Structures, Biosynthesis and Biological Implications of Phosphorylcholine Substituted Antigens

Home , Brugia pahangi, Dimethylethanolamine, Megathura crenulata, Phosphorylcholine

Aus dem Biochemischen Institut

Leiter: Prof. Dr. Klaus T. Preissner

Fachbereich Humanmedizin der Justus-Liebig-Universität Gießen

Immunomodulation by nematodes:

Structures, biosynthesis and biological implications of phosphorylcholine substituted antigens

Habilitationsschrift zur Erlangung der Venia legendi des Fachbereichs Humanmedizin der Justus-Liebig-Universität Gießen

vorgelegt von Dr. rer. nat. Günter Lochnit

Gießen, 2003

Dedicated to all people suffering from nematode infections.

Abbreviations A. ceylanicum Ancylostoma ceylanicum AHC Adenosylhomocysteine A. suum Ascaris suum A. lumbricoides Ascaris lumbricoides A. viteae Acanthocheilonema viteae BCR B cell receptor B. malayi Brugia malayi B. pahangi Brugia pahangi CDP cytidine diphosphate C. elegans Caenorhabditis elegans Cer ceramide CPH ceramide pentahexoside C. vicina Calliphora vicina DAG diacylglycerol ESI-IT-MS electrospray-ionization ion-trap mass spectrometry FITC fluorescein isothiocyanate Fuc L-fucose Gal D-galactose GalNAc 2-acetamido-2-deoxy-D-galactose Glc D-glucose GlcNAc 2-acetamido-2-deoxy-D-glucose GLC/MS gas-liquid chromatography mass spectrometry GSL glycosphingolipid H. contortus Haemonchus contortus Hex hexose HexNAc N-acetylhexosamine HF hydrogen fluoride HPLC high-performance liquid chromatography HPTLC high-performance thin-layer chromatography HSA human serum albumin IFN-γ interferon gamma Ig immunoglobulin IL interleukin Ins inositol LPS lipopolysaccharide L. sigmodontis Litomosoides sigmodontis LSIMS liquid secondary-ion mass spectrometry MALDI-TOF-MS matrix-assisted laser desorption-ionization time-of-flight mass spectrometry Man D-mannose MHC major histocompatibility complex M. libycus Meriones libycus N. americanus Necator americanus N. brasiliensis Nippostrongylus brasiliensis NB-DNJ N-butyl deoxynojirimycin NN-DNJ N-nonyl deoxynojirimycin NMR nuclear magnetic resonance O. volvulus Onchocerca volvulus O. gibsoni Onchocerca gibsoni

O. moubata Ornithodorus moubata P phosphate PAF platelet activating factor PBMC peripheral blood mononuclear cell(s) PC phosphorylcholine PDMP DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride PE phosphorylethanolamine Ptd phosphatidyl Ptd-C phosphatidylcholine Ptd-E phosphatidylethanolamine RER rough endoplasmic reticulum SAM S-adenosyl-methionine SM sphingomyelin S. digitata Setaria digitata S. stercoralis Strongyloides stercoralis T. cruzi Trypanosoma cruzi T. canis Toxocara canis Th T-helper TNF-α tumor necrosis factor alpha TLR toll-like receptor T. spiralis Trichinella spiralis T. trichiura Trichuris trichiura W. bancrofti Wuchereria bancrofti

1 Introduction ...... 1

1.1 Immune evasion strategies of pathogens ...... 2

1.2 Phylogeny and morphology of nematodes...... 3

1.3 Model organisms...... 7

1.3.1 Caenorhabditis elegans, a free living nematode ...... 7

1.3.2 Ascaris suum, an intestinal parasite ...... 8

1.3.3 Acanthocheilonema viteae, a model for filariasis ...... 9

1.4 Phosphorylcholine as an immunomodulatory substituent ...... 9

1.5 Aims of the work...... 13

2 Results and discussion ...... 14

2.1 Antigens with phosphorylcholine epitopes ...... 14

2.1.1 A. suum glycosphingolipids reflect the high glycosylation potential of nematodes...... 14

2.1.2 Phosphorylcholine-substituted glycosphingolipids are phylogenetic markers of nematodes...... 19

2.1.3 C. elegans secretes a putative aspartyl protease carrying a novel phosphorylcholine epitope ...... 21

2.1.4 A. suum shows a widespread distribution of the PC-epitope..22

2.1.5 The expression of this epitope is developmentally regulated in C. elegans ...... 26

2.2 Biosynthesis of phosphorylcholine containing molecules.....28

2.2.1 C. elegans exhibits the classical Kennedy pathway ...... 28

2.2.2 Indications for a complex choline metabolism in nematodes..30

2.2.3 Inhibitors of the choline metabolism and glycosphingolipid biosynthesis have anthelminthic potential...... 33

2.3 Biological implications of phosphorylcholine epitopes...... 39

2.3.1 Zwitterionic glycosphingolipids induce proinflammatory cytokines...... 39

2.3.2 The modulation of the host´s innate and adaptive immune responses by zwitterionic glycosphingolipids can be dissected into PC-dependent and PC-independent effects ...... 40

2.3.3 PC-substituted antigens can bind to the PAF-receptor and are internalized ...... 42

3 Summary...... 45

4 Perspectives...... 46

5 Literature...... 47

6 Supplement...... 62

6.1 Complete list of own publications...... 62

6.2 Submitted publications ...... 64

6.3 Acknowledgements ...... 65

Introduction 1

1 Introduction Parasitism is one of the most interesting biological interactions between organisms. Parasites causing high morbidity and mortality represent early forms of cooperativity, whereas those resulting in low mortality and persisting infections reflect a progressive adaptation of the pathogen to its host. Most parasites infecting higher vertebrates are lower organisms, i.e., invertebrates. These pathogens are confronted with one of the most complex biological systems, the immune system. The central question is, therefore, of the mechanisms that have evolved to invade the host and to evade the host’s immune system to persist for a long time. One fourth of the world’s population is chronically infected by nematodes (roundworms) [1], which are referred to as helminths together with cestodes (tapeworms) and trematodes (flukes). Nematodes can parasitize the eye, mouth, tongue, alimentary canal, liver, lungs or body cavity. The intestinal parasite A. lumbricoides occurs at high levels (1400 million infections/year) under poor hygienic conditions and is often found together with Trichuris trichiura (1000 million infections/year) [2]. The infection results in malnutrition and retardation of growth in children, where infection rates in endemic regions can reach 90% causing pneumonitis, asthma, diarrhea, nausea, abdominal pain and anorexia. Control strategies have focused on the interruption of transmission of infective eggs by water, food and human hands. Drugs used include levamisole, pyrantel, mebendazole, albendazol and ivermectin. Hookworms (A. duodenale and N. americanus) must be classified as one of the most destructive helminth parasites (1300 million infections/year) [3]. A. duodenale occurs mainly in Asia, whereas N. americanus is the predominant species in the New World. The adult stages live in the small intestine and feed on blood and tissue. This results in severe blood loss, the so-called “hookworm anaemia”. Infection can occur by swallowing larvae or skin penetration by the infective larvae. Anthelmintics used include mebendazole, thiabendazole and pyrantel. Strongyloides stercoralis (200 million infections/year) is localized in the duodeno- jejunal region, where it burrows deeply into the mucosa [4]. The life cycle consists of two phases, the parasitic and the free-living one. In the parasitic generation, the adults are always female and parthenogenetic. Males appear in the free-living part of the cycle. Larvae passed in the feces differentiate under favourable external conditions into mature male and female worms, whereas under unfavourable conditions they can undergo direct development into infective larvae. The clinical symptoms are abdominal pain, diarrhea and urticaria. Interestingly, the number of deaths is quite low compared to the large number of infected people. Ascaris and Trichuris cause approximately 70,000 deaths/year and hookworm infections 65,000 deaths/year. The high morbidity but low mortality is a characteristic of long-lasting nematode infections. Obviously, nematodes have developed subtle mechanisms to escape the host’s immune response and to prevent an immune protection over a long period. This is demonstrated by the quick reinfection after anthelminthic treatment. Infected children and pregnant women have the highest risk of malignancy resulting in growth retardation and anaemia. Since most of the anthelminthic drugs used at present have teratogenic potential, they cannot be applied to these groups. Furthermore, some drugs affect only single

Introduction 2 stages of the worms. This shows the urgent need for new drugs with less lower side- effects and a broader application spectrum, which may target of all nematode stages. Knowledge of these adaptation mechanisms could provide valuable information towards the development of new therapeutical strategies. Various model systems, such as Ascaris suum, an intestinal nematode, the filariid Acanthocheilonema viteae and the free living nematode Caenorhabditis elegans, as the best studied metazoan organism so far, have been established for these studies. In the last ten years the small hapten phosphorylcholine (PC) became the center of interest, due to its immunomodulatory capacity. It has turned out that this epitope plays a central role in the development of nematodes, their reproduction and survival as parasites. The present thesis summarizes the work on the structural analysis of PC substituted nematode antigens, their biosynthesis and biological implications.

