Kelm, Sørge
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Kelm, Sørge
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Kelm, Sørge
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Kelm, Soerge
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GND
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Item type:Publication, The evolutionary history of sialylation - Perspectives from fish genomes(2004-07-21); ; ; Sialic acids are key determinants of carbohydrate structures that play important roles in a variety of biological functions. In order to obtain a better understanding how sialylation developed during evolution we investigated two completely sequenced teleost genomes (Takifugu rubripes and Danio rerio) with emphasis on the proteins involved in biosynthesis and recognition of sialic acids. The analysis revealed the existence of all key enzymes crucial for sialic acid metabolism including putative orthologues for almost all 20 sialyltransferases (STs) known for mammalian species. A thorough phylogenetic analysis of all ST sequences has provided strong evidence that the development of the major ST branches occurred well before the diversification into protostomes and deuterostomes strongly suggesting that the defining characteristics of STs have not changed much in 1000 million years. Interestingly, from the relationship between the ST subfamilies it can be deduced that the evolution of the different ST lineages was driven by the availability of appropriate acceptor substrates.In contrast to STs, the largest family of sialic acid-recognizing lectins found in mammals, the siglecs, seems to be a relatively new invention in evolution. Analysis of the two teleost genomes revealed only one unambiguous orthologue of the known mammalian siglecs showing highest sequence similarity to mammalian Siglec-4, the myelin-associated glycoprotein (MAG). Although the low sequence similarity in the cytoplasmic tail, different splice variants and the existence of several motifs implicated in signal transduction suggest alternative biological functions for this part of the protein, the high conservation of genomic organization, binding specificity and expression of Siglec-4 from fish to mammals emphasizes an indispensable role for this protein in all vertebrates in contrast to other Siglecs which maintained a high degree of structural and possibly functional flexibility during evolution.doctoral thesis261 160 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Structure and Function Relationship of Trans-Sialidases from Trypanosoma congolense(2015-11-13); ; ; The study presented here addresses structural and functional relations of trans-sialidases (TS) from the African parasite Trypanosoma congolense and their biochemical characterisation. TS are unusual enzymes found in the flagellate protozoan parasite Trypanosoma and catalyse the stereo and region specific transfer of terminal sialic acid from donor sialo-glycoconjugates to terminal galactose residues of suitable acceptor substrates, resulting in alpha-2,3-sialylated glycoconjugates. Major research on trypanosomal TS has been done on Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. However, only little has been known about TS from the African Trypanosoma congolense TS, the prevalent causative agent of African animal Trypanosomiasis (AAT) in livestock and domestic animals also termed nagana.doctoral thesis376 374 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, CD33-Related Siglecs in Early Recognition of Tumour Cells and Viruses by Mononuclear Phagocytes(2011-01-25); ; ; The sialic acid binding immunoglobulin-like lectins (siglecs) comprise a family of receptors that are differentially expressed on leukocytes and other immune cells. Their molecular properties and the presence of tyrosine-based motifs suggest that they could be involved in fine-tuning the immune responses. Peripheral blood derived monocytes express siglec-3, -5, -7, -9, and -10 (1-10% monocyte population). Upon differentiation to macrophages a general trend of decrease in siglec-3, -5 and -9 and increase for siglec-7 expression was observed. The level of expression and degree of change during differentiation varied between individual donors. It is of special interest that siglec expression on mononuclear phagocytes is further regulated by different cytokines (IFN-gamma: increased siglec-1 and decrease siglec-5, -7 and -9; IL-4: increased siglec-7 and 9 and decrease siglec-1). Six month monocyte surface siglec expression analysis of the 15 volunteers revealed that, siglec-9 shows the most diverse expression pattern as compared to the other monocyte limited siglecs, and these changes in expression pattern has direct relation with serum CRP