic keratoconjunctivitis (EKC), and potentially fatal acute respiratory distress syndrome, in healthy adults. Species D adenoviruses are especially diverse, accounting for more than 50% of total adenovirus diversity and many instances of disease, including outbreaks of EKC. They can also be attractive as a basis for therapeutic viruses due to low rates of seroprevelance in the population and favourable immunogenicity profiles. However, little is known about most members of this species or the mechanisms which can make them either pathogenic or therapeutically useful. The work presented herein seeks to better understand how species D adenoviruses infect cells. We therefore investigate their fiber proteins, which mediate primary cell surface receptor binding. We identify previously unknown adenovirus receptor interactions and examine these results in the context of developing new therapeutic adenovirus vectors