1.1 Immune evasion strategies of pathogens Most parasitic infections are chronic and persistent, despite eliciting potent anti- pathogen immune responses. This implies that the parasites must have developed equally potent mechanisms of immune evasion [5]. Since a number of mechanisms may be used by the parasite in parallel or are restricted to certain life-cycle stages, most of them are incompletely characterized. Several potential immunomodulatory mechanisms have been proposed, as shown in the following examples. During the filarial genome project, a homologue of the cytokine macrophage migration inhibitory factor (MIF) was sequenced as one of the most abundantly expressed genes. It is proposed to alter inflammatory responses and possibly contribute to the Th2 cytokine bias observed in these infections [6]. Furthermore, the filarial genome project revealed several transforming growth factor ß (TGF-ß) homologues in Brugia spp., which have been discussed as immunomodulatory agents [7, 8]. Serpins, i.e. serine protease inhibitors which are known as inhibitors of a number of immunological activities, including the complement system, IL-1 activation, as well as lytic and apoptotic pathways, have been characterized in Brugia malayi [9]. Other inhibitors of cysteine and aspartyl proteases are involved in major histocompatibility complex (MHC) II antigen processing pathways in human B-cells whilst neutrophil´s protease inhibitors may further contribute to evasion strategies [8]. An inhibitor of cysteine proteases (cystatin) from A. viteae downregulates T-cell proliferation and enhances IL-10 production [10]. Secreted metalloproteinases of the hookworm Necator americanus cleave eotaxin and inhibit the eotaxin-mediated recruitment of eosinophils to sites of inflammation [11]. Cleavage of immunoglobins or complement proteins is a further strategy of immune evasion. Numerous proteases have been described, which e. g. cleave immunoglobin (Ig)G or IgA to yield fragments blocking activation of complement, phagocyte attack mediated by intact IgG or IgM and/or degranulation of eosinophils. A neutrophil inhibitory factor was further found in hookworms, that blocked the adhesion of activated human neutrophils to vascular endothelial cells and inhibited the release of H2O2 [12]. Helminth C-type lectins have been found in C. elegans, Toxocara canis, N. americanus, Nippostrongylus brasiliensis, Ancylostoma ceylanicum, A. suum and Haemonchus contortus, where they are often major surface and secretory products. These molecules can compete with host lectins for binding to ligands involved in selectin-mediated inflammation [13, 14, 15]. In hematophagous

Introduction 3 hookworms these lectins, together with anticoagulant peptides, also inhibit blood clotting by binding to coagulation factors, thus assisting feeding. As major surface molecules they can further acquire host glycoproteins for masking purposes. An additional mechanism of immune evasion is the neutralization of free radicals generated by the oxidative burst of leukocytes by superoxide dismutases found in a range of parasitic nematodes [16]. Glutathione S-transferases secreted by parasitic helminths can neutralize lipid hydroperoxides [17]. Molecular mimicry [18] is a further immune evasion mechanism of parasites. Immune evasion strategies of protozoan parasites belong to the best understood investigated mechanisms. Protozoa often multiply within cells, show a high variation of their surface antigens, antigen shedding and modulation of the host´s immune response [19]. Plasmodium spp. causing malaria evades the human immune response by changing its surface antigens during the different life cycle stages. Development of an effective immune response is further hampered by highly polymorphic and variable stage-specific surface proteins [20]. Antigens with multiple repeats acting as superantigens induce a thymus-independent humoral response that downregulates antibody isotype maturation and production of high-affinity antibodies. The release of acute-phase proteins and their binding to lymphoid cells, thus inhibiting lymphocyte proliferation, is discussed as one explanation for the observed immunosuppression. Trypanosomes, responsible for sleeping sickness, establish the infection by the antigenic variation of the variant surface glycoprotein. The glycophosphatidyl inositol anchor of these proteins can overactivate macrophages, which then become suppressor macrophages that produce tumor necrosis factor (TNF)-α and induce CD8+ cells to secrete interferon (IFN)-γ. This results in a decrease in interleukin (IL)-2 receptor expression and IL-2 synthesis, impairing the proliferative T-cell response. Repetitive polyclonal B-cell activation by antigen variation with subsequent production of IgM antibodies, which can interfere with the binding of inhibitory IgG molecules, prevents elimination of the parasites. Due to their habitat within the mononuclear phagocytes, Leishmania spp. have developed several mechanisms to escape the proteolytic processes and oxidative burst of their host cells. Further strategies include the cleavage of CD4 and MHC II class molecules, downregulation of macrophage co-stimulatory molecules, inhibition of the chemotaxis of neutrophils and monocytes, shedding of complement complexes and suppression of IL-12 production.

1.2 Phylogeny and morphology of nematodes In the past few years the advances in molecular phylogenetics have dramatically changed the traditional tree of life (see Fig. 1 A). Some traditional clades were split (e.g. the Aschelmintha) into highly divergent new branches, whilst formerly strongly distant organisms have been gathered into new groups (e.g. Nematozoa with Arthropoda, Platyhelmintha with Mollusca and Annelida) [21]. About 92% of named parasites belonging to the Protostoma and 87% are in the Ecdysozoa. Most of the remaining parasites are found within the phylum Nematoda, in which the majority of species (60%) are parasitic. The parasitic nematodes can be divided into plant- and animal-specific species (for their phylogenetic relationships see Fig. 1 B; for the classical taxonomy see Fig. 2). Together with the free living species, nematodes represent four out of five animals on the planet, thus reflecting their great success in evolution.

Introduction 4

Vertebrata Cephalochordata Urochordata Deuterostoma Hemichordata Echinodermata Cycliophora Bryozoa Entoprocta Platyhelmintha Brachiopoda Phoronida Nemertea Lophotrochozoa Annelida Echiura Mollusca Sipuncula Protostoma Gnathostomulida Syndermata

Nematozoa Gastrotricha Cephaloryncha Ecdysozoa Onychophora Tardigrada Arthropoda

Rhombozoa Orhonectida Chaetognatha

Fig. 1 Phylogenetic relationships within the Bilatera [21].

Nematodes exhibit a high degree of structural uniformity in basic anatomical organization [22]. They have an elongated cylindrical shape with a body musculature consisting entirely of longitudinal fibers (see Fig. 3). The size of nematodes can vary dramatically. Most of the free-living nematodes are of microscopical size, as are many of the parasitic species invading the blood or lymph channels of their hosts. The intestinal worms are generally larger and some, living in tissue habitats, can reach enormous lengths. The pharynx is one of the most characteristic features of nematodes. Since these worms have a high internal pressure and an intestinal wall comprizing only one cell layer, the intestine can be only filled by a pumping organ. The pharynx consists of muscle, supporting, nerve and gland cells. The epithelial cells of the intestine bear at the inner surface a bacillary layer similar to microvilli. In males, the reproductive system opens into the rectum forming a cloaca. The nervous system contains typically a nerve ring and associated ganglia at the anterior end, major dorsal and ventral nerve cords, together with smaller subdorsal and subventral cords, and a smaller set of ganglia in the posterior region. Interestingly, the nerves do not branch to the muscles, instead, branches of the muscles reach the nerves.

Introduction 5 this work. in detail Species Ancylostoma duodenale Necator americanus Trichuris trichiura anradtselegans Caenorhabditis Strongyloides stercoralis contortus Haemonchus Nippostrongylus brasiliensis Brugia malayi Wuchereria bancrofti Onchocerca volvulus Acanthocheilonema viteae Dirofilaria immitis suum Ascaris lumbricoides Ascaris canis Toxocara spiralis Trichinella Family Bold species are discussed Bold species are discussed Rhabditidae Ancylostomatidae Onchocercidae Ascarididae Trichinellidae Trichuridae Strongyloididae Trichostrongylidae Heligmosomatidae Order Rhabditida Strongylida Spirurida Ascaridida Trichocephalida Rhabditia (Secernentea) Eoplia (Adenophorea) Phylum Class Nematoda Fig. 2 of prominent nematodes. Taxonomic classification

Introduction 6

Fig. 3 Morphology of nematodes [22, 23]. Body architecture of C. elegans (A) and cross section of a female A. suum (B). BL, basal lamina; CSM, contractile portion of muscle cells; CU, cuticle; E, embryo; EMU external musculature of uterus; HYP, hypodermis; I, intestine; IE, intestinal epithelium; MI, microvilli of brush border; NU, nuclei; O, ovary; PS, pseudocelomic space; U, uterus; UE, uterine epithelium.

The muscles, therefore, consist of the contractile and a non-contractile portion. Sense organs comprize the amphids at the head functioning as chemoreceptors, phasmids at the posterior end, cephalic papillae around the mouth as tactile sensors and caudal papillae around the cloaca, which are probably involved in copulation. The males of some species have spicules for holding the vulva open during copulation. Eye spots (ocelli) are found in some free-living nematodes. The excretory system is quite diverse amongst the nematodes and may comprize a glandular and a canal system playing a role in osmoregulation and the ecdysis of the larva. The body wall consists of a cuticle, hypodermis and muscle layer. The cuticle

Introduction 7 shows a triple-layered outer membrane and three inner layers. The outer covering is considered to be a “modified cell membrane” and is referred to as the epicuticle, followed by a cortical, medial and basal zone overlaying the hypodermis. The life cycles can range from very simple to extremely complex. Most nematodes are dioecious, producing eggs with tough resistant coverings. The monoecious species are either parthenogenetic or self-fertilizing hermaphrodites. Most nematodes are oviparous, but some are ovoviviparous or viviparous. The female reproductive system consists usually of two ovaries, orientated in opposite directions. The ovaries are formed by an epithelial layer and a germinal cord. The ovaries pass into the uteri. Sperms are stored in the lower part, where fertilization occurs. The uteri lead into the vagina. The egg contains a well-developed shell to protect the enclosed larva or embryo in different environments. It can consist of three to five layers comprizing lipoproteins, chitin, lipids and uterine as well as rectal secretions. Microfilariae of the ovoviviparous Filaroidea are enclosed by a sheath, which can be regarded as a modified egg-shell. The growth of nematodes is accompanied, like that of the arthropods, by ecdysis. There are always four molts. The life cycles of parasitic nematodes can be very heterogeneous. The simplest as in A. suum omits any intermediate host (Fig. 4A). Embryonated eggs are swallowed, larvae hatch in the intestine, and migrate through different tissues and organs, before returning to the intestine. Others are dependent on intermediate hosts, such as, insects, crustaceans or molluscs. In Mermithoidea, the larval stage is parasitic, whereas the adult is free- living. Another type is the alternation of parasitic and free-living generations as in species of the genus Strongyloides spp.