levels. Furthermore, co-culturing monocytes with viruses revealed that mere presence of viral particles affects monocyte surface siglec expression profile. In addition, the viral dose and time of incubation contribute to these changes. The alteration in the siglec expression pattern in response to pathogens/tumour cells could be part of the host defence system. K562 erythroleukemia tumour cells were able to elicit early sialic acid (Sia) dependent cytokine secretion by mononuclear phagocytes. And this early cytokine secretion by mononuclear phagocytes upon co-culture with K562 surface Sia derivatives was confirmed with real time IL-6 mRNA synthesis. Furthermore, an accumulation of siglec-7 was observed at the cellular synapses between these two cell types. As siglecs are the only Sia recognising receptors present on innate immune system cells, and they could be involved in the observed Sia mediated activation processes. Here it is shown that siglec-7 could plays an important role in controlling the Sia mediated activation of mononuclear phagocytes by K562 tumour cells, as in presence of anti siglec-7 mAb activation of mononuclear phagocytes is reduced by 30-40%.doctoral thesis263 134 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Biochemical characterisation of trans-sialidases from Trypanosoma congolense(2013-12-18); ; ; The work compiled here aimed at biochemically characterising trans-sialidase (TS) genes from Trypanosoma congolense, the most prevalent causative agent of animal African trypanosomiasis, also called nagana. Using published partial TS sequences as starting queries, database at the Welcome Trust Sanger Institute (WTSI) was queried and TS1 genes identified. This is a multi-copy gene group comprising 11 members. Members were cloned and recombinant protein expressed in fibroblasts and purified proteins assayed for enzyme activities (Publication 1/Chapter 1). All TS1 variants were found to be active TS enzymes, transferring Sia from donor fetuin to the lactose acceptor substrate and could resialylate asialofetuin up to 50%. Use of synthetic substrates revealed poor specific activities. Further searches on WTSI using TconTS1 gene variants as queries revealed 3 other TS genes, TconTS2, TconTS3 and TconTS4, all conserving the critical amino acids required in activity. A further 3 distantly related genes, this latter group is presumed inactive due to lack of conservation of critical amino acids (Publication 2/Chapter 3). TS/sialidase ratios confirmed the former to be active trans-sialidases but with differing specific activities. A mouse monoclonal antibody raised from native proteins probably containing other TconTS and not only TconTS1 reacted with both TconTS1 and TconTS2, two of the proteins with the highest specific activities. Incidentally, the epitope for the antibody is localised on the lectin domain (LD) in both enzymes. Phylogenetic analysis using the LD grouped TconTS1 and TconTS2 together while the use of catalytic domain (CD) grouped them separately. This suggests a possible role for the LD in enzyme activities and hence pathology of nagana. To gain knowledge on the role of TS in pathogenesis of nagana, blood glycoconjugates were employed as substrates and activities of TS gene characterised (Manuscript/Chapter 4). All TconTS proteins transferred Sia from serum-bound glycoconjugates to lactose. In absence of lactose, TconTS1 and TconTS2 released free Sia from the serum-glycoconjugates, possibly explaining the high amounts of free Sia observed in blood and serum of animals suffering nagana. TconTS3 showed an unidentified product peak in presence of serum-bound Sia and lactose, while 3 unidentified signals probably representing serum-glycoconjugates inherent in serum were altered by TconTS2. Collectively, the above indicate the possibility of acceptor and donor preferences and show that expressing more than one TS gene at a time could be beneficial to the parasite. Since TS genes are expressed in two different hosts systems; the Glossina insect vector and the mammalian hosts with different pH systems, pH optima for the enzymes was studied. TconTS2 exhibited a wide pH optima that would make it active in both host systems.doctoral thesis289 157 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Trans-Sialidase from :i:Trypanosoma congolense:/i: - Isolation, Characterisation and Molecular Biology(2003-02-06); ; ; :i:Trypanosoma congolense:/i: is the agent of Nagana, the trypanosomiasis in African ruminants. During its insect stage the parasite expresses an enzyme called trans-sialidase (TS), which enables the parasites to transfer sialic acids from the environment onto trypanosomal surface molecules and is believed to play an important role in