Publications by authors named "Sørge Kelm"

39 Publications

Diversity of trypanosomes in humans and cattle in the HAT foci Mandoul and Maro, Southern Chad-A matter of concern for zoonotic potential?

PLoS Negl Trop Dis 2021 Jun 9;15(6):e0009323. Epub 2021 Jun 9.

Centre for Biomolecular Interactions Bremen, Department of Biology and Chemistry, University of Bremen, Bremen, Germany.

Background: African trypanosomes are parasites mainly transmitted by tsetse flies. They cause trypanosomiasis in humans (HAT) and animals (AAT). In Chad, HAT/AAT are endemic. This study investigates the diversity and distribution of trypanosomes in Mandoul, an isolated area where a tsetse control campaign is ongoing, and Maro, an area bordering the Central African Republic (CAR) where the control had not started.

Methods: 717 human and 540 cattle blood samples were collected, and 177 tsetse flies were caught. Trypanosomal DNA was detected using PCR targeting internal transcribed spacer 1 (ITS1) and glycosomal glyceraldehyde-3 phosphate dehydrogenase (gGAPDH), followed by amplicon sequencing.

Results: Trypanosomal DNA was identified in 14 human samples, 227 cattle samples, and in tsetse. Besides T. b. gambiense, T. congolense was detected in human in Maro. In Mandoul, DNA from an unknown Trypanosoma sp.-129-H was detected in a human with a history of a cured HAT infection and persisting symptoms. In cattle and tsetse samples from Maro, T. godfreyi and T. grayi were detected besides the known animal pathogens, in addition to T. theileri (in cattle) and T. simiae (in tsetse). Furthermore, in Maro, evidence for additional unknown trypanosomes was obtained in tsetse. In contrast, in the Mandoul area, only T. theileri, T. simiae, and T. vivax DNA was identified in cattle. Genetic diversity was most prominent in T. vivax and T. theileri.

Conclusion: Tsetse control activities in Mandoul reduced the tsetse population and thus the pathogenic parasites. Nevertheless, T. theileri, T. vivax, and T. simiae are frequent in cattle suggesting transmission by other insect vectors. In contrast, in Maro, transhumance to/from Central African Republic and no tsetse control may have led to the high diversity and frequency of trypanosomes observed including HAT/AAT pathogenic species. Active HAT infections stress the need to enforce monitoring and control campaigns. Additionally, the diverse trypanosome species in humans and cattle indicate the necessity to investigate the infectivity of the unknown trypanosomes regarding their zoonotic potential. Finally, this study should be widened to other trypanosome hosts to capture the whole diversity of circulating trypanosomes.
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http://dx.doi.org/10.1371/journal.pntd.0009323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8224965PMC
June 2021

[Suitability of biological acellular dermal matrices as a skin replacement].

Handchir Mikrochir Plast Chir 2020 Dec 30;52(6):533-544. Epub 2020 Jul 30.

University of Bremen CBIB, Faculty of Biology and Biochemistry.

Introduction:  Tissue defects are associated with loss of epidermal and dermal components of the skin. For full-thickness tissue defects, dermal equivalents are useful to enable rapid wound closure. Split-thickness skin grafts are associated with loss of tissue elasticity resulting in scar contractures that can impair joint mobility. Synthetic collagen matrices and allogeneic acellular dermal matrices (ADM) are commercially available and could serve as skin tissue replacement. The aim of this study was to investigate whether ADM of different dermal layers or bioartificial matrices can serve as cutaneous replacement. For this purpose, cellular migration, differentiation and the inflammatory reaction were studied in an established skin organ model.

Materials And Methods:  Human split-thickness skin grafts were transplanted onto ADM (Epiflex, DIZG, Berlin, Germany), de-epidermized dermis (DED) or an artificial collagen-elastin matrix (Matriderm, Dr. Suwelack, Billerbeck, Germany). Epithelial migration was studied using an established skin culture model at the air-liquid interface. In addition, the effect of tissue from different dermal compartments, e. g. papillar and reticular dermis, on epithelial migration was compared. Epithelial resurfacing and differentiation of matrices as well as the inflammatory reaction were studied using histological, immunohistochemical and biochemical analyses.

Results And Conclusion:  Significantly more epithelial outgrowth area was found on DED (2.54 mm ± 0.43 mm, mean ± SEM) compared to papillary ADM (1.32 mm ± 0.44 mm, p = 0.039), to reticular ADM (no horizontal growth, p < 0.0001) and collagen-elastin matrix (0.78 mm ± 0.11 mm,  0.0056) measured by fluorescence microscopy over 10 days presumably due to the presence of pro-migratory basement membrane residues on DED. Reepithelialization was significantly higher ( < 0.002) on papillary dermis compared to ADM of reticular origin. In contrast to the biological matrices, a complete horizontal penetration was found in the macroporous collagen-elastin matrix. Pro-inflammatory mediators varied depending on the human skin donor and matrix. In summary, the biochemical structure of the matrix' surface and its origin influenced the epithelial behaviour with regard to migration, differentiation and inflammatory response.
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http://dx.doi.org/10.1055/a-1200-1189DOI Listing
December 2020

Lung Surfactant Accelerates Skin Wound Healing: A Translational Study with a Randomized Clinical Phase I Study.

Sci Rep 2020 02 13;10(1):2581. Epub 2020 Feb 13.

University of Bremen, Competence Center for Clinical Trials Bremen, Bremen, Germany.

Lung surfactants are used for reducing alveolar surface tension in preterm infants to ease breathing. Phospholipid films with surfactant proteins regulate the activity of alveolar macrophages and reduce inflammation. Aberrant skin wound healing is characterized by persistent inflammation. The aim of the study was to investigate if lung surfactant can promote wound healing. Preclinical wound models, e.g. cell scratch assays and full-thickness excisional wounds in mice, and a randomized, phase I clinical trial in healthy human volunteers using a suction blister model were used to study the effect of the commercially available bovine lung surfactant on skin wound repair. Lung surfactant increased migration of keratinocytes in a concentration-dependent manner with no effect on fibroblasts. Significantly reduced expression levels were found for pro-inflammatory and pro-fibrotic genes in murine wounds. Because of these beneficial effects in preclinical experiments, a clinical phase I study was initiated to monitor safety and tolerability of surfactant when applied topically onto human wounds and normal skin. No adverse effects were observed. Subepidermal wounds healed significantly faster with surfactant compared to control. Our study provides lung surfactant as a strong candidate for innovative treatment of chronic skin wounds and as additive for treatment of burn wounds to reduce inflammation and prevent excessive scarring.
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http://dx.doi.org/10.1038/s41598-020-59394-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7018835PMC
February 2020

Matrix Metalloproteinase-3 is Key Effector of TNF-α-Induced Collagen Degradation in Skin.

