Thesis (doctoral)--Lund University, 1992. / Added t.p. with thesis statement inserted.

The sialidases are a group of glycosyl hydrolases that specifically remove terminal sialic acid (Neu5Ac) residues from various glycans. In the two common human pathogenic bacteria Streptococcus pneumoniae and Pseudomonas aeruginosa, these enzymes have been shown to be key virulence factors directly involved in bacterial colonization and infection. However, little is known about their detailed structural and mechanistic features and lack of this information significantly slows down the progress of new drug discovery targeting these enzymes. Therefore, we embarked structural and kinetic studies towards the three distinct sialidases (designated as NanA, NanB and NanC) from S. pneumoniae, as well as the putative sialidase (designated as PaNA) from P. aeruginosa. Full-length NanA failed to crystallize due to the presence of some natively disordered regions. The catalytic domain of NanA (CNanA) was therefore subcloned, which was crystallized and the structure was determined to 1.5 Å. CNanA exists as a dimer with close contacts between the two monomers. The second pneumococcal sialidase NanB only shares 24% sequence identity with NanA. Crystal structure of NanB was also determined to 1.7 Å, which exhibits a multi-domain monomeric architecture. In general, the core catalytic domain of both CNanA and NanB adopts the classic six- bladed β-propeller fold (or called sialidase fold), with a set of highly conserved residues stacking around the proposed active sites. NanC is a close homologue of NanB, sharing over 50% sequence identity. However, NanC crystallization is not successful so far. To compare the three sialidases in more detail, a computational NanC model was made based on the structure of NanB. Mapping of the active sites of CNanA and NanB was achieved using Neu5Ac2en, a general sialidase inhibitor as the probe. Although sharing many common features, NanA, NanB and NanC present different topologies around the catalytic centre, give these enzymes a high level of diversity in enzymatic kinetics, substrate specificity and catalytic properties. NMR studies show that NanA acts as a classic hydrolytic sialidase; while NanB is found to be an intermolecular trans-sialidase like the leech sialidase; NanC, however, handles multiple catalytic roles efficiently, which include releasing Neu5Ac2en from α2,3- sialyllactose and hydration of Neu5Ac2en to Neu5Ac with high efficiency. S. pneumoniae thus expresses NanA, NanB and NanC for disparate but cooperative roles. Such a working pattern of three sialidases in one microbe is unusual in nature, which might be essential for pneumococcal pathogenesis at various stages. Based on the crystal structures of CNanA and NanB, preliminary work towards S. pneumoniae sialidases inhibitor design is under way, in which, a variety of techniques, such as the fluorescence-based thermal shift assay, NMR spectroscopy, computational docking and X-ray crystallography, are incorporated in. The crystal structure of PaNA was determined to 1.9 Å. This protein appeared to be a unique trimer in crystal that is associated, in part, by the immunoglobulin-like trimerization domain around a three-fold crystallographic axis. The core catalytic domain of PaNA also presents the conserved sialidase fold. Surprisingly, no sialidase activity was detected with this enzyme. In addition, two key catalytic residues including one of the arginine in the arginine triad and the acid/base catalyst aspartic acid are missing in PaNA. In silico docking suggests that Phe129 may confer substrate selectivity towards pseudaminic acid, which is a specific carbohydrate superficially similar to Neu5Ac, but with different stereochemistry at the C-5 position. Site-directed mutagenesis further confirmed that mutation of Phe129 to alanine could turn PaNA into a poor sialidases. Moreover, the crystal structure of PaNA also indicates that His45, Tyr21 and Glu315 may form a charge relay to compensate the missing aspartic acid. Subsequent mutagenesis and NMR kinetic studies proved His45-Tyr21-Glu315 to be a novel charge relay taking the role of the acid/base catalyst. Therefore, PaNA could be a pseudaminidase with structural and mechanistic variations. This enzyme, together some other uncharacterized fellow proteins, might form a novel subclass in the sialidase superfamily. The various findings in the current projects provide meaningful insights towards several sialidases that have been linked to bacterial virulence, which may contribute to a more intensive understanding of S. pneumoniae and P. aeruginosa pathogenesis.