maintaining the pathogenicity of the parasites. Thus far, only two TS proteins and their sequences have been characterised in detail, one from the American trypanosome :i:T. cruzi:/i: and one from the African trypanosome :i:T. b. brucei:/i:. :p:This study describes the purification and characterisation of two TS forms from the African trypanosome :i:T. congolense:/i:. The purification of the two TS forms using a combination of anion exchange chromatography, isoelectric focusing, gel filtration and, subsequently, antibody affinity chromatography resulted, in both cases, in the isolation of a 90 kDa monomer on SDS-PAGE which was identified as TS using micro-sequencing. Monoclonal antibody 7/23, which bound and partially inhibited TS activity, was found in both cases to bind to a 90 kDa protein. Both TS forms possessed sialidase and transfer activity, but markedly differed in their activity ratios. Interestingly, GARP, the surface glycoprotein, was co-purified with TS-form 1 suggesting an association between both proteins. :p: This study also describes the identification of two partial TS gene sequences from :i:T. congolense:/i: utilising a PCR-based approach. Both :i:T. congolense:/i: TS genes, TS1 and TS2, which share only 50 % identity with each other, show significant similarity with known viral, bacterial and trypanosomal sialidases and TS. In both partial sequences most of the critical active site residues common to other trypanosomal sialidases and TS are conserved. Moreover, a peptide sequence derived from the native, active TS from :i:T. congolense:/i: was found within the sequence of TS1.doctoral thesis278 130 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Interaction of Siglec-4 with naturally occurring and synthetic glycoconjugates(2011-06-14); ; ; The aim of this work has been to provide insights into the structure-function relationship of Siglec-4 binding naturally occurring sialic acids as well as synthetic sialic acid derivatives. Structural information of the Siglec binding site and its interactions with glycoconjugates were obtained from homology modeling of the sialic acid binding domain and molecular docking calculations with several sialosides. Furthermore, the interaction of chemically synthesized sialic acid derivatives with improved inhibitory potential for Siglec-4 as well as the interactions of Siglec-2 and Siglec-4 with oligo- and even polyvalent sialoglycoconjugates occurring in bovine milk are described. Finally, the potential of enzymatic sialylation using trans-sialidases from Trypanosoma congolense has been investigated demonstrating that trans-sialidases can sialylate glycoproteins like asialofetuin to sufficiently high density for Siglec-4 recognition.doctoral thesis226 105 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Diversity of trypanosomes, evidence of potential zoonotic species in humans, cattle, and tsetse in Chad: the Mandoul and Maro sleeping sickness foci(2021-04-06); ; ; Molecular studies of circulating trypanosomes in Chad remain insufficient, particularly in a priming time of the disease elimination involving tsetse control and human screenings and treatments campaigns. Trypanosomes are responsible for Human African Trypanosomiasis (HAT) and Animal African Trypanosomiasis (AAT). Trypanosoma brucei gambiense and T. b. rhodesiense are the species responsible for the HAT forms (i.e. sleeping sickness). In contrast, T. b. brucei, T. congolense, and T. vivax are the most pathogenic African livestock species. The parasites are transmitted by bites of infected tsetse, flies of the genus Glossina, the most prominent vector for AAT and HAT. This study aimed to investigate the circulating trypanosomes, including potential zoonotic species in two HAT/AAT foci in Southern Chad, Mandoul and Maro. As for tsetse habitat, the Mandoul HAT/AAT focus represents an isolated ecological island. Tsetse control started in 2014 and human screenings and treatments campaigns were ongoing before 2014. In contrast, the Maro HAT/AAT focus is an area bordering the Central African Republic (CAR) where anti-tsetse campaigns had not started when this study was undertaken and represented an open area for tsetse reinvasions. 