Int J Mol Sci 2019 Oct 22;20(20). Epub 2019 Oct 22.

Digestive Disease Center and Copenhagen Wound Healing Center, Bispebjerg Hospital, University of Copenhagen, 2400 Copenhagen, Denmark.

Inflammatory processes in the skin augment collagen degradation due to the up-regulation of matrix metalloproteinases (MMPs). The aim of the present project was to study the specific impact of MMP-3 on collagen loss in skin and its interplay with the collagenase MMP-13 under inflammatory conditions mimicked by the addition of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Skin explants from MMP-3 knock-out (KO) mice or from transgenic (TG) mice overexpressing MMP-3 in the skin and their respective wild-type counterparts (WT and WTT) were incubated ex vivo for eight days. The rate of collagen degradation, measured by released hydroxyproline, was reduced ( < 0.001) in KO skin explants compared to WT control skin but did not differ ( = 0.47) between TG and WTT skin. Treatment with the MMP inhibitor GM6001 reduced hydroxyproline media levels from WT, WTT and TG but not from KO skin explants. TNF-α increased collagen degradation in the WT group ( = 0.0001) only. More of the active form of MMP-13 was observed in the three MMP-3 expressing groups (co-incubation with receptor-associated protein stabilized MMP-13 subforms and enhanced detection in the media). In summary, the innate level of MMP-3 seems responsible for the accelerated loss of cutaneous collagen under inflammatory conditions, possibly via MMP-13 in mice.
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http://dx.doi.org/10.3390/ijms20205234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829232PMC
October 2019

Widespread co-endemicity of Trypanosoma species infecting cattle in the Sudano-Sahelian and Guinea Savannah zones of Cameroon.

BMC Vet Res 2019 Oct 16;15(1):344. Epub 2019 Oct 16.

TOZARD Research Laboratory, P.O. Box 59, Bambili-Tubah, Bamenda, Cameroon.

Background: African animal trypanosomosis remains the major constraint of livestock production and livelihood of pastoral communities in Cameroon. Despite several decades of vector and parasite control efforts, it has not been eradicated. Alternative and sustainable control strategies require a sound knowledge of the local species, strains and vectors. In the Sudano-Sahelian and Guinea Savannah of Cameroon the prevalence and genetic diversity of trypanosomes infecting cattle was investigated by microscopy of cattle blood buffy coat and molecular methods using generic primers targeting parts of the internal transcribed spacer 1 (ITS-1) and encoded glycosomal glyceraldehyde 3-phosphate dehydrogenase-gene (gGAPDH).

Results: A total of 1176 randomly chosen cattle from five divisions in the Sudano-Sahelian and Guinea Savannah of Cameroon were examined. The overall prevalence of trypanosomes by microscopy was 5.9% (56/953) in contrast to 53.2% (626/1176) when molecular tools were used. This indicated a limited sensitivity of microscopy in subclinical infections with frequently low parasitemia. Three trypanosome species were identified by light microscopy: T. vivax (2.3%), T. brucei (3.7%) and T. congolense (3.0%), whereas five were identified by PCR, namely T. grayi/T. theileri (30.8%), T. vivax (17.7%), T. brucei (14.5%) and T. congolense (5.1%). Unexpected cases of T. grayi (n = 4) and T. theileri (n = 26) were confirmed by sequencing. Phylogenetic analysis of the gGAPDH revealed the presence of T. vivax, clade A and T. vivax clade C, which were co-endemic in the Faro et Deo division. T. grayi/T. theileri were the predominant species infecting cattle in tsetse free areas. In contrast, T. vivax, T. brucei and T. congolense were more abundant in areas where the Glossina-vectors were present.

Conclusions: The abundance of pathogenic trypanosomes in tsetse infested areas is alarming and even more, the occurrence of T. vivax, T. brucei, T. congolense, T. theileri and T. grayi in tsetse-free areas implies that tsetse control alone is not sufficient to control trypanosomosis in livestock. To implement control measures that reduce the risk of spread in tsetse free areas, close monitoring using molecular tools and a thorough search for alternative vectors of trypanosomes is recommended.
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http://dx.doi.org/10.1186/s12917-019-2111-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796345PMC
October 2019

Genetic diversity of trypanosome species in tsetse flies (Glossina spp.) in Nigeria.

Parasit Vectors 2019 Oct 14;12(1):481. Epub 2019 Oct 14.

Centre for Biomolecular Interactions, Department of Biology and Chemistry, University of Bremen, Bremen, Germany.

Background: Trypanosomes cause disease in humans and livestock in sub-Saharan Africa and rely on tsetse flies as their main insect vector. Nigeria is the most populous country in Africa; however, only limited information about the occurrence and diversity of trypanosomes circulating in the country is available.

Methods: Tsetse flies were collected from five different locations in or adjacent to protected areas, i.e. national parks and game reserves, in Nigeria. Proboscis and gut samples were analysed for trypanosome DNA by molecular amplification of the internal transcribed spacer 1 (ITS1) region and part of the trypanosome specific glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene.

Results: The most abundant Trypanosoma species found in the tsetse gut was T. grayi, a trypanosome infecting crocodiles. It was ubiquitously distributed throughout the country, accounting for over 90% of all cases involving trypanosomes. Trypanosoma congolense was detected in gut samples from all locations except Cross River National Park, but not in the proboscis, while T. brucei (sensu lato) was not detected at all. In proboscis samples, T. vivax was the most prominent. The sequence diversity of gGAPDH suggests that T. vivax and T. grayi represent genetically diverse species clusters. This implies that they are highly dynamic populations.