1.3 Model organisms

1.3.1 Caenorhabditis elegans, a free living nematode In 1965, Sidney Brenner selected C. elegans as a model system to study animal development and behavior [24]. The short life cycle, small size, ease of laboratory cultivation and genetic manipulation has made this worm to the best understood metazoic organism today. The C. elegans population consists mainly of hermaphrodites and only a small number of males. The life cycle consists of 14 hours embryogenesis and 36 hours of postembryonic development through four larval stages (see Fig. 4 B). The hermaphrodite produces sperm at the late L4 stage, then returning to the production of oocytes as an adult. Progeny are restricted to 300-350 individuals due to the limited number of sperm, but can be raised to 1000 by mating with males. Under unfavourable food conditions the nematode can undergo dauer larva formation at the second molt. Dauer larvae can survive for months. The hermaphrodite consists of 959 cells with 302 nerve cells deriving from four somatic and one germ-line cell. The complete wild-type cell lineage from the fertilized egg to the adult was determined by observation of cell division and cell migrations in living animals due to the transparency of the body [25]. The genome was completely sequenced in 1998, indicating approximately 20,000 genes [26]. The large number of laboratories working on C. elegans has led to the establishment of several platforms providing the scientific community with information and material. The WormBase contains information on genomic sequences, mutants, phenotypes, strains, RNA interferance (RNAi) experiments and gene homologies. The ORFeome project provides all open reading frames in referential vectors allowing convenient transfer in different expression systems. A DNA microarray service is offered by the Sanger Institute, Cambridge England and a complete E. coli RNAi feeding library covering all

Introduction 8

ORFs by the MRC. Since this nematode shows high conservation in anatomy, genetics and development, C. elegans can be regarded as an excellent model even for parasitic species [22, 27, 28, 29].

A B

C Adults Fig. 4 Life cycles of model nematodes.

L4 (A) A. suum adults (1) produce eggs (2), which develop to the infective stage (3). Mf After the eggs were swallowed (4), larvae hatch (5) and migrate via the liver into the lungs (6). By way of the trachea Meriones libycus the larvae reach the intestine, where Ornithodorus moubata they mate. (B) C. elegans can develop from L1 larvae either to adult worms or dauer larvae. (C) A. viteae is maintained

L1 in the tick O. moubata as intermediate L3 and the jird M. libycus as final host. Microfilariae (Mf) are taken up by the tick and develop to infectious larvae L3. In the jird they develop within 6 weeks L2 to adults.

1.3.2 Ascaris suum, an intestinal parasite There has been much debate on the taxonomic status of human and pig Ascaris. Scanning electron microscopy and biochemical differences have finally indicated two separate species, A. lumbricoides and A. suum, respectively. Due to their close relationship, they are regarded as sibling species [30]. The parasite shows a simple life cycle (see Fig. 4 A) [2]. Eggs are excreted in the feces, with the infective larvae developing within 9-13 days. After the eggs are swallowed, the larvae hatch in the duodenum. The L2 larvae burrow into the mucosa, penetrate blood vessels and appear in the liver after 6 hours. In the liver they develop to the L3 stage within a few days. They then migrate to the heart and lungs. After seven days they break into the

Introduction 9 alveoli and reach the intestine via the trachea, where they molt again, reach adulthood, mate and produce eggs. Ascaris has been used as the prototypic nematode model organism for decades, due to its large size (females 200-350 nm, males 150-310 mm), in biochemical and neurophysiological studies. Since large quantities of worms can be obtained at the abattoir, isolation of biomolecules is easily feasible. In vitro cultivation of adult nematodes is possible and also generation of early adults from eggs in vitro has been reported [31, 32, 33]. Due to its large size and well known anatomy, this nematode is an excellent model for immunofluorescence and immunohistochemical studies.

1.3.3 Acanthocheilonema viteae, a model for filariasis Due to the problems of cultivating human filariids like Onchocerca volvulus or Wuchereria bancrofti in animal models, A. viteae, a rodent filariid, has become one of the most studied model systems for filariasis [34, 35]. A. viteae contains PC- substituted antigens similar to those of the human filarids [36, 37, 38]. Adults recovered from jirds can be maintained for days in cell culture medium, thus allowing metabolic studies. The jird, Meriones libycus, is usually infected by subcutaneous injection of L3 larvae obtained from the tick Ornithodorus moubata. Microfilariae appear in the blood circulation after 42-65 days after infection. Ticks were infected by allowing them to feed on the shaved abdomen of anesthetized jirds. After two molts they develop within 6 weeks to the infective stage (see Fig 4 C).

1.4 Phosphorylcholine as an immunomodulatory substituent PC has been recognized as a structural component in both prokaryotic and eukaryotic pathogens. First detected in the Gram-positive bacterium Streptococcus pneumoniae in 1967 [39]; it was later found in both Gram-negative bacteria and many important disease-causing parasites, such as, protozoa [40, 41], and gastrointestinal [42, 43] and filarial nematodes (for reviews see [441, 45]). It was further found in the cestode Bothriocephalus scorpii [46] and trematode Schistosoma mansoni [43]. PC can be attached to teichoic acid [47], lipoteichoic acid [48], lipopolysaccharide [49], glycolipids (this thesis) and glycoproteins [50]. Work on PC- epitope bearing bacteria, however, has shown, that this modification can act as a double-edged sword, facilitating colonization and invasiveness but also being a target for innate and adaptive immune responses [45, 49, 51, 52, 53]. An immunosuppressive activity of PC-antigens was first reported from Trichinella spiralis in infected mice during the muscle phase of the life cycle [54, 55, 56, 57, 58, 59]. Immunolocalization studies revealed the presence of these epitopes in different internal structures [60, 61, 62, 63]. The PC-moieties are attached via complex-type N-glycans to antennae predominantly comprised of lacdiNAc (GalNAcß1-4GlcNAc) [64, 65] (see Fig. 5). Somatic antigen-bound PC-epitopes were also found in Brugia [66] in addition to those on the circulating filarial antigen. These antigens were found to suppress the T cell proliferative response towards phytohemagglutinin in a dose- dependent manner [67]. There is evidence for N- and O-glycan linked PC-epitopes in these parasites [68, 69]. Similar results have also been reported for O. gibsoni excretory-secretory (ES) products [70]. The N-glycan structures of O. volvulus and O.

1 Own publications are indicated in bold.

Introduction 10 gibsoni containing PC resemble those from A. viteae [71] (see Fig. 5). The first mass- spectrometrical localization of a PC-epitope linked via an N-glyan was performed by Haslam et al. [72] for C. elegans. It could be demonstrated, that PC is linked - similarly as for the glycosphingolipid-based epitope - to C6 of GlcNAc, substituting a trimannosyl fucosylated core structure (Fig. 5). A second, but different PC-epitope from C. elegans was published by Cipollo et al., postulating a PC-substituted Man5- structure (Fig. 5) [73]. A PC-substituted ES product has also been described for Wuchereria bancrofti [74]. Likewise, PC-epitopes were reported for Dictyocaulus viviparus [75], N. brasiliensis [76, 77] and A. suum [78].

A ±GlcNAcß6

Manα6±Fucα6 ±GlcNAcß2 PC 1- 2 Manß4GlcNAcß4GlcNAc ±GlcNAcß4-Manα3 GlcNAcß2

B Manα6 Manα6 PC 3 Manα3 Manß4GlcNAcß4GlcNAc Manα3

PC6 Manα6±Fucα6 GlcNAcß Manß4GlcNAcß4GlcNAc Manα3

C Manα6±Fucα6

PCx GalNAc0-4ß4GlcNAcß1-4 Manß4GlcNAcß4GlcNAc Manα3

D Manα6±Fucα6

PCx HexNAcß4(GlcNAcß)0-5 Manß4GlcNAcß4GlcNAc Manα3

Fig. 5 Published PC-substituted N-glycan structures from A. viteae (A), C. elegans (B), T. spiralis (C) and O. volvulus and O. gibsoni (D).

Most of our current knowledge on the biological implications of PC-substituted proteins results from investigations on ES-62, a secretory product from the filariid A.

Introduction 11 viteae [79, 80, 81]. This glycoprotein of tetrameric structure [82] revealed homology to aminopeptidases [38, 83]. Sensitivity towards N-glycosidase F treatment indicated the presence of PC-substituted N-glycans [84, 85] on ES-62. Mass spectrometric analysis revealed partially fucosylated trimannosyl core structures, carrying between one and four additional N-acetylglucosamine residues substituted with PC [86] (see Fig. 5). Further PC-epitopes have been found in the egg, and on uterine and intestinal membranes of A. viteae [36, 37]. ES-62 was found to be stage-specifically expressed and secreted in the post-L3 stages, whilst mRNA was also found in L1 and L3 stages [87]. Filariasis is characterized by a suppressed, anti-inflammatory T-helper (Th2) type of immune response with reduced IFN-γ, increased IL-10 and greatly elevated IgG4 antibody levels [88]. There was early evidence, that PC might be this immunomodulatory component [89]. Later it was found, that ES-62 at concentrations found in filariasis patients indeed prevented proliferation of B lymphocytes, beeing associated with ligation to the B-cell receptor (BCR) [90], in a PC-dependent manner by anergizing B cells [91], whereas at high concentrations an activation was observed. Similar suppressive effects were also found for T cells [91, 92].

sIg PC Ig-α/β PM R Lyn Shc Ras SHP1 X Raf Grb2 Sos ? MEK

Erk Pac1 Nucleus Erk

Fig. 6 Uncoupling of the B-cell receptor (BCR) from the RasErk MAPkinase cascade [79]. The protein kinase Lyn phosphorylates the ITAMs of Ig-α and Ig-ß after ligation of the BCR. Shc, a Ras adaptor protein binds to the phosphorylated ITAMs and is phosphorylated. This leads to the recruitment of the Grb2Sos complex and activation of Ras, which in turn initiates the Erk MAPkinase cascade. ES-62 induces, after binding to a receptor, via an unknown mechanism, the negative regulators SHP-1, a tyrosine phosphatase, and PAC-1, a nuclear MAPkinase dual phosphatase. This prevents binding of Shc to the ITAMs and terminates Erk signals.