889 humans and 540 cattle were recruited, and 177 tsetse were caught to investigate the circulating trypanosome species in both foci. During the surveys on humans, organised in 2017 and 2018 in nomadic and sedentary settlements, questionnaires related to demographic data, anthropometric measurements, health and nutritional status, literacy, and knowledge on parasitic vector diseases were recorded, besides the blood and urine sample collections. Also, cattle blood samples were collected, and questionnaires linked to the settlement systems (nomadic or sedentary), common animal diseases, and animal health status, were filled, and the PCVs measured. During the entomological surveys, alive tsetse were dissected in wings, legs, and proboscis, and gut tissues. As an outcome, the surveyed human groups showed different responses on the social determinant of health (SDOH), on which the vulnerability to infectious diseases and non-communicable diseases depend. The low education level and the lack of sensitisation hampered the understanding of vector-borne parasitic diseases and the preventive measures to be adopted. Interestingly, only a few people are obese, or in overweight range in these areas, with few who presented a high glycemia and high blood pressure. Looking at the entomological data, tsetse are rare in Mandoul where only one fly was caught. In contrast, in Maro, their presence was farther noticed. Extracted DNA samples screened using nested PCR targeting the internal transcribed spacer I (ITS1) supported by the glycosomal glyceraldehyde- 3-phosphate dehydrogenase (gGAPDH)-PCR, and sequencing revealed different trypanosomes diversity according to focus. In Mandoul, there is no evidence of T. b. gambiense and T. congolense neither in humans nor in cattle. Since only one fly was collected here, no statement on presence of trypanosomes in tsetse can be made. However, an unknown Trypanosoma species (Trypanosoma-sp.-129-H) was identified in one person, and a high prevalence of T. theileri in cattle, besides few T. vivax and T. simiae. In contrast, in Maro, T. b. gambiense was identified in humans, as well as evidence for T. congolense DNA. Furthermore, in this focus, sample from cattle and tsetse showed evidence for a high diversity of trypanosomes. This includes pathogenic, non-pathogenic, and unknown species. The diversity of trypanosome species in Maro is linked to the relatively high number of tsetse observed. Consequently, tsetse are vectoring trypanosomes in this focus. This is influenced mainly by the missing tsetse control, besides pastoralist transhumance activities from and to CAR. Whereas in Mandoul, the campaigns are ongoing since 2014 (i.e., three years before and during the surveys). They have reduced the tsetse populations and the pathogenic trypanosome species. However, the other species identified in this focus could be transmitted by other vectors such as tabanid and Stomoxys. Regarding the evidence of T. congolense in humans, it was also confirmed when performing the ELISA assay to investigate the anti-TconTS (anti-T. congolense Trans-sialidase), an enzyme produced during infection with this species. The assay showed a high number of people having an immune response against this parasite exposure. It could be due to a current or cleared infection. The evidence of zoonotic trypanosomes in humans need to be monitored in the field, and the pathogenicity studied in case of non-transient infection. For this purpose, loop-mediated isothermal amplification (LAMP) techniques were optimised for both human and animal pathogenic Trypanosoma species. The assays were successfully tested in the field for the identification of T. congolense in cattle. This thesis gives an insight into the circulating trypanosomes in the Mandoul and the Maro HAT/AAT foci in these cited hosts. It provides a better understanding of the genetic of trypanosomes to support elimination goals. In conclusion, there is evidence of the diversity of trypanosomes and emerging zoonotic species in the study sites. Thus, we suggest widening the study to other mammalian hosts, vectors, and involving other areas, as well as joining efforts with CAR in terms of tsetse control operation and disease elimination in these areas. We also suggest investigating the potentials of emerging zoonotic trypanosomes.doctoral thesis624 252 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Interaktionen von CD22 mit Milchglykoproteinen und mögliche Auswirkungen auf die Regulation der B-Zell-Aktivierung(2004-08-25); ; ; The development of the neonatal immune system is well balanced by breast milk components leading to an appropriate maturation of the immune system. Poorly balanced support of the developing immune system may lead to autoimmune diseases or allergies. Different properties of milk formula components could be the reason for deviations from this balance. Little is known about the molecular mechanisms leading to effects of milk components on the immune system. One mechanism known to regulate B-cell receptor-mediated cell activation is the inhibitory effect of CD22, which sialic acid-dependent interaction on the B-cell surface seems to be critical for its negative regulatory function. The results presented in this work provide a differentiated