Conclusions: The prevalence of animal pathogenic trypanosomes throughout Nigeria emphasises the role of protected areas as reservoirs for livestock trypanosomes. The genetic diversity observed within T. vivax and T. grayi populations might be an indication for changing pathogenicity or host range and the origin and consequences of this diversity has to be further investigated.
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http://dx.doi.org/10.1186/s13071-019-3718-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6792248PMC
October 2019

Novel specific human and mouse stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10) antibodies for biochemical and immunohistochemical analyses.

Wound Repair Regen 2019 07 7;27(4):309-323. Epub 2019 Mar 7.

Wound Repair Unit, Centre for Biomolecular Interactions Bremen, Department of Biology and Biochemistry, University of Bremen, Bremen, Germany.

Matrix metalloproteinases (MMP) are a family of more than 25 zinc-dependent enzymes that are centrally involved in cellular migration, tissue remodeling, cancer invasion and metastasis. Besides degrading extracellular matrix proteins, MMPs are crucial for growth factor and cytokine release and activation. At the same time, they can inactivate inflammatory mediators and enzymes themselves through protein degradation. Subclasses of MMPs include collagenases, gelatinases, stromelysins, membrane-bound MMPs, and others. With regard to the stromelysin subfamily, three members exist, e.g., stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), and stromelysin-3 (MMP-11). MMP-3, and MMP-10 share extensive similarities at the amino acid level that made it difficult to develop specific antibodies distinguishing between MMP-3 and MMP-10. Scrutinizing published data on and performing different analyses with detection of both stromelysins with commercially available or lab-made antibodies showed ambiguous results with regard to specificity of antibodies used to date. We developed new specific antibodies against the most divergent parts of the active forms of both proteins. We assessed the specificity of our novel specific anti-human and anti-mouse MMP-3 and MMP-10 antibodies in cell lysates and different human and murine skin tissues. Tests analyzing specificity of the novel antibodies included Western immunoblotting, immunofluorescence, and immunohistochemistry on paraffin sections. Analyses demonstrated specific detection of respective protein for human or mouse samples except for the anti-human MMP-3 antibody. The aim of this summary was to call attention the MMP research community to distinguish clearly between both enzymes. Our new specific anti-mouse MMP-3 and both MMP-10 antibodies allow us to address this detection problem and to enable comparative studies between both stromelysins with regard to their respective location and function in the tissue.
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http://dx.doi.org/10.1111/wrr.12704DOI Listing
July 2019

Enhancing vector refractoriness to trypanosome infection: achievements, challenges and perspectives.

BMC Microbiol 2018 11 23;18(Suppl 1):179. Epub 2018 Nov 23.

Molecular Department, Vector and Vector Borne Diseases Institute, Tanzania Veterinary Laboratory Agency, Majani Mapana, Off Korogwe Road, Box, 1026, Tanga, Tanzania.

With the absence of effective prophylactic vaccines and drugs against African trypanosomosis, control of this group of zoonotic neglected tropical diseases depends the control of the tsetse fly vector. When applied in an area-wide insect pest management approach, the sterile insect technique (SIT) is effective in eliminating single tsetse species from isolated populations. The need to enhance the effectiveness of SIT led to the concept of investigating tsetse-trypanosome interactions by a consortium of researchers in a five-year (2013-2018) Coordinated Research Project (CRP) organized by the Joint Division of FAO/IAEA. The goal of this CRP was to elucidate tsetse-symbiome-pathogen molecular interactions to improve SIT and SIT-compatible interventions for trypanosomoses control by enhancing vector refractoriness. This would allow extension of SIT into areas with potential disease transmission. This paper highlights the CRP's major achievements and discusses the science-based perspectives for successful mitigation or eradication of African trypanosomosis.
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http://dx.doi.org/10.1186/s12866-018-1280-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251094PMC
November 2018

High-affinity heterotetramer formation between the large myelin-associated glycoprotein and the dynein light chain DYNLL1.

J Neurochem 2018 12 26;147(6):764-783. Epub 2018 Nov 26.

Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland.

The close association of myelinated axons and their myelin sheaths involves numerous intercellular molecular interactions. For example, myelin-associated glycoprotein (MAG) mediates myelin-to-axon adhesion and signalling via molecules on the axonal surface. However, knowledge about intracellular binding partners of myelin proteins, including MAG, has remained limited. The two splice isoforms of MAG, S- and L-MAG, display distinct cytoplasmic domains and spatiotemporal expression profiles. We used yeast two-hybrid screening to identify interaction partners of L-MAG and found the dynein light chain DYNLL1 (also termed dynein light chain 8). DYNLL1 homodimers are known to facilitate dimerization of target proteins. L-MAG and DYNLL1 associate with high affinity, as confirmed with recombinant proteins in vitro. Structural analyses of the purified complex indicate that the DYNLL1-binding segment is localized close to the L-MAG C terminus, next to the Fyn kinase Tyr phosphorylation site. The crystal structure of the complex between DYNLL1 and its binding segment on L-MAG shows 2 : 2 binding in a parallel arrangement, indicating a heterotetrameric complex. The homology between L-MAG and previously characterized DYNLL1-ligands is limited, and some details of binding site interactions are unique for L-MAG. The structure of the complex between the entire L-MAG cytoplasmic domain and DYNLL1, as well as that of the extracellular domain of MAG, were modelled based on small-angle X-ray scattering data, allowing structural insights into L-MAG interactions on both membrane surfaces. Our data imply that DYNLL1 dimerizes L-MAG, but not S-MAG, through the formation of a specific 2 : 2 heterotetramer. This arrangement is likely to affect, in an isoform-specific manner, the functions of MAG in adhesion and myelin-to-axon signalling. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Read the Editorial Highlight for this article on page 712.
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http://dx.doi.org/10.1111/jnc.14598DOI Listing
December 2018

Molecular screening of tsetse flies and cattle reveal different Trypanosoma species including T. grayi and T. theileri in northern Cameroon.

Parasit Vectors 2017 12 29;10(1):631. Epub 2017 Dec 29.

TOZARD Research Laboratory, P.O. Box 59, Bambili-Tubah, Bamenda, Cameroon.

Background: African trypanosomes are mainly transmitted through the bite of tsetse flies (Glossina spp.). The present study investigated the occurrence of pathogenic trypanosomes in tsetse flies and cattle in tsetse fly-infested areas of Northern Cameroon.