Introduction 12

The interference with proliferation of B cells, however, resulted in the activation of several tyrosine kinases like Lyn, Syk and Blk, and Erk2, an isoform of MAPkinase, and modulation of PKC isoform expression [90, 93, 94]. Desensitization further resulted in a failure to activate the phosphoinositide 3-kinase (PI-3-kinase) and Ras- MAPK pathways via the BCR [93]. Anergy of T cells was found to be associated with disruption of TCR coupling to the phospholipase D, PKC, PI-3-kinase and RasMAPK signalling cascades [91, 92]. Later, it was found that ES-62 induces the activation of two negative regulatory molecules, the tyrosine phosphatase SHP-1 and the MAPkinase phosphatase PAC-1, which dephosphorylate the immunoreceptor tyrosine-based activation motifs (ITAMS) on Ig-ß, thus preventing the recruitment of other signalling molecules, and MAPkinase (see Fig. 6) [95]. Until now, it remains unclear, however, whether PC acts on the cell surface or after internalization. In addition to the biological activities outlined above, ES-62 has further effects on antibody and cytokine responses. It induces a Th2 antibody response as indicated by the production of IgG1 antibodies [91] in an IL-4-dependent manner [96]. This shift towards a Th2 response might be due to the induction of IL-10 by ES-62 [91]. IL-10 downregulates IFN-γ, a cytokine necessary for antibody-class switching to IgG2a, an antibody characteristic for Th1 responses. In macrophages, it suppresses the production of IL-12, a key cytokine in the development of Th1 responses, and that of the proinflammatory cytokines TNF-α and IL-6 [97]. Investigations on the effects of ES-62 on dendritic cells revealed an induction of maturation of dendritic cells with the capacity to induce a Th2 response [98].

Introduction 13

1.5 Aims of the work Previous studies on the serological behavior of nematode-derived, immunoreactive lipids indicated that the glycolipids of nematodes share characteristic phylum specific antigenic epitopes recognized by infection sera of humans and animals [99, 100]. These glycolipids showed properties indicative of an unusual non-carbohydrate substituent. Initial chemical characterizations revealed the presence of phosphate and an organic base, probably choline. Since such modifications on glycolipids have not been reported for vertebrates, a nematode-specific biosynthetic route could be postulated. Parasite-specific biosynthetic pathways are of special interest, since they are potential targets for the development of novel anthelminthics. The first part of our investigations was, therefore, to elucidate the structures of these glycolipids and to identify the unusual substituent. Further studies should clarify, whether these structures are nematode-specific or restricted to a limited number of nematode species. Since these structures were found to be highly antigenic, immunohistochemical and immunofluorescence studies were performed to localize these epitopes within the nematodes. Another approach was to establish methods to identify the enzymes and metabolic intermediates involved in the biosynthesis of these antigens. These investigations should form the basis for the search for specific inhibitors interfering with their biosynthesis and their analysis for use as novel nematicides. The biological implications are a further aspect of these antigens in host-parasite interaction. Although such highly antigenic epitopes seem to be unfavourable for a parasite, these structures might have fundamental functions during the development of the nematodes themselves or for their survival within the host. We, therefore, investigated the receptor involved in the binding of these antigens on immune cells; if they were taken up, the next question was as to which cytokines were induced in these cells and whether the cells were influenced in terms of immunoregulation and/or immunosuppression. To investigate the structures, biosynthesis and biological implications of PC- substituted glycolipids, several model systems were established and investigated. The intestinal nematode A. suum was used due to its ease of accessibility and well- known anatomy, which makes this nematode an excellent model for immunohistochemical studies and source of large amounts of material for the isolation of antigenic biomolecules. The free living nematode C. elegans was selected to make use of the enormous genetic and developmental data available, and its ease of cultivation and manipulation. In this context, an axenic C. elegans culture was established to test inhibitors for nematode development and for biosynthetic analysis. Investigations were further extended to O. volvulus, a parasite causing river blindness, and to Litomosoides sigmodontis and A. viteae, two model systems for filariasis.

Results 14

2 Results and discussion

2.1 Antigens with phosphorylcholine epitopes

2.1.1 A. suum glycosphingolipids reflect the high glycosylation potential of nematodes Serodiagnosis is an important tool in the diagnosis of infectious diseases, the analysis of the development of infection and control of treatment. A prerequisite is, therefore, the isolation and identification of species specific antigens. In this context, the main focus has been laid on glycoconjugates, in particular, glycoproteins [101]. For parasitic nematodes, a high serological cross-reactivity has been observed, e.g. between A. suum and T. canis as well as between N. americanus and A. lumbricoides [102, 103, 104]. This cross-reactivity was also observed at the level of glycolipids, when we compared glycolipids from the model organisms A. suum, N. brasiliensis and L. sigmodontis (carinii) [100]. The neutral glycolipid fraction of all three species showed very similar patterns after separation on high-performance thin-layer chromatography (HPTLC) plates. A fast and slow migrating group of glycolipids could be detected. The high polarity and characteristic migration behavior of the slow migrating group resembled that of the amphoteric glycolipids from Calliphora vicina pupae [105]. Indeed, we could show by different chemical spray reagents that this group of glycolipids contained phosphate, choline and, in part, free amino groups. To determine, whether lipid-bound, antigenic determinants existed in these model organisms, the neutral glycolipid fractions were analyzed by HPTLC immunostaining using the corresponding homologous and heterologous antisera. Intriguingly, all species tested showed an identical staining pattern, indicating the presence of structurally similar, if not identical glycolipid structures. Sensitivity towards treatment with endoglycoceramidase revealed the presence of glycosphingolipids. Treatment with hydrogen fluoride (HF), which cleaves phosphomono- and diester bonds, resulted in the elimination of immunoreactivity and abolished aslo the polar character of the glycosphingolipids. Thus, it could be concluded, that the antigenic determinant was linked via a phosphoester-linkage to the glycosphingolipid backbone. Carbohydrate constituent analysis revealed the presence of glucose (Glc), fucose (Fuc), mannose (Man), galactose (Gal), N- acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). For the characterization and structural analysis of these glycosphingolipids we developed a separation protocol and chemical methods for the preliminary determination of the amphoteric glycosphingolipids [106], using A. suum as model organism. The separation revealed the presence of neutral, amphoteric and acidic glycosphingolipids. Since we expected that the amphoteric glycosphingolipids corresponded, with respect to their carbohydrate backbone, to the neutral structures, we structurally elucidated these glycolipids first. After separation of the acidic glycosphingolipids from the neutral fraction by anion- exchange chromatography, we could subdivide the neutral fraction into a neutral and neutral-zwitterionic fraction by silica-gel chromatography [107, 108]. High performance liquid chromatography (HPLC) separation of the neutral fraction revealed the presence of ceramide mono-, di, tri- and pentahexosides. The carbohydrate structures were elucidated by matrix-assisted laser desorption- ionization time-of-flight mass spectrometry (MALDI-TOF-MS), carbohydrate composition analysis, liquid secondary-ion mass spectrometry (LSIMS), nuclear

Results 15 magnetic resonance spectroscopy (NMR), gas-liquid chromatography-mass spectrometry (GLC/MS), methylation analysis and sequential exoglycosidase treatments. The results revealed that the carbohydrate structures were based on the so-called arthro-series, which had been previously described for the insects C. vicina and Lucilia caesar [109, 110, 111]. The four glycosphingolipids elucidated reflected a biosynthetic pathway with the pentasaccharide Galα1-3GalNAcß1-4GlcNAcß1- 3Manß1-4Glc as the most complex structure (see Fig. 7). Not only the carbohydrate backbone, but also the ceramide moiety of the neutral glycosphingolipids from A. suum showed remarkable peculiarities. Cerebronic acid (C24h:0) was found to be the dominating fatty acid, whilst the sphingoid bases comprized iso-branched C17 sphingosine, sphinganine and phytosphingosine. During our initial characterization of the amphoteric glycosphingolipids, we could already show that this fraction consisted of two major compounds (components A and C) and several minor components [106]. Both major compounds contained for phosphate and choline, the latter also a free amino group. By silica-gel column chromatography, we could isolate components A and C and two fractions, B1 and B2, comprizing mixtures of minor compounds [112]. HPTLC immunostaining with a phosphorylcholine-specific antibody showed a positive reaction with all fractions, whereas HF treatment abolished the immunoreactivity. Also, HF-treatment of components A and C resulted in a single glycosphingolipid with migration properties similar to the ceramide pentahexoside from the neutral fraction. Since staining for amino groups was also negative after HF treatment, we concluded that component C contained a further phosphate-linked substituent, most probably ethanolamine. This could be confirmed by its identification as the 9-fluorenyl-methoxycarbonyl derivative. Choline was further identified after derivatization with pentafluoropropionic acid anhydride and LSIMS analysis. Consistent with these data, carbohydrate composition analysis revealed the presence of Glc, Gal and GalNAc for both components and, in addition, Man for component A. Following HF treatment, GlcNAc was found as an additional monosaccharide residue in both compounds. Due to its lability under strong alkaline conditions, the phosphorylethanolamine (PE) substituent was found to be eliminated during permethylation, thus resulting in similar methylation analysis data for both components. This confirmed the arthro-series pentasaccharide core structure of the neutral fraction for both components A and C. Nevertheless, the GlcNAc residue was only detected after HF treatment, to allow the localization of the PC-substituent to this monosaccharide residue. A methylation step converting PE to PC, and subsequent methylation analysis, allowed the assignment of the PE- substituent to the Man residue. For the exact localization of the substitution positions, HF treatments were performed either before or after the permethylation step and indicated that the amphoteric substituents PC and PE were bound at C6 of GlcNAc and C6 of Man, respectively (cf. Fig. 7). These structural data could be further confirmed by NMR. Component C represented the first glycosphingolipid carrying two zwitterionic substituents. Mass spectrometric analyses showed characteristic fragmentation behavior of these glycosphingolipids in MALDI-TOF-MS due to the loss of the trimethylamine group of choline and formation of a cyclic phosphotriester in the case of component A and loss of ethanolamine in the case of component C. This fragmentation pattern can, therefore, be regarded as a fingerprint to identify molecules substituted with these zwitterionic substituents. The development of new mass spectrometric techniques, such as, electrospray and the use of ion-traps allowing repetitive fragmentation steps, nowadays provide the possibility to deduce much more structural information from mass spectrometric data.