Abstract not available

The sialyltransferase family is a group of enzymes that transfer sialic acid from donor CMP-Neu5Ac onto suitable carbohydrate chains of glycoproteins and glycolipids. In vertebrates, sialylation is implicated in many physiological and pathobiological processes, including nervous and immune system development and functioning, pathogen-host interaction, cancer cell proliferation and apoptosis. However, the complexity of the sialylation pathway and limitation of genetic and in vivo approaches interferes with functional analyses in mammalian organisms. We use Drosophila because of its simplified physiology and reduced genetic redundancy to characterize the evolutionarily conserved function of sialylation and to reveal its relationship to the role of sialic acids in humans. This dissertation focuses primarily on Drosophila sialyltransferase, DSIAT, so far the only sialyltransferase described in protostomes. Gene targeting of the DSIAT endogenous locus with a DSIAT-HA tagged version uncovered its remarkably dynamic stage- and cell-specific expression. I found that the expression of DSIAT is developmentally regulated and is restricted to motor neurons and cholinergic interneurons within the central nervous system of Drosophila. To reveal the role of DSIAT in development and functioning of fly nervous system I performed characterization of neurological phenotypes of DSIAT knockout flies, also generated by gene targeting approach. I observed that DSIAT mutant larvae are sluggish and have abnormal neuromuscular junction (NMJ) morphology. Electrophysiological analysis of mutant larval NMJ showed altered evoked NMJ activity. It was also observed that DSIAT knockout adult flies are paralyzed when are exposed to higher temperatures. Longevity assays showed that DSIAT adult mutants have significantly reduced life span. I used genetic interaction analysis to identify possible sialylated targets in Drosophila and found that ?-subunit of voltage gated sodium channel is a potential sialylated protein in the fly nervous system. All these data strongly supports the hypothesis that DSIAT plays an important role for neural transmission and development in Drosophila. This research work establishes Drosophila as a useful model system to study sialylation which may shed light on related biological functions in higher organisms including humans.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Oct. 9, 2008). Includes bibliographical references (p. 89-101).

In nature, proteins with weak binding affinity often use a multivalency approach to enhance protein affinity via an avidity effect. Interested in this multivalency approach, we have isolated a carbohydrate binding module (CBM) that recognises sialic acid (known as a CBM40 domain) from both Vibrio cholerae (Vc) and Streptococcus pneumoniae (Sp) NanA sialidases, and generated multivalent polypeptides from them using molecular biology. Multivalent CBM40 constructs were designed either using a tandem repeat approach to produce trimeric or tetrameric forms that we call Vc3CBM and Vc4CBM, respectively, or through the addition of a trimerization domain derived from Pseudomonas aeruginosa pseudaminidase to produce three trimeric forms of proteins known as Vc-CBMTD (WT), Vc-CBMTD (Mutant) and Sp-CBMTD). Due to the position and flexibility of the linker between the trimerization domain and the CBM40 domain, site directed mutagenesis was employed to introduce a disulphide bond between the monomers at positions S164C and T83C of the CBM40 domain in order to promote a stable orientation of the binding site for easier access of sialic acids. Data from isothermal titration calorimetry (ITC) reveals that interaction of multivalent CBM40 proteins with α(2,3)-sialyllactose was mainly enthalpy driven with entropy contributing unfavorably to the interaction suggesting that these proteins establish a strong binding affinity to their ligand minimizing dissociation to produce stable multivalent molecules. However, using surface plasmon resonance (SPR), a mixed balance of entropy and enthalpy contributions was found with all constructs as determined by Van't Hoff plots. This proved that binding does not occur through a simple protein-ligand interaction but through disruption of hydrophobic and/or ionic hydration that provide the driving force to the process. Interestingly, the valency of multiple-linked polypeptides also plays an important part in the protein stabilization. However, little is known about their detailed structure when in multivalent form, as attempts to crystallize the whole protein molecule of Vc-CBMTD (WT) failed due to linker and domain flexibility. Only the trimerization domain (TD) part from Pseudomonas aeruginosa pseudaminidase was successfully crystallized and structure was determined to 3.0 Å without its CBM40 domain attached. In this thesis, we have also reported on the potential anti-influenza and anti- parainfluenza properties of these proteins, which were found to block attachment and inhibit infection of several influenza A and parainfluenza virus strains in vitro. As widely mentioned in literature, terminal sialic acids on the cell surface of mammalian host tissue provide a target for various pathogenic organisms to bind. Levels of viral inhibition were greatest against A/Udorn/72 H3N2 virus for Vc4CBM and Vc3CBM constructs with the lowest EC50 of 0.59 µM and 0.94 µM respectively, however most of the multivalent proteins tested were also effective against A/WSN/33 H1N1 and A/PR8/34 H1N1 subtypes. For parainfluenza virus, all constructs containing V. cholerae sialidase CBM40 domain showed great effect in inhibiting virus infection during cell protection assay. The best EC50 values were 0.2 µM from Vc-CBMTD (WT) followed by 1.17 µM from Vc4CBM and 1.78 µM from Vc-CBMTD (Mutant) which was against hPIV2, hPIV3 and hPIV5 infections respectively. Only a construct from S. pneumoniae sialidase known as Sp-CBMTD showed negligible effect on cell protection. All constructs were further tested for cytotoxicity in mammalian cell culture as well as undergoing an inhibition study on viral replication proteins. For the in vivo study, we also demonstrated the effectiveness of Vc4CBM to protect cotton rats and mice from hPIV3 and Streptococcus pneumoniae infections, when given intranasally in advance or on the day of infection. Therefore, these novel multivalent proteins could be promising candidates as broad-spectrum inhibitors or as a prophylactic treatment for both influenza and parainfluenza associated diseases.