Results: Trypanosomes were identified using nested polymerase chain reaction (PCR) analysis of internal transcribed spacer 1 (ITS1) region, both by size estimation and sequencing of PCR products. Apparent density indices recorded in Gamba and Dodeo were 3.1 and 3.6 tsetse flies per trap and day, respectively. Trypanosoma prevalence infection rate for the tsetse fly gut (40%) and proboscis (19%) were recorded. Among the flies where trypanosomes were detected in the gut, 41.7% were positive for T. congolense and 14.6% for T. brucei ssp., whereas in the proboscis 36% harboured T. congolense and 62% contained T. vivax. T. grayi was highly prevalent in tsetse fly gut (58%). The most common mixed infections were the combination of T. congolense and T. grayi. Trypanosome prevalence rate in cattle blood was 6%. Among these, T. vivax represented 26%, T. congolense 35%, T. brucei ssp. 17% and T. theileri 17% of the infections. Surprisingly, in one case T. grayi was found in cattle. The mean packed cell volume (PCV) of cattle positive for trypanosomes was significantly lower (24.1 ± 5.6%; P < 0.05) than that of cattle in which trypanosomes were not detected (27.1 ± 4.9%). Interestingly, the occurrence of T. theileri or T. grayi DNA in cattle also correlated with low PCV at pathological levels.

Conclusion: This molecular epidemiological study of Trypanosoma species in Northern Cameroon revealed active foci of trypanosomes in Dodeo and Gamba. These findings are relevant in assessing the status of trypanosomosis in these regions and will serve as a guide for setting the priorities of the government in the control of the disease.
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http://dx.doi.org/10.1186/s13071-017-2540-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5747950PMC
December 2017

Siglec-7 restores β-cell function and survival and reduces inflammation in pancreatic islets from patients with diabetes.

Sci Rep 2017 04 5;7:45319. Epub 2017 Apr 5.

Centre for Biomolecular Interactions Bremen, University of Bremen, Germany.

Chronic inflammation plays a key role in both type 1 and type 2 diabetes. Cytokine and chemokine production within the islets in a diabetic milieu results in β-cell failure and diabetes progression. Identification of targets, which both prevent macrophage activation and infiltration into islets and restore β-cell functionality is essential for effective diabetes therapy. We report that certain Sialic-acid-binding immunoglobulin-like-lectins (siglecs) are expressed in human pancreatic islets in a cell-type specific manner. Siglec-7 was expressed on β-cells and down-regulated in type 1 and type 2 diabetes and in infiltrating activated immune cells. Over-expression of Siglec-7 in diabetic islets reduced cytokines, prevented β-cell dysfunction and apoptosis and reduced recruiting of migrating monocytes. Our data suggest that restoration of human Siglec-7 expression may be a novel therapeutic strategy targeted to both inhibition of immune activation and preservation of β-cell function and survival.
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http://dx.doi.org/10.1038/srep45319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381285PMC
April 2017

Intra- or extra-exosomal secretion of HDGF isoforms: the extraordinary function of the HDGF-A N-terminal peptide.

Biol Chem 2017 06;398(7):793-811

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Hepatoma-derived growth factor (HDGF) is a protein with diverse intracellular functions. Moreover, after non-conventional secretion, extracellular HDGF is able to influence different signaling pathways, leading for example to induction of processes like epithelial-mesenchymal transition (EMT) and cell migration. Intriguingly, in recent proteome studies, HDGF was also found secreted by special microvesicles called exosomes. Recently, we demonstrated the existence of two new HDGF isoforms (B and C). These isoforms are involved in different cellular processes than HDGF-A. Along this line, in the present study we discovered that full length HDGF-A clearly is located inside of exosomes, whereas the isoforms HDGF-B and HDGF-C are found exclusively on the outer surface. Furthermore, while HDGF-B and HDGF-C seem to use exosomes mediated pathway exclusively, HDGF-A was found also as unbound protein in the conditioned media. The new finding of an intra- or extra-exosomal localisation of protein splice variants opens a fascinating new perspective concerning functional diversity of HDGF isoforms. Dysregulation of HDGF expression during cancer development and tumor progression is a commonly known fact. With our new findings, unraveling the potential functional impact according to physiological versus pathophysiologically altered levels and compositions of intra- and extra-exosomal HDGF has to be addressed in future studies.
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http://dx.doi.org/10.1515/hsz-2016-0315DOI Listing
June 2017

Structural characterisation of high affinity Siglec-2 (CD22) ligands in complex with whole Burkitt's lymphoma (BL) Daudi cells by NMR spectroscopy.

Sci Rep 2016 11 3;6:36012. Epub 2016 Nov 3.

Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland, 4222, Australia.

Siglec-2 undergoes constitutive endocytosis and is a drug target for autoimmune diseases and B cell-derived malignancies, including hairy cell leukaemia, marginal zone lymphoma, chronic lymphocytic leukaemia and non-Hodgkin's lymphoma (NHL). An alternative to current antibody-based therapies is the use of liposomal nanoparticles loaded with cytotoxic drugs and decorated with Siglec-2 ligands. We have recently designed the first Siglec-2 ligands (9-biphenylcarboxamido-4-meta-nitrophenyl-carboxamido-Neu5Acα2Me, 9-BPC-4-mNPC-Neu5Acα2Me) with simultaneous modifications at C-4 and C-9 position. In the current study we have used Saturation Transfer Difference (STD) NMR spectroscopy to monitor the binding of 9-BPC-4-mNPC-Neu5Acα2Me to Siglec-2 present on intact Burkitt's lymphoma Daudi cells. Pre-treatment of cells with periodate resulted in significantly higher STD NMR signal intensities for 9-BPC-4-mNPC-Neu5Acα2Me as the cells were more susceptible to ligand binding because cis-binding on the cell surface was removed. Quantification of STD NMR effects led to a cell-derived binding epitope of 9-BPC-4-mNPC-Neu5Acα2Me that facilitated the design and synthesis of C-2, C-3, C-4 and C-9 tetra-substituted Siglec-2 ligands showing an 88-fold higher affinity compared to 9-BPC-Neu5Acα2Me. This is the first time a NMR-based binding study of high affinity Siglec-2 (CD22) ligands in complex with whole Burkitt's lymphoma Daudi cells has been described that might open new avenues in developing tailored therapeutics and personalised medicine.
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http://dx.doi.org/10.1038/srep36012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5093622PMC
November 2016

Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies.