Results 16

In order to establish the required experimental conditions, carbohydrate moieties of components A and B were first used as reference compounds [113]. Therefore, we successfully applied the off-line nanoelectrospray ionization-ion trap technology to the structural elucidation of oligosaccharides derived from the minor glycosphingolipids of A. suum fractions B1 and B2. In the positive ion-mode, this technique gives information on their monosaccharide sequences and the localization of non-carbohydrate substituents, such as, PC or PE, whereas the negative ion-mode provides information on the substitution positions of the monosaccharides resulting from ring-cleavages [113]. This method allows the analysis of selected structures from a mixture in a very short time with high sensitivity and without the need of a prior derivatization.

A pentasaccharide core arthro-series core GlcNAcß6Galß6 PC6 PE6 Galα3GalNAcß4GlcNAcß3Manß4Glcß1Ceramide Gal/GalNAcß3Galß3 Fucα3 Fucα2 Gal2 elongation motifs

B HSO33Galß1Ceramide Galα2myoIns1-P-1Ceramide

O OH N 24

Ceramide = O 17 OH Fig. 7 Summarized ceramide structure and glycan motifs found in A. suum glycosphingolipids. Sphingoid base may also represent iso-branched C17 sphinganine or phytosphingosine. (A) Neutral and neutral zwitterionic arthro-series glycolipids; (B) acidic glycolipids.

Since the minor glycosphingolipids of A. suum exhibited a high structural heterogeneity, we released the oligosaccharides by endoglycoceramidase treatment from the ceramide moiety and separated the resultant glycans by two-dimensional HPLC [114], thus leading to homogeneous oligosaccharide species for most of the components. As for the major components A and C, the PC residue was found to be located at C6 of the GlcNAc residue of the arthro-series core structure (see Fig. 7). The smallest components detected were tri- and tetrasaccharides containing PC- and, in part, additional PE-substituents. Oligosaccharide chains with extended pentasaccharide core structures, however, represented the dominant species, which carried additional ß-galactosyl residues linked to the formerly unsubstituted α- galactosyl residue. These ß-galactose branches could be further substituted by GalNAc, GlcNAc and/or Gal residues (Fig. 7). A third group of oligosaccharides

Results 17 comprized structures which are characterized by a fucosylation of the arthro-series core. The fucosyl residue was always located at C3 of the GlcNAc from the corresponding tetra- and pentasccharides. One component showed an additional galactosyl residue located at C2 of this fucose, which, to our knowledge, represents the first glycan structure from nematodes containing an internal fucose moiety. Mass spectrometric data indicated the presence of further minor structures comprizing up to 17 monosaccharide residues [114]. The neutral and zwitterionic glycosphingolipid structures from A. suum indicated a close relationship to those of insects, but remarkably different to those from Annelida [115], Cestoda [116, 117, 118, 119] and Trematoda [120, 121, 122]. These phylogenetic relationships was later confirmed by DNA sequence homologies [21] (see Fig. 1). Zwitterionic glycosphingolipids have been structurally characterized from various members of the invertebrate phyla, including the identification of the monosaccharide-amphoteric moiety: in the Sarcomastigophora (Flagellata) as Man- phosphorylethanolamine [123]; in the Annelida as Gal-phosphorylcholine [115, 124, 125, 126]; in the Arthropoda (Crustacea) as Glc-phosphonoethanolamine [127]; in the Arthropoda (Insecta) as GlcNAc-phosphorylethanolamine [105, 128, 129]; in the Mollusca (freshwater Bivalvia) as Man-phosphorylethanolamine [130] and in the Mollusca (marine Gastropoda) as Gal-phosphonoethanolamine [131, 132]. Iso- branched sphingoid bases were normally restricted to some atypical bacteria and protozoa [133, 134], but have also been found in arthopods, such as, Parafontaria laminata armigera [135]. The acidic fraction of A. suum was found to contain two glycolipids. The component AFII was phosphate-positive, whilst component AFI could be stained with azure A, a cationic dye specific for sulfate residues. Structural analysis by our group [136] and others [137] revealed AFII to be an unusual phosphoinositolglycosphingolipid Galα2Ins-P-1-ceramide and AFI a 3-sulfogalactosylcerebroside HSO3-3Galß1- ceramide (Fig. 7). Sulfatides represent a class of acidic glycosphingolipids containing one or two sulfate esters as substituents on their oligosaccharide chain. So far, about 20 different structures isolated from various tissues of vertebrates, echinoderms and microorganisms have been established [138]. Sulfatides are major lipid constituents of the brain, kidney and gastric mucosa, i.e., in organs characterized by cells with ´high traffic membranes´. In the brain, sulfatides play an important role in myelination [139, 140]. Other functions are the adaption to conditions of high osmolarity [141, 142], blood coagulation [143] and protection of the gastric mucosa against self- digestion [144]. Phosphoinositol-containing glycolipids (phyto- or mycolipids) have been found to be widespread in plants [145, 146, 147, 148, 149, 150], yeast [151, 152, 153, 154, 155], protozoa [156, 157], fungi [158, 159, 160] and the lugworm [161]. In human peripheral nerve tissue, a phosphoinositolgalactosylcerebroside (Ins2-P-3GalCer) is next to sulfatide the most abundant acidic glycolipid present [162]. Even less is known concerning the function(s) of the phosphoinositol-containing glycosphingolipids, although they are widely distributed in nature. It has been speculated that these structures may function as anchors for membrane proteins in a way similar to the description for glycosyl-phosphatidylinositol anchors [163], but with a higher stability due to the increased rigidity of the ceramide moiety.

Results 18

A B

C D

E F

Fig. 8 Immunohistochemical localisation of AF II- and AF I-epitopes from A. suum [136]. Cryosections (5 µm) of adult worms were incubated with the AF II-specific antiserum (a, b) or the monoclonal anti-sulphatide antibody SNH.1 (c-f). Positive immunoreaction were observed in the intestine (a, b), hypodermis and contractile zone of the somatic muscle cells (c), the external musculature layer of the uterus (d) and the ovaries (e, f). BL, basal lamina; CG cytoplasmic granules; CSM, contractile portion of somatic muscle cell; CU, cuticle; DR, droplet; E, embryos; EMU, external musculature of uterus; HYP, hypodermis; IE, intestinal epithelium; INT, intestine; LC, lateral cord; MI, microvilli of brush border; NSM, non-contractile portion of somatic muscle cell(s); NU, nuclei; O, ovary; OO, oocytes; OE, ovarial epithelium; PS, pseudocoelomic space; RA, rachis; SA, sarcoplasm; SP, spermatozoa; U, uterus; UE, uterine epithelium. Calibration scale represents 35 µm.

Results 19

Immunohistochemical localization studies of the glycolipid-bound antigenic determinants in A. suum with a polyclonal antiserum against AF II and an anti- sulfatide monoclonal antibody against AF I revealed the presence of the AF II-epitope in the intestine, whereas the AF I-epitope was found in the hypodermis, contractile zone of somatic muscle cells and the external musculature of the uterus [136] (Fig. 8). These investigations thus revealed the most complex glycolipid glycan structures from nematodes reported so far and provided significant new information on the glycome of these organisms. Since the glycan motifs found in A. suum differ from those of the respective host, they have to be synthesized by the nematode itself. Following the dogma of “one linkage - one enzyme”, a large number of glycosyltransferases must be postulated. Knowledge of parasitic helminth glycosphingolipid structures and enzymes involved in their biosynthesis is a prerequisite for both the study of their biological relevance for the nematode itself and for its survival in the host as well as for the development of specific inhibitors as potential new nematicides.

2.1.2 Phosphorylcholine-substituted glycosphingolipids are phylogenetic markers of nematodes PC has been found to be the structural basis for the serological cross-reactivity of zwitterionic glycolipids from various parasitic nematodes. Zwitterionic glycolipids of L. sigmodontis and A. suum reacted with homologous and heterologous infection sera derived from animals experimentally infected with A. suum, L. sigmodontis and N. brasiliensis. In addition, zwitterionic glycolipids from L. sigmodontis were recognized by sera from humans infected with O. volvulus, B. malayi, Loa loa, and S. stercoralis [99, 100]. A. suum zwitterionic glycosphingolipids were also recognized by infection sera from human (A. lumbricoides), horse (Parascaris equorum) and raccoon (Baylisascaris procyonis) [23]. In order to substantiate respective serological cross-reactivities by structural data, our studies were, therefore, extended to the neutral and zwitterionic glycosphingolipids from the human filariid O. volvulus, the buffalo filariid S. digitata and the rodent filariid L. sigmodontis [164], using the methods and instrumentation described above. HPTLC immunostaining of the neutral fraction with an arthro-series-specific antiserum raised against the ceramide pentahexoside from A. suum [23], revealed the presence of the corresponding ceramide tri- and pentahexosides in all these species. The zwitterionic fractions exhibited a band with migration properties similar to those of component A from A. suum, as indicated by the immunoreactivity with the PC-specific monoclonal antibody TEPC-15. Additionally, a faster migrating band was recognized, whereas components with higher polarity were only detected in S. digitata and L. sigmodontis microfilariae. Subsequent structural analyses of the neutral glycosphingolipids from O. volvulus revealed ceramide mono- and dihexosides structurally identical to those from A. suum. The zwitterionic fraction comprized the arthro-series-based ceramide tri-, tetra- and pentahexosides substituted with PC at C6 of GlcNAc. The ceramide moiety analysis showed the presence of the 2-hydroxylated C22:0, C23:0 and C24:0 fatty acids and C17 sphingosine as major constituents [164]. HPTLC immunostaining with the arthro-series specific antiserum and the PC-specific monoclonal antibody TEPC-15 revealed the presence of zwitterionic glycosphingolipids based on the artho-series and the corresponding neutral ceramide

Results 20 pentahexoside also in A. viteae [165]. Nano-electrospray ion-trap mass spectrometry coupled with HPLC separation on a silica-gel column allowed further structural characterization of these glycolipids. The mass spectra indicated the presence of PC- substituted ceramide tri- and pentahexosides. Fragmentation data confirmed the carbohydrate sequences of HexNAc-Hex-Hex for the trihexoside and Hex-HexNAc- HexNAc-Hex-Hex for the pentasaccharide and allowed the localization of the PC- substituent to the innermost HexNAc. The deduced ceramide mass increments corresponded to the compositions found for A. suum [112, 166].