perspective for the regulation of the immune system by milk glycoproteins/-peptides and clearly demonstrate species-dependent differences between milk proteins. Human caseins were identified as potential inhibitors of the sialic acid-dependent interaction of CD22 with IgM. They inhibited better than undigested camel caseins, human whey proteins and bovine caseins. Proteolytic digestion of caseins reduced the inhibitory potencies, whereas the inhibitory potencies of digested whey proteins increased. Most of the sialylated proteins and CD22-specific binding partners were identified in human caseins by lectin blotting. After affinity chromatography of human caseins, caseins (k-casein) and the MFGM proteins clusterin and lactoferrin were identified as isolated ligands of CD22. In B-cell activation assays the Ca-mobilisation was clearly reduced in presence of digested caseins and whey proteins, but sialylation appears to reduce these effects strongly. Obviously, the interaction of milk glycoproteins/-peptides lead to different effects than the sialic acid derivative BPC-Neu5Ac, which reduces the inhibitory function of CD22. This finding indicate a mechanism independent of the sialic acid binding site for the milk glycoproteins/-peptides.doctoral thesis224 77 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Identifikation und Charakterisierung von Perlucin Spleißvarianten der marinen Abalone Haliotis laevigata.(2014-04-25); ; ; Mother of pearl is a polymer of significance for material sciences due to its fascinating properties. A frequently investigated protein in mother of pearl from the Australian Abalone Haliotis laevigata is the protein Perlucin. Despite its importance relatively little is known about the role of Perlucin in detail in the biomineralisation of mother of pearl. After SDS-PAGE and staining with Coomassie the native protein from H. laevigata shows a heterogenic band pattern (Blank et al., 2001 [1] und Weiss et al., 2000 [2]) postulated to be an expression of Perlucin isoform variability. Focus of the submitted thesis is to test this hypothesis by synthesizing cDNA from the mantle epithelium of the Australian Abalone H. laevigata. Assembling degenerated primer based on the amino acid sequence of the native Perlucin of H. laevigata (Mann et al., 2000 [3]) seven previously unknown splice variants of the protein have been identified. These splice variants differ mainly in the number of a ten amino acid long peptide (repeat) with zero, two, three, five, six or eight repetitions at the C-terminus (of the protein). Three of the seven splice variants have been characterised in detail: Perlucin-R0 (no repeat), Perlucin-R5 (five repeats) and Perlucin-R8 (eight repeats). The splice variants as well as the Repeat-peptide have been identified in the organic matrix of the shell from H. laevigata using MALDI-ToF MS. For subsequent experiments the Perlucin splice variants were fused with the maltose-binding protein (MBP) using a bacterial expression system. After tandem purification via the N-terminal His-Tag and a C-terminal Strep-Tag very pure MBP-Perlucin splice variants were obtained. Native Perlucin can lead to a faster precipitating of CaCO3 (Weiss et al., 2000 [2]) consisting of a heterogeneous protein mixture potentially containing the here newly identified splice variants. The here presented thesis demonstrates that the different variants expressed as MBP fusion proteins in E. coli showed strong differences in their influence on precipitating CaCO3. These differences might be due to a splice variant specific formation of large protein aggregates influenced by the number of the 10mer peptide. In the presence of the MBP-Perlucin splice variants, the precipitation of CaCO3 leads to precipitates with well-defined surface structures, which might grew first followed by a tree structure, but the passing into a very regular layer-by-layer structural pattern. The newly discovered Perlucin splice variant could potentially have multiple functions during the mineralisation of CaCO3. First, they could act as a nucleation-seed by oligomerisation and formation of aggregates of up to 1 µm and second, they could have a modulatory effect on the mineralisation of CaCO3, influenced by the differing length of the repeat-region. Phylogenetic comparison of H. laevigata Perlucin with other related proteins from the GenBank database showed that all Perlucins differ substantially from C-type lectins of nematodes, snakes, fish and human. The phylogenetic study in this thesis defined five proteins as Perlucin. Based on a subsequent phylogenetic classification in the C-type lectin group, these five proteins can be assigned to the Tetranectin-family and Collectins.doctoral thesis291 110