PLoS One 2016 30;11(8):e0161778. Epub 2016 Aug 30.

Centre for Biomolecular Interactions Bremen, University of Bremen, Bremen, Germany.

Aims: Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples.

Methods: The established PAXgeneTM blood collection method (Qiagen) was compared with the more recent TempusTM collection and storing system. RNA from blood samples collected by both systems was extracted on columns with the corresponding Norgen and PAX RNA extraction Kits. RNA quantity and quality was compared photometrically, with Ribogreen and by Real-Time PCR analyses of various reference genes (PPIA, β-ACTIN and TUBULIN) and exemplary of SIGLEC-7.

Results: Combining different sampling methods and extraction kits caused strong variations in gene expression. The use of PAXgeneTM and TempusTM collection systems resulted in RNA of good quality and quantity for the respective RNA isolation system. No large inter-donor variations could be detected for both systems. However, it was not possible to extract sufficient RNA of good quality with the PAXgeneTM RNA extraction system from samples collected by TempusTM collection tubes. Comparing only the Norgen RNA extraction methods, RNA from blood collected either by the TempusTM or PAXgeneTM collection system delivered sufficient amount and quality of RNA, but the TempusTM collection delivered higher RNA concentration compared to the PAXTM collection system. The established Pre-analytix PAXgeneTM RNA extraction system together with the PAXgeneTM blood collection system showed lowest CT-values, i.e. highest RNA concentration of good quality. Expression levels of all tested genes were stable and reproducible.

Conclusions: This study confirms that it is not possible to mix or change sampling or extraction strategies during the same study because of large variations of RNA yield and expression levels.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161778PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004844PMC
August 2017

Two new isoforms of the human hepatoma-derived growth factor interact with components of the cytoskeleton.

Biol Chem 2016 May;397(5):417-36

Hepatoma-derived growth factor (HDGF) is involved in diverse, apparently unrelated processes, such as cell proliferation, apoptosis, DNA-repair, transcriptional control, ribosome biogenesis and cell migration. Most of the interactions of HDGF with diverse molecules has been assigned to the hath region of HDGF. In this study we describe two previously unknown HDGF isoforms, HDGF-B and HDGF-C, generated via alternative splicing with structurally unrelated N-terminal regions of their hath region, which is clearly different from the well described isoform, HDGF-A. In silico modeling revealed striking differences near the PHWP motif, an essential part of the binding site for glycosaminoglycans and DNA/RNA. This observation prompted the hypothesis that these isoforms would have distinct interaction patterns with correspondingly diverse roles on cellular processes. Indeed, we discovered specific associations of HDGF-B and HDGF-C with cytoskeleton elements, such as tubulin and dynein, suggesting previously unknown functions of HDGF in retrograde transport, site directed localization and/or cytoskeleton organization. In contrast, the main isoform HDGF-A does not interact directly with the cytoskeleton, but via RNA with messenger ribonucleoprotein (mRNP) complexes. In summary, the discovery of HDGF splice variants with their discrete binding activities and subcellular distributions opened new avenues for understanding its biological function and importance.
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http://dx.doi.org/10.1515/hsz-2015-0273DOI Listing
May 2016

Carbohydrate Recognition Specificity of Trans-sialidase Lectin Domain from Trypanosoma congolense.

PLoS Negl Trop Dis 2015 16;9(10):e0004120. Epub 2015 Oct 16.

Centre for Biomolecular Interactions Bremen, Faculty for Biology and Chemistry, University Bremen, Bremen, Germany; Africa Centre of Excellence for Neglected Tropical Diseases and Forensic Biotechnology, Ahmadu Bello University, Zaria, Nigeria; Institute for Glycomics, Griffith University Gold Coast Campus, Queensland, Australia.

Fourteen different active Trypanosoma congolense trans-sialidases (TconTS), 11 variants of TconTS1 besides TconTS2, TconTS3 and TconTS4, have been described. Notably, the specific transfer and sialidase activities of these TconTS differ by orders of magnitude. Surprisingly, phylogenetic analysis of the catalytic domains (CD) grouped each of the highly active TconTS together with the less active enzymes. In contrast, when aligning lectin-like domains (LD), the highly active TconTS grouped together, leading to the hypothesis that the LD of TconTS modulates its enzymatic activity. So far, little is known about the function and ligand specificity of these LDs. To explore their carbohydrate-binding potential, glycan array analysis was performed on the LD of TconTS1, TconTS2, TconTS3 and TconTS4. In addition, Saturation Transfer Difference (STD) NMR experiments were done on TconTS2-LD for a more detailed analysis of its lectin activity. Several mannose-containing oligosaccharides, such as mannobiose, mannotriose and higher mannosylated glycans, as well as Gal, GalNAc and LacNAc containing oligosaccharides were confirmed as binding partners of TconTS1-LD and TconTS2-LD. Interestingly, terminal mannose residues are not acceptor substrates for TconTS activity. This indicates a different, yet unknown biological function for TconTS-LD, including specific interactions with oligomannose-containing glycans on glycoproteins and GPI anchors found on the surface of the parasite, including the TconTS itself. Experimental evidence for such a scenario is presented.
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http://dx.doi.org/10.1371/journal.pntd.0004120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608562PMC
April 2016

Splice variants of perlucin from Haliotis laevigata modulate the crystallisation of CaCO3.

PLoS One 2014 13;9(5):e97126. Epub 2014 May 13.

Centre for Biomolecular Interactions Bremen, Department of Biology and Chemistry, University Bremen, Bremen, Germany.