As CeOv Sd Ls Av

GlcNAcß3Manß4Glcß-Cer + + + + +

PC-6 GlcNAcß3Manß4Glcß-Cer + + +

PC-6 PE-6 GlcNAcß3Manß4Glcß-Cer +

GalNAcß4GlcNAcß3Manß4Glcß-Cer +

PC-6 GalNAcß4GlcNAcß3Manß4Glcß-Cer + + +

PC-6 PE-6 GalNAcß4GlcNAcß3Manß4Glcß-Cer +

Galα3GalNAcß4GlcNAcß3Manß4Glcß-Cer + + + + +

PC-6 Galα3GalNAcß4GlcNAcß3Manß4Glcß-Cer + + + + + + Component A

PC-6 PE-6 Galα3GalNAcß4GlcNAcß3Manß4Glcß-Cer + Component C

PC-6 Galß3Galα3GalNAcß4GlcNAcß3Manß4Glcß-Cer + +

Table 1 Occurrence of selected glycosphingolipids within the nematodes A. suum (As), C. elegans (Ce), O. volvulus (Ov), S. digitata (Sd), L. sigmodontis (Ls) and A. viteae (Av).

These studies showed that arthro-series glycosphingolipids carrying, in part, a PC substituent, represent highly conserved antigens among parasitic nematodes (Table 1). To determine, whether these structures also occur in free living nematodes, S. Gerdt established C. elegans as a model in our laboratory. The short life cycle, small size, and ease of laboratory cultivation and genetic manipulation has made this worm

Results 21 the best understood eukaryotic organism today. Because of the anatomically and developmentally conserved phylogeny of the phylum Nematoda [22], C. elegans has been studied and analyzed as a prototypic model for parasitic nematodes. Analysis of the neutral glycosphingolipids from a mixed population of C. elegans, obtained by the isolation protocol developed for A. suum, revealed the presence of ceramide mono-, di- and trihexosides [167] based on the arthro-series. Ceramide moiety analysis indicated 2-hydroxylated C22:0 and C24:0 fatty acids and iso- as well as anteiso-branched C17 sphingosines as major constituents. HPTLC immunostaining with TEPC-15 of the zwitterionic fraction revealed one major and several minor species. Detailed analysis of these structures, performed by S. Gerdt et al., showed the presence of ceramide tetra-, penta- and hexahexosides [168]. For all three major structures PC was found to be located to the GlcNAc of the arthro- series core. The largest structure comprised an additional ß-galactosyl residue at C3 of the α-Gal of the pentasaccharide core (see Fig. 7). Immunochemical analysis of the zwitterionic fractions derived from different life-cycle stages of C. elegans revealed only minor quantitative differences. These data displayed the close structural coincidence with the equivalent structures from parasitic nematodes.

2.1.3 C. elegans secretes a putative aspartyl protease carrying a novel phosphorylcholine epitope Nematodes release enzymes and metabolic products, which together with cuticular material, are collectively termed excretory/secretory (ES) products. During the cultivation of C. elegans in axenic medium, we observed, starting from the culture supernatant, a single PC-positive protein band in Western-blot analysis, with an apparent molecular weight of 40 kDa (Lochnit et al., submitted). In synchronized cultures, starting only with eggs obtained by sodium hypochlorite treatment of adult worms, the protein was first detectable in the culture medium after 24 hours and increased in amount, thereafter, with cultivation time. Excreted proteins were separated by 2-dimensional gel electrophoresis. The gels were either electroblotted or stained with Coomassie blue. The PC-modified protein at 40 kDa was visualized on the PVDF-membrane with the TEPC-15 antibody, revealing two major and one minor isoelectric forms (Fig. 9). The corresponding spots were excised from the gel and the protein was in-gel digested with trypsin. The tryptic peptides were subjected to nano-HPLC coupled to electrospray ionization ion-trap mass spectrometry (ESI-IT- MS) to identify the protein by peptide mass fingerprint mapping and sequence information obtained from ESI-IT-MS2 fragmentation. Using this approach, this protein could be demonstrated to be homologous to the 41.84 kDa aspartyl protease ASP-6 of C. elegans, the amino acid sequence of which is characterized by the presence of a single N-glycosylation site. Lectin blotting studies of the obtained protein with wheat germ agglutinin revealed the presence of GlcNAc residues. N- glycosidase F treatment abolished this staining, whilst reactivity with the PC-specific antibody TEPC-15 was not affected. Hence, the PC-epitope does not appear to be linked via a N-glycan to the protein backbone. It can, therefore, be speculated that the PC epitope is bound either via an O-glycan or directly to an amino acid. This could be the first indication of a further PC-epitope on (glyco)proteins of C. elegans besides the published PC-substituted Man5-structure [73]. The detailed structural analysis of the PC-epitope is currently in progress. Affinity chromatography of C. elegans total worm homogenate on immobilized pepstatin columns has revealed the presence of several aspartyl proteases [169]. Nevertheless, none of these proteins was PC-positive. Using a commercially available kit, Pepstatin-inhibitable aspartyl

Results 22 protease activity could be demonstrated for these proteins, whereas respective proteolytic activity was very low in the culture medium. One explanation for these observations could reside in the unknown substrate specificity of the secreted aspartyl protease. The data could further indicate the presence of two isoforms of ASP-6, a unmodified and probably proteolytically active one and a second, secreted and PC-modified form with different, if present, enzymatic activity.

pI 10 3 10 3

A B

50 kDa 1 1 2 2 40 kDa

30 kDa

Fig. 9 2-D dimensional gel electrophoresis of the proteins from C. elegans axenic culture medium [170]. Separated proteins were either stained with Coomassie (A) or detected immunochemically by western-blott using the PC-specific antibody TEPC-15 (B). Analyzed proteins are marked by boxes.

Proteolytic enzymes of parasites have been ascribed numerous functions, such as, food digestion, penetration of host tissue barriers, anticoagulation, extracorporal digestion, proteolytic cleavage of surface-bound immunoglobulin and inactivation of complement and cytotoxic mediators expressed by host leukocytes [171, 172, 173]. C. elegans crude extracts exhibit strong proteolytic activity at acidic pH, which is almost completely inhibited by pepstatin and to a much smaller extent by leupeptin, whereas at neutral and alkaline pH only low proteolytic activity was detected [174]. The predominance of aspartyl proteases was confirmed by affinity purification and identification of five proteins [169]. At the cDNA level six aspartyl proteases have been identified showing high homology to those of a number of mammalian parasites [175]. The assumed importance of aspartyl proteases for the survival of mammalian parasites makes them excellent targets for anti-parasitic drugs.

2.1.4 A. suum shows a widespread distribution of the PC-epitope For the immunolocalization studies of zwitterionic glycosphingolipids in A. suum rabbit polyclonal antibodies were raised by injecting the zwitterionic fractions A, B1, B2 and C [23]. The four hyperimmune sera obtained exhibited complete serological cross-reactivity with all the above zwitterionic fractions on HPTLC immunostaining and the resultant staining patterns were identical to that of the PC-specific

Results 23 monoclonal antibody TEPC-15. The cross-reactivity could not be eliminated by affinity purification of the polyclonal antisera on Celite 545 with the different glycolipid antigens as ligands, which indicated the immunological dominance of the PC-epitope and predominance of resultant anti-PC antibodies. Inhibition assays of the four polyclonal antisera-immunoreactivities using PC, phosphodimethylaminoethanol and PE revealed competition only in the case of PC. Immunization of a rabbit with a preparation containing the A. suum neutral ceramide pentahexoside resulted in a polyclonal antiserum recognizing the neutral glycolipids ceramide trihexoside, ceramide tetrahexoside and ceramide pentahexoside (CPH) of A. suum, as well as, the zwitterionic glycosphingolipids both before and after removal of the PC-residue by hydrofluoric acid treatment. The staining patterns exhibited by the monoclonal antibody TEPC-15 and the polyclonal, monospecific A, B1, B2 and C hyperimmune sera defined a comparably, restricted distribution of immunofluorescence within the tissues and organs of the adult female worm. For cryosections, the body wall of cuticle, hypodermis and somatic muscle cells was characterized by labelling of the following structures: all three zones and the epicuticular layer of the cuticle; the syncytial hypodermis; and, the basal lamina surrounding the somatic muscle cells. The intestine displayed a strong fluorescent signal for the basal lamina and a weak signal for the poorly preserved intestinal epithelium and microvilli of the brush border. The female reproductive tract exhibited the following immunostaining pattern: ovarial epithelial cell longitudinal striations and basal lamina of the ovary; the external musculature and basal lamina of the uterus, which could not be clearly differentiated by this technique; the uterine epithelium, which demonstrated one of the few differences between monoclonal and polyclonal, monospecific antibodies, in that, it was labelled only by the latter; and the embryos and embryonic layers of eggs, whereby a discrimination of the latter was not possible by this method. To investigate the glycolipid nature of the PC antigenic determinant in the tissues and organs of the female worm, a pretreatment of cryosections with organic solvents was undertaken. This resulted in the incomplete reduction of specific immunofluorescence in all organs tested, as exemplified, by the cuticle. Proteinase K-pretreatment resulted, besides a loss in tissue integrity, resulted in the complete elimination of specific immunofluorescence in all organs tested. Taken together, these results indicated that all organs expressed glycolipid- and protein-bound PC in different proportions. To examine the distribution and protein, i.e., non-lipid, nature of the PC-epitope in the tissues and organs of the female adult worm at a higher resolution, TEPC-15- incubation and FITC-immunofluorescence were performed on processed, paraffin- embedded sections. The results confirmed the premise, that protein-bound PC was similarly distributed as glycolipid-bound PC in the adult female A. suum. The body wall was characterized by immunostaining the following structures: the epicuticle, and the median and basal zones of the cuticle; the fibrillar hypodermis; and, the basal lamina and sarcoplasm of the somatic muscle cells. In addition, the thickened basal lamina/basement membrane was also PC-positive. The intestine manifested specific immunolabelling on the surface of the basal lamina, the intestinal epithelium, apart from the nuclei and terminal web, and the microvilli of the brush border. The reproductive tract revealed the following sites of immunoreactivity: the undifferentiated external musculature and basal lamina of the uterus; uterine epithelial cells, excluding the nuclei; apparent secretion of PC-positive material present on the epithelial cell-plasma membrane, in the uterine cavity and covering