Abstract not available

No description available.

No description available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Erythropoietin is a hematopoietic hormone and the main physiological function is the induction of erythropoiesis. Recombinant DNA technology enabled cloning and expression of rhEPO gene to produce recombinant human erythropoietin (rhEPO). It is a sialoglycoprotein composed of 165 amino acids chain and about 40% of the molecule consists of carbohydrates, important for biological activity due to the presence of sialic acid residues at the termini of chains affecting its half-life. In the present study an alternative in vitro cell culture-based assay using TF-1 cell line was validated, to be used in conjunction with a reversed-phase liquid chromatography method with fluorescence detection (F-RP-LC) validated to determine the content of sialic acids. The values obtained for the sialic acids were higher than 126.83 ng/μg, and the biotechnology-derived products were subjected to the cell culture bioassay giving potencies 2.91% ± 0.85 lower related to the bioassay in normocythaemic mice, with significant correlation calculated by the Pearson coefficient (r = 0.9947). In parallel, it was determined the content/potency of the products by the validated reversed-phase and size-exclusion liquid chromatography methods that showed mean results 3.14% higher and 2.87% lower, respectively, compared to the in vitro bioassay. It was demonstrated that in vitro cell culture bioassay represent a valid alternative to the in vivo bioassay for the potency assessment of rhEPO, in the context of the reduction and/or replacement of animals. Likewise, correlation of the results obtained with the physicochemical methods, represents advances for the characterization of the biomolecule, which can be applied to the production steps and for the quality control, contributing to assure the batch-to-batch consistency of the bulk and the finished biological products of rhEPO. / A eritropoietina é um hormônio hematopoiético cuja principal função fisiológica é a indução da eritropoiese. Através da tecnologia do DNA recombinante foi possível a clonagem e expressão do gene para produzir a eritropoietina humana recombinante (rhEPO). É uma sialoglicoproteína composta por cadeia de 165 aminoácidos e, aproximadamente, 40% da molécula é constituída de carboidratos, importantes para a atividade biológica devido à presença de resíduos de ácidos siálicos nas extremidades das cadeias, que influenciam na meia-vida da biomolécula. No presente estudo foi validado ensaio alternativo baseado na cultura da linhagem de células TF-1 in vitro, e método por cromatografia líquida por fase reversa com detecção por fluorescência para determinação de ácidos siálicos, para avaliação em conjunto da potência de rhEPO. Determinaram-se teores de ácidos siálicos acima de 126,83 ng/μg e os produtos biotecnológicos foram submetidos ao bioensaio por cultura de células fornecendo potências 2,91% ± 0,85 menores em relação ao ensaio biológico em camundongos normocitêmicos, com correlação significativa calculada pelo coeficiente de correlação de Pearson (r = 0,9947). Paralelamente, determinou-se o teor/potência dos produtos pelas metodologias validadas por cromatografia líquida por fase reversa e por exclusão molecular, que forneceram média de resultados 3,14% maior e 2,87% menor, respectivamente, em relação ao bioensaio in vitro. Demonstrou-se que o bioensaio por cultura de células in vitro, constitui-se em alternativa ao ensaio biológico in vivo para a avaliação de potência de rhEPO, no contexto da redução e/ou substituição do uso de animais. Do mesmo modo, a correlação com os resultados fornecidos pelos métodos físico-químicos, representa avanços para caracterização da biomolécula, que podem ser aplicados nas etapas do processo de produção e para o controle de qualidade, contribuindo para garantir a consistência lote-a-lote da solução concentrada e dos produtos biológicos acabados de rhEPO.