Perlucin is one of the proteins of the organic matrix of nacre (mother of pearl) playing an important role in biomineralisation. This nacreous layer can be predominately found in the mollusc lineages and is most intensively studied as a compound of the shell of the marine Australian abalone Haliotis laevigata. A more detailed analysis of Perlucin will elucidate some of the still unknown processes in the complex interplay of the organic/inorganic compounds involved in the formation of nacre as a very interesting composite material not only from a life science-based point of view. Within this study we discovered three unknown Perlucin splice variants of the Australian abalone H. laevigata. The amplified cDNAs vary from 562 to 815 base pairs and the resulting translation products differ predominantly in the absence or presence of a varying number of a 10 mer peptide C-terminal repeat. The splice variants could further be confirmed by matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF MS) analysis as endogenous Perlucin, purified from decalcified abalone shell. Interestingly, we observed that the different variants expressed as maltose-binding protein (MBP) fusion proteins in E. coli showed strong differences in their influence on precipitating CaCO3 and that these differences might be due to a splice variant-specific formation of large protein aggregates influenced by the number of the 10 mer peptide repeats. Our results are evidence for a more complex situation with respect to Perlucin functional regulation by demonstrating that Perlucin splice variants modulate the crystallisation of calcium carbonate. The identification of differentially behaving Perlucin variants may open a completely new perspective for the field of nacre biomineralisation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0097126PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019660PMC
December 2014

Biochemical diversity in the Trypanosoma congolense trans-sialidase family.

PLoS Negl Trop Dis 2013 5;7(12):e2549. Epub 2013 Dec 5.

Centre for Biomolecular Interactions Bremen, Faculty for Biology and Chemistry, University Bremen, Bremen, Germany.

Trans-sialidases are key enzymes in the life cycle of African trypanosomes in both, mammalian host and insect vector and have been associated with the disease trypanosomiasis, namely sleeping sickness and nagana. Besides the previously reported TconTS1, we have identified three additional active trans-sialidases, TconTS2, TconTS3 and TconTS4, and three trans-sialidase like genes in Trypanosoma congolense. At least TconTS1, TconTS2 and TconTS4 are found in the bloodstream of infected animals. We have characterised the enzymatic properties of recombinant proteins expressed in eukaryotic fibroblasts using fetuin as model blood glycoprotein donor substrate. One of the recombinant trans-sialidases, TconTS2, had the highest specific activity reported thus far with very low sialidase activity. The active trans-sialidases share all the amino acids critical for the catalytic reaction with few variations in the predicted binding site for the leaving or acceptor glycan. However, these differences cannot explain the orders of magnitudes between their transfer activities, which must be due to other unidentified structural features of the proteins or substrates selectivity. Interestingly, the phylogenetic relationships between the lectin domains correlate with their specific trans-sialylation activities. This raises the question whether and how the lectin domains regulate the trans-sialidase reaction. The identification and enzymatic characterisation of the trans-sialidase family in T. congolense will contribute significantly towards the understanding of the roles of these enzymes in the pathogenesis of Animal African Trypanosomiasis.
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http://dx.doi.org/10.1371/journal.pntd.0002549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3855035PMC
July 2014

C-4 modified sialosides enhance binding to Siglec-2 (CD22): towards potent Siglec inhibitors for immunoglycotherapy.

Angew Chem Int Ed Engl 2013 Mar 25;52(13):3616-20. Epub 2013 Feb 25.

Centre for Biomolecular Interactions Bremen, Department of Biology and Chemistry, University of Bremen, 28334 Bremen, Germany.

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http://dx.doi.org/10.1002/anie.201207267DOI Listing
March 2013

Characterisation and metabolism of astroglia-rich primary cultures from cathepsin K-deficient mice.

Biol Chem 2012 Sep;393(9):959-70

School of Engineering and Science, Research Center MOLIFE, Molecular Life Science, Jacobs University Bremen, Campus Ring 1, D-28759 Bremen, Germany.

Cathepsin K is important for the brain, because its deficiency in mice is associated with a marked decrease in differentiated astrocytes and changes in neuronal patterning in the hippocampus as well as with learning and memory deficits. As cathepsin K activity is most prominent in hippocampal regions of wild type animals, we hypothesised alterations in astrocyte-mediated support of neurons as a potential mechanism underlying the impaired brain functions in cathepsin K-deficient mice. To address this hypothesis, we have generated and characterised astroglia-rich primary cell cultures from cathepsin K-deficient and wild type mice and compared these cultures for possible changes in metabolic support functions and cell composition. Interestingly, cells expressing the oligodendrocytic markers myelin-associated glycoprotein and myelin basic protein were more frequent in astroglia-rich cultures from cathepsin K-deficient mice. However, cell cultures from both genotypes were morphologically comparable and similar with respect to glucose metabolism. In addition, specific glutathione content, glutathione export and γ-glutamyl-transpeptidase activity remained unchanged, whereas the specific activities of glutathione reductase and glutathione-S-transferase were increased by around 50% in cathepsin K-deficient cultures. Thus, lack of cathepsin K in astroglia-rich cultures appears not to affect metabolic supply functions of astrocytes but to facilitate the maturation of oligodendrocytes.
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http://dx.doi.org/10.1515/hsz-2012-0145DOI Listing
September 2012

Purification and biochemical characterization of a lysosomal α-fucosidase from the deuterostomia Asterias rubens.

Biochimie 2012 May 17;94(5):1199-205. Epub 2012 Feb 17.

Protein Biochemistry and Molecular Biology Laboratory, Department of Biochemistry, University of Hyderabad, Hyderabad 500 046, India.

In vertebrates, mannose 6-phosphate receptors [MPR300 (Mr 300 kDa) and MPR46 (Mr 46 kDa)] are highly conserved transmembrane glycoproteins that mediate transport of lysosomal enzymes to lysosomes. Our studies have revealed the appearance of these putative receptors in invertebrates such as the molluscs and deuterostomes. Starfish tissue extracts contain several lysosomal enzyme activities and here we describe the affinity purification of α-fucosidase. The purified enzyme is a glycoprotein that exhibited a molecular mass of ∼56 kDa in SDS-PAGE under reducing conditions. It has also cross-reacted with an antiserum to the mollusc enzyme suggesting antigenic similarities among the two invertebrate enzymes. LC-MS/MS analysis of the proteolytic peptides of the purified enzyme in combination with de novo sequencing allowed us to do partial amino acid sequence determination of the enzyme. These data suggest that this invertebrate enzyme is homologous to the known mammalian enzyme. The purified enzyme exhibited a mannose 6-phosphate dependent interaction with the immobilized starfish MPR300 protein. Our results demonstrate that the lysosomal enzyme targeting pathway is conserved even among the invertebrates.
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http://dx.doi.org/10.1016/j.biochi.2012.02.007DOI Listing
May 2012

Interaction of HRP-2 isoforms with HDGF: chromatin binding of a specific heteromer.