Results 24 the contained eggs; and, the embryos and the apparent inner lipid layer, but not intermediate chitinous layer, of the eggs themselves (Fig. 10).

A CZ EP C EP MZ CU CZ BZ HYP BL HYP MZ BM BZ BL CSM SA MYO CSM

B D EP CZ

MZ BL

SA BZ HYP

CSM

E BL G E

U MI TW UE PS IE IE PS BL TW EMU/BL MI

OE OO

F H NU EMU/BL

U MI

Fig. 10 Localization of the PC-epitope in A. suum [23].

Results 25

I K CU EP CSM MZ UE EMU/BL HYP BZ CZ

U BL BL

E SA MYO

J EP L TW MI

CU HYP

BL SA IE MYO NU BL CSM UE PS E EMU/BL U

M N EP

CU CZ UL MZ CL BZ BZ LL EMU/BL HYP E BL SA UE

CSM

Fig. 10 continued A. suum sections were treated as paraffin-embedded, Goldner trichrome-stained sections (a, e, g); as Tissue-Tek OCT compound-embedded, monoclonal antibody TEPC-15 (b-d, f, h)- or polyclonal anti-B1 antiserum (i)-incubated and FITC immunofluorescence-stained cryosections; as paraffin-embedded, TEPC-15 (j-m)- incubated and FITC immunofluorescence-stained sections; and as paraffin- embedded, anti-arthro series antiserum (n)-incubated and chromogen AEC-stained section. Section (c) was pretreated with organic solvent. BL, basal lamina; BM, basement membrane, BZ, basal zone; CL, chitinous layer; CSM, contractile portion of somatic muscle cell(s); CU, cuticle; CZ, cortical zone; E, embryo(s); EMU, external musculature of uterus; EP, epicuticle; HYP, hypodermis; I, intestine; IE, intestinal epithelium; LL, lipid layer; MI, microvilli of brush border; MYO, myofibrils of obliquely striated, coelomyarian muscle cells; MZ, median zone; NU, nuclei; O, ovary; OE, ovarial epithelium; OO, oocyte(s); PS, pseudocoelomic space; SA, sarcoplasm; TW, terminal web; U, uterus; UE, uterine epithelium; UL, uterine layer. Calibration scale represents 50µm (a-f, h-n) and 100 µm (g), respectively.

Results 26

A reasonable inference to the finding of a restricted distribution of the PC-epitope in the tissues and organs of the studied female worm would be an expected, parallel location of the backbone arthro-series glycoconjugate, as PC- and PE-modified zwitterionic glycolipids of A. suum are based on the arthro-carbohydrate series of neutral glycosphingolipids. To assess this supposition, the binding of the affinity- purified, polyclonal anti-arthro series antibodies to processed, paraffin-embedded sections was investigated. The pattern of immunolabelling closely paralleled that for both the glycolipid- and protein-bound PC-epitope in the cuticle, hypodermis, somatic muscle cells, intestine and female reproductive tract. The only additional immunostaining by anti-arthro-series antibodies was of a fine, membranous network in the oocytes of the ovary [23]. Parallel immunohistochemical staining of anti-arthro series antibodies-treated and FITC-immunofluorescence-visualised cryosections, however, revealed the presence of arthro-series glycoconjugates solely in the thickened basal lamina/basement membrane underlying the cuticle and hypodermis. Chloroform/methanol pretreatment abolished the antibody binding completely, to suggest the restriction of antigens of the glycolipid-bound arthro-series to these tissue compartments. This has been the first report for PC-epitopes located at the cuticular surface of the adult nematode. In A. suum lung-stage larvae, PC-epitopes appeared to be confined to internal membranous structures and the lining of the intestinal tract [78]. Anti-PC antibodies raised against O. gibsoni eggs revealed the presence of PC-epitopes in B. malayi egg-bearing regions and the intestines of adult worms [66]. In the case of N. brasiliensis, PC-antigens of the L3 infective larvae, adult worms and eggs were restricted to internal structures of the parasite, especially, the gonads and intestinal tract [77]. In the filarial parasite A. viteae, PC-epitopes were restricted to internal structures, such as, the egg as well as uterine and intestinal membranes, but not on the cuticle, whereas PC-negative antigens were predominantly expressed on the cuticle [36]. Stage-specific expression of the PC-epitope has also been observed in T. spiralis, being abundant on internal structures of the muscle larva and the adult worm, but absent from the fetal larva [61, 62]. PC-antigens in the cuticle of muscle larvae were restricted to the inner layer, whereas this epitope was absent from fetal larvae and adult worms. As a first approximation, the membrane-associated tissue and organ distribution of the PC-epitope in L1 muscle larvae of T. spiralis [63] would appear to resemble the pattern in the adult female nematode, A. suum.

2.1.5 The expression of this epitope is developmentally regulated in C. elegans Investigations on the localization of the PC epitope in C. elegans were accomplished at defined stages of embryonic and postembryonic development on freeze-cracked, fixed worms [168] by S. Gerdt in our laboratory. Embryogenesis is divisible into two phases of approximately equal length: cell proliferation and organogenesis, as exemplified by the gastrula, bean and comma stages of development, to yield a spheroid of embryonic cells; and morphogenesis, as exemplified by the threefold “pretzel” stage, whereby elongation of the embryo to the L1 larva occurs. At the early gastrula stage diffuse labeling of the outer cell layer and at the bean stage labelling of the dorsally located hypodermal precursors were observed, using the PC-specific antibody TEPC-15. In the comma stage, embryos the hypodermis was labelled, whilst with ongoing development and differentiation the labeling was progressively restricted to the lateral hypodermal seam cells (see Fig. 11). Because of the random nature of the axes of exposed surfaces available for investigation following the

Results 27 freeze-cracking procedure, it was not possible to observe the complete pattern of PC- epitopes in a single preparation. Instead, with an accumulated series of freeze- cracked preparations from postembryonic, developmental stages, it was possible to define those cells, tissues and organs expressing the PC-epitope. In L1 and L2 larvae, the PC-epitope was located in the basement membrane of all organs, in particular, that of the digestive tract and the boundary between body wall muscle and hypodermis, as well as the cytoplasm of somatic muscle cells and interventing desmosomes. In the L3, L4 larvae and adults, the epitope was additionally located in the basement membrane of the reproductive tract, whereby this included staining around the nuclei in the syncytial ovary. To distinguish between lipid- and protein- bound PC-epitopes, the different stages were pretreated with chloroform/methanol to extract lipids. Embryos were characterized by lipid-bound PC-epitopes in the external cell layer until the comma stage, thereafter, during progressive, temporal expression in the lateral hypodermal cells, it appeared to be protein bound. Postembryonic stages were distinguished by lipid-bound PC in all internal organs, while organic solvent treatment unmasked protein-bound PC in the basal layer(s) of the cuticle.

123

456

789

Fig. 11 Immunofluorescence localization of PC-epitopes during embryonic and postembryonic development of C. elegans [44, 168]. 1, Diffuse labelling of the outer cell layer of the early gastrula stage; 2, labelling of the dorsally located hypodermal precursors at the bean stage; 3, labelling of the hypodermis of the comma stage; 4-6, restricted labelling of lateral hypodermal seam cells in three phases of the developing threefold stage; 7, labelling of the body wall basement membrane between hypodermis and muscle and body wall muscle cell (arrowhead) in L2 larva; 8, restricted labelling to basal layers of the cuticle in chloroform/methanol treated L2 larva; 9, labelling of the intestinal basement membrane and cellularized germ cells within the developing loop of the gonadal ovary (arrowheads) in late L3 larva.

The asssumed restriction of PC-epitopes to the seam cells during embryogenesis makes this epitope a potential seam cell-differentiation marker, as indicated by the

Results 28 corresponding labeling with a monoclonal antibody [176] specific for the 20 seam cells of AB lineage, whereas in postembryogenesis it can be regarded as a basement-membrane marker, suggested by a similar localization of UNC-52, a C. elegans homolog of perlecan [168].