FEBS J 2012 Mar 12;279(5):737-51. Epub 2012 Jan 12.

Department of Biochemistry, Centre for Biomolecular Interactions Bremen (CBIB), University of Bremen, Germany.

Hepatoma-derived growth-factor-related protein 2 (HRP-2) belongs to a family with five additional members: hepatoma-derived growth factor (HDGF); lens epithelium-derived growth factor; and the HDGF-related proteins -1, -3 and -4. Very little is known regarding the function of HRP-2 in particular. This study shows for the first time heteromer formation of different members of the HRP family; HDGF and HRP-2. In addition, we discovered a previously unknown splice variant of HRP-2 mRNA encoding for a protein with a 53-amino acid deletion in its hath region. This HRP-2 isoform c interacts preferentially with a processed form of HDGF probably because of the loss of an α helix of HRP-2. Furthermore, in contrast to other isoforms of HRP-2, isoform c binds to chromatin similar to its most closely related family member lens epithelium-derived growth factor with potential consequences regarding its function in HIV integration. Interestingly, only the new HRP-2 isoform c and a processed form of HDGF are displaced from condensed mitotic metaphase chromatin. In conclusion, these observations provide a new perspective for understanding the biological functions of HDGF and related proteins.
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http://dx.doi.org/10.1111/j.1742-4658.2011.08464.xDOI Listing
March 2012

From a library of MAG antagonists to nanomolar CD22 ligands.

ChemMedChem 2012 Jan 11;7(1):134-43. Epub 2011 Oct 11.

Institute of Molecular Pharmacy, Pharmacenter, University of Basel, Basel, Switzerland.

Siglec-2, also known as CD22, is involved in the regulation and survival of B-cells and has been successfully targeted in cell depletion therapies with antibody-based approaches. Sialic acid derivatives, already known to bind with high affinity to myelin-associated glycoprotein (MAG, Siglec-4), were screened for their binding affinity for CD22 by surface plasmon resonance. The best compound identified was further modified with various hydrophobic substituents at the 2-, 5-, and 9-positions of the sialic acid scaffold, leading to nanomolar derivatives, of which ligand 17 b shows the most promising pharmacodynamic and pharmacokinetic profiles. Isothermal titration calorimetry measurements demonstrate that the binding is enthalpy driven. Interestingly, the thermodynamic fingerprints reveal an excellent correlation between gains in enthalpy and compensation by increased entropy costs. Moreover, 17 b exhibits a residence time in the range of a few seconds, clearly prolonged relative to residence times typically observed for carbohydrate-lectin interactions. Finally, initial tests regarding drug-like properties of 17 b demonstrate the required high plasma protein binding yet a lack of oral availability, although its distribution coefficient (log D) is in the required range.
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http://dx.doi.org/10.1002/cmdc.201100407DOI Listing
January 2012

A novel type of macrothrombocytopenia associated with a defect in α2,3-sialylation.

Am J Pathol 2011 Oct 22;179(4):1969-77. Epub 2011 Aug 22.

Max Planck Institute for Molecular Biomedicine, Münster, Germany.

We describe a novel type of human thrombocytopenia characterized by the appearance of giant platelets and variable neutropenia. Searching for the molecular defect, we found that neutrophils had strongly reduced sialyl-Lewis X and increased Lewis X surface expression, pointing to a deficiency in sialylation. We show that the glycosylation defect is restricted to α2,3-sialylation and can be detected in platelets, neutrophils, and monocytes. Platelets exhibited a distorted structure of the open canalicular system, indicating defective platelet generation. Importantly, patient platelets, but not normal platelets, bound to the asialoglycoprotein receptor (ASGP-R), a liver cell-surface protein that removes desialylated thrombocytes from the circulation in mice. Taken together, this is the first type of human thrombocytopenia in which a specific defect of α2,3-sialylation and an induction of platelet binding to the liver ASGP-R could be detected.
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http://dx.doi.org/10.1016/j.ajpath.2011.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3181377PMC
October 2011

Biochemical characterization of trans-sialidase TS1 variants from Trypanosoma congolense.

BMC Biochem 2011 Jul 30;12:39. Epub 2011 Jul 30.

Centre for Biomolecular Interactions Bremen, Department of Biology and Chemistry, University of Bremen, Germany.

Background: Animal African trypanosomiasis, sleeping sickness in humans and Nagana in cattle, is a resurgent disease in Africa caused by Trypanosoma parasites. Trans-sialidases expressed by trypanosomes play an important role in the infection cycle of insects and mammals. Whereas trans-sialidases of other trypanosomes like the American T. cruzi are well investigated, relatively little research has been done on these enzymes of T. congolense.

Results: Based on a partial sequence and an open reading frame in the WTSI database, DNA sequences encoding for eleven T. congolense trans-sialidase 1 variants with 96.3% overall amino acid identity were amplified. Trans-sialidase 1 variants were expressed as recombinant proteins, isolated and assayed for trans-sialylation activity. The purified proteins produced α2,3-sialyllactose from lactose by desialylating fetuin, clearly demonstrating their trans-sialidase activity. Using an HPLC-based assay, substrate specificities and kinetic parameters of two variants were characterized in detail indicating differences in substrate specificities for lactose, fetuin and synthetic substrates. Both enzymes were able to sialylate asialofetuin to an extent, which was sufficient to reconstitute binding sites for Siglec-4. A mass spectrometric analysis of the sialylation pattern of glycopeptides from fetuin revealed clear but generally similar changes in the sialylation pattern of the N-glycans on fetuin catalyzed by the trans-sialidases investigated.

Conclusions: The identification and characterization of a trans-sialidase gene family of the African parasite T. congolense has opened new perspectives for investigating the biological role of these enzymes in Nagana and sleeping sickness. Based on this study it will be interesting to address the expression pattern of these genes and their activities in the different stages of the parasite in its infection cycle. Furthermore, these trans-sialidases have the biotechnological potential to be used for enzymatic modification of sialylated glycoconjugates.
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http://dx.doi.org/10.1186/1471-2091-12-39DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3173295PMC
July 2011

Impact of strong and weak lipid-protein interactions on the structure of a lipid bilayer on a gold electrode surface.

Chemphyschem 2011 Apr 25;12(6):1066-79. Epub 2011 Mar 25.

Center of Interface Science (CIS), Department of Pure and Applied Chemistry, Carl von Ossietzky University of Oldenburg, Oldenburg, Germany.

A lipid bilayer deposited on an electrode surface can serve as a benchmark system to investigate lipid-protein interactions in the presence of physiological electric fields. Recoverin and myelin-associated glycoprotein (MAG) are used to study the impact of strong and weak protein-lipid interactions on the structure of model lipid bilayers, respectively. The structural changes in lipid bilayers are followed using electrochemical polarization modulation infrared reflection-absorption spectroscopy (PM IRRAS). Recoverin contains a myristoyl group that anchors in the hydrophobic part of a cell membrane. Insertion of the protein into the 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC)-cholesterol lipid bilayer leads to an increase in the capacitance of the lipid film adsorbed on a gold electrode surface. The stability and kinetics of the electric-field-driven adsorption-desorption process are not affected by the interaction with protein. Upon interaction with recoverin, the hydrophobic hydrocarbon chains become less ordered. The polar head groups are separated from each other, which allows for recoverin association in the membrane. MAG is known to interact with glycolipids present on the surface of a cell membrane. Upon probing the interaction of the DMPC-cholesterol-glycolipid bilayer with MAG a slight decrease in the capacity of the adsorbed lipid film is observed. The stability of the lipid bilayer increases towards negative potentials. At the molecular scale this interaction results in minor changes in the structure of the lipid bilayer. MAG causes small ordering in the hydrocarbon chains region and an increase in the hydration of the polar head groups. Combining an electrochemical approach with a structure-sensitive technique, such as PM IRRAS, is a powerful tool to follow small but significant changes in the structure of a supramolecular assembly.
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http://dx.doi.org/10.1002/cphc.201100036DOI Listing
April 2011

Secretion of hepatoma-derived growth factor is regulated by N-terminal processing.

Biol Chem 2010 Dec;391(12):1401-10

Center for Biomolecular Interactions Bremen, Department of Biochemistry, University of Bremen, Germany.

Hepatoma-derived growth factor (HDGF) was first purified as a growth factor secreted by hepatoma cells. It promotes angiogenesis and has been related to tumorigenesis. To date, little is known about the molecular mechanisms of HDGF functions and especially its routes or regulation of secretion. Here we show that secretion of HDGF requires the N-terminal 10 amino acids and that this peptide can mediate secretion of other proteins, such as enhanced green fluorescent protein, if fused to their N-terminus. Our results further demonstrate that cysteine residues at positions 12 and 108 are linked via an intramolecular disulfide bridge. Surprisingly, phosphorylation of serine 165 in the C-terminal part of HDGF plays a critical role in the secretion process. If this serine is replaced by alanine, the N-terminus is truncated, the intramolecular disulfide bridge is not formed and the protein is not secreted. In summary, these observations provide a model of how phosphorylation, a disulfide bridge and proteolytic cleavage are involved in HDGF secretion.
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http://dx.doi.org/10.1515/BC.2010.147DOI Listing
December 2010

Design, synthesis, biological evaluation, and modeling of a non-carbohydrate antagonist of the myelin-associated glycoprotein.

Bioorg Med Chem 2010 Oct 17;18(20):7239-51. Epub 2010 Aug 17.

Institute of Molecular Pharmacy, Pharmacenter, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.

Broad modifications of various positions of the minimal natural epitope recognized by the myelin-associated glycoprotein (MAG), a blocker of regeneration of neurite injuries, produced sialosides with nanomolar affinities. However, important pharmacokinetic issues, for example, the metabolic stability of these sialosides, remain to be addressed. For this reason, the novel non-carbohydrate mimic 3 was designed and synthesized from (-)-quinic acid. For the design of 3, previously identified beneficial modifications of side chains of Neu5Ac were combined with the replacement of the ring oxygen by a methylene group and the substitution of the C(4)-OH by an acetamide. Although docking experiments to a homology model of MAG revealed that mimic 3 forms all but one of the essential hydrogen bonds identified for the earlier reported lead 2, its affinity was substantially reduced. Extensive molecular-dynamics simulation disclosed that the missing hydrogen bond of the former C(8)-OH leads to a change of the orientation of the side chain. As a consequence, an important hydrophobic contact is compromised leading to a loss of affinity.
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http://dx.doi.org/10.1016/j.bmc.2010.08.027DOI Listing
October 2010

Examination of the biological role of the alpha(2-->6)-linked sialic acid in gangliosides binding to the myelin-associated glycoprotein (MAG).

J Med Chem 2009 Feb;52(4):989-1004

Institute of Molecular Pharmacy, Pharmacenter, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.

The tetrasaccharide 1, a substructure of ganglioside GQ1b alpha, shows a remarkable affinity for the myelin-associated glycoprotein (MAG) and was therefore selected as starting point for a lead optimization program. In our search for structurally simplified and pharmacokinetically improved mimics of 1, modifications of the core disaccharide, the alpha(2-->3)- and the alpha(2-->6)-linked sialic acid were synthesized. Biphenylmethyl and (S)-lactate were identified as suitable replacements for the alpha(2-->6)-linked sialic acid. Combined with a core modification and the earlier found aryl amide substituent in the 9-position of the alpha(2-->3)-linked sialic acid, high affinity MAG antagonists were identified. All mimics were tested in a competitive target-based binding assay, providing relative inhibitory potencies (rIP). Compared to the reference tetrasaccharide 1, the rIPs of the most potent antagonists 59 and 60 are enhanced nearly 400-fold. Their K(D)s determined in surface plasmon resonance experiments are in the low micromolar range. These results are in semiquantitative agreement with molecular modeling studies. This new class of glycomimetics will allow to validate the role of MAG in the axon regeneration process.
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http://dx.doi.org/10.1021/jm801058nDOI Listing
February 2009