2.2 Biosynthesis of phosphorylcholine containing molecules

2.2.1 C. elegans exhibits the classical Kennedy pathway In addition to the choline-bearing molecules found in vertebrates, e.g. phosphatidylcholine (Ptd-C), sphingomyelin (SM), acetylcholine and platelet- activating factor (PAF) as well as their metabolic intermediates, nematodes contain PC/PE-substituted glycosphingolipids and (glyco)proteins. Whereas the biosynthesis of the former is quite well understood [177], that of the latter remains unclear. Neither the donor for the PC- and PE-substituents nor the involved transferases have been identified so far, although the addition of PC or PE to glycosphingolipids and (glyco)proteins might represent a novel target for chemotherapy against these parasites. It has been speculated, as to whether CDP-choline and CDP-ethanolamine could act as donors for the transfer of the zwitterionic substituents. In the case of Ptd-C there exist, in general, two biosynthetic routes, the Kennedy pathway and the stepwise methylation of phosphatidylethanolamine (Ptd-E) [177]. Many organisms have been shown to possess both pathways. The Kennedy pathway (see Fig. 12) involves uptake of mainly exogenous choline, its phosphorylation by choline kinase in the cytosol, activation of phosphorylcholine in the ER and transfer of activated choline from the donor CDP-choline to diacylglycerol (DAG). Alternatively, cells can become independent of exogenous choline by methylation of phosphorylethanolamine (PE). Nevertheless, utilization of these pathways can vary dramatically between species, as has been shown for rat and chicken neurons in cell culture [178]. In chicken neurons phosphatidylcholine is synthesized mainly via methylation of PE, whereas in the rat cells stepwise methylation of Ptd-E plays a major role (see Fig. 12). For biosynthetic studies we established the axenic culture of C. elegans [179]. Cultivated in a completely chemically defined medium, the worms need approximately 6-7 days to complete their life cycle. Since the medium contains high amounts of detergents, even hydrophobic compounds can be administered to the worms. In contrast to monoxenic cultures with Escherichia coli as food source, this cultivation avoids the presence of a metabolically interfering organism. Results from feeding and inhibition experiments can, therefore, be directly attributed to C. elegans. A two-step, phase-separation protocol was established in our laboratory for the analysis of metabolic intermediates [165]. A Bligh & Dyer partition [180] was followed by a butanol extraction of the dried methanol/water phase. Water soluble metabolites, such as, choline, phosphorylcholine and CDP-choline were recovered from the water phase, whereas the phospholipids were found in the chloroform- and zwitterionic glycosphingolipids in the butanol-phases. The metabolites were further analyzed by HPTLC/autoradiography and HPLC/liquid scintillation counting. To elucidate, as to whether the classical Kennedy pathway is also present in nematodes, we prepared microsomal fractions from C. elegans and incubated them with radioactive CDP-choline for 15 min. Analysis of the products revealed labelled phosphatidylcholine and, to a lower extent, sphingomyelin (Lochnit and Geyer, submitted). The choline phosphotransferase activity could be inhibited by farnesol

Results 29

(10-160 µM) in a dose-dependent manner to approximately 60%. This is in accordance with the mammalian cholinephosphotransferase [181]. Analysis of C. elegans worms cultured in the presence of either radiolabelled choline, phosphorylcholine or S-adenosyl-methionine revealed the presence of the metabolic intermediates phosphorylcholine and CDP-choline, i.e., characteristic metabolites of the Kennedy pathway (see Fig. 12). Furthermore, some additional radiolabelled molecules could be detected by HPTLC/autoradiography in the butanol phase. Identification of these metabolites is currently in progress. This study is, therefore, the first proof for the presence of the Kennedy pathway in nematodes.

PC-GSLs PC-(glyco)proteins X SM DAG

X Ceramide

ATP ADP CTP PPi DAG CMP Choline PC CDP-choline Ptd-C Phosphoryl- Choline Choline- choline-Cytidylyl- Phospho- Kinase Transferase transferse

AHC AHC Kennedy-Pathway AHC

SAM SAM SAM

Me2E Ptd-Me E P-Me2E 2 AHC AHC AHC

SAM SAM SAM MeE P-MeE Ptd-MeE AHC AHC AHC

SAM SAM SAM

ATP ADP CTP PPi DAG CMP CDP-ethanol- Ethanolamine PE Ptd-E amine

CO2

Ptd-S

Fig. 12 Biosynthetic pathways of choline metabolism. Metabolic pathways known from higher vertebrates are indicated with black arrows, whilst potential nematode-specific pathways are given in red. The Kennedy pathway is marked by a box. AHC, adenosyl-homocysteine; CDP-C, cytidine 5´- diphosphorylcholine; CDP-E, cytidine 5´-diphosphorylethanolamine; CMP, cytidine 5´-monophosphate; DAG, diacylglycerol; GSL, glycosphingolipid; PC, phosphorylcholine; PE, phosphorylethanolamine; Ptd-C, phosphatidylcholine; Ptd-E, phosphatidylethanolamine; Ptd-S, phosphatidylserine; SAM, S-adenosylmethionine; SM, sphingomyelin.

Results 30

2.2.2 Indications for a complex choline metabolism in nematodes Investigations on the biosynthesis of PC-substituted glycoproteins have been focused mainly on ES-62 from A. viteae in the group of B. Harnett (University of Strathclyde, Glasgow). Here, the PC-epitope was found to be linked via a N-glycan to the protein backbone, as shown by its sensitivity towards N-glycosidase F treatment [85]. Furthermore, in vitro cultivation of adult worms in the presence of either tunicamycin, an inhibitor of N-glycosylation and transport of activated sugars [182, 183, 184, 185], or 1-deoxymannojirimycin, an inhibitor of glycan processing in the cis Golgi, resulted in PC-negative ES-62 [50, 186, 187]. These data indicated the Golgi lumen to be the site of PC attachment. Similar results have been obtained for the excretory-secretory products of B. pahangi [68]. Mass spectrometric analysis of ES- 62 N-glycans has revealed the presence of partially fucosylated trimannosyl core structures, carrying between one and four additional N-acetylglucosamine residues substituted with PC [86] (see Fig. 5). It had been speculated, that CDP-choline might be the donor for PC-moieties in ES- 62, analogous to the biosynthesis of phosphatidylcholine via the Kennedy pathway (see Fig. 12). Indeed, choline kinase, the first enzyme in the Kennedy pathway, was found to be necessary for the transfer of PC to ES-62, as shown by its inhibition with hemicholinium-3 [188]. Conversely, hexadecylphosphorylcholine [189], an inhibitor of CTP:phosphorylcholine cytidylyltransferase in mammalian cells, had no effect on this transfer. Since there was also no interference with the biosynthesis of Ptd-C, this inhibitor is probably not effective in nematodes. These data suggested that the transfer of PC might require a metabolite of the Kennedy pathway for phospholipid biosynthesis. In collaboration with B. Harnett (University of Strathclyde) we, therefore, investigated whether the end-product of this pathway, Ptd-C, and/or SM could act as PC donors [165]. Adult female A. viteae were pulsed for 5 min with [3H]choline and pulsed-chased for a series of 10 min time intervals. By a combination of HPLC and HPTLC analyses of appropriate parasite extracts, all components of the Kennedy pathway of Ptd-C biosynthesis (choline, PC, CDP-choline and Ptd-C) were detected as radiolabelled products within 10 min of chase. Conversely, SM was not labelled until 50 min of chase. SDS-PAGE/fluorographic analysis of the detergent extract of A. viteae showed ES-62 to be [3H]choline labelled 20 min into the chase (see Fig. 13). As ES- 62 is radiolabelled before SM, this ruled out the latter metabolite as a PC donor for the parasite glycoprotein. To investigate whether Ptd-C could be the donor of PC, we took advantage of the existence of a second pathway of phosphatidylcholine biosynthesis: phosphatidylserine can be decarboxylated to form Ptd-E, which in turn can be methylated with S-adenosylmethionine (SAM) as methyl donor to produce Ptd-C (see Fig. 12). This means, that Ptd-C can be biosynthetically radiolabelled by employing radioactive serine. If Ptd-C is the PC donor for ES-62, then when using this approach, the PC substituents of ES-62 should be also radiolabelled. The results, however, revealed that unlike utilising [3H]choline as radiolabel [85], there was no radioactivity incorporated into the ES-62 PC-glycan during 96 h of culture in the presence of [3H]serine. HPTLC analysis clearly displayed evidence of substantial biosynthetic labelling of Ptd-C within 24 hours. Thus, although it was possible to label the polar head group of Ptd-C via [3H]serine, radiolabelling of the PC-glycan of ES-62 did not occur [165].

Results 31

We also attempted to promote labelling of ES-62 by employing [3H]SAM as a methyl group donor since this would be similarly expected to radiolabel Ptd-C (see Fig. 12). Unlike the results obtained with [3H]serine, we did, in fact, achieve labelling of the parasite glycoprotein but it took ~24 h for radiolabelled ES-62 to appear in the culture medium (see Fig. 13). This is in significant contrast to radiolabelling with [3H]choline which required only 2-3 h [187]. Ptd-C, however, was labelled ~20 hours earlier than ES-62. This suggested that the radioactivity incorporated into ES-62 might not be due to direct transfer from Ptd-C but might rather involve other intermediates. This would be consistent with the reduction in radiolabelled Ptd-C between 4 and 20 h seen in the extracts from worms cultured with [3H]SAM. As a final experiment, we also attempted labelling of ES-62 with [14C]ethanolamine, which again should be incorporated into Ptd-C via the non-Kennedy pathway of synthesis. The result we obtained was similar to that when using [3H]SAM, i.e., labelling was not observed until 24-48h culture in vitro.

Choline CDP-C Ptd-C PC ES-62 SM

10 20 50 min 4 h 24 h

SAM

Ptd-C ES-62 X

Fig. 13 Evaluation of potential PC donors for ES-62 biosynthesis. Radioactive labelling with choline resulted in rapid formation of phosphorylcholine (PC), CDP-choline and phosphatidylcholine (Ptd-C), when detergent extracts of A. viteae were analyzed, whereas sphingomyelin (SM) was synthesized significantly later than ES-62, thus excluding SM as a donor for PC. Ptd-C could be excluded as a direct donor by analogous labelling experiments with S-adenosylmethionine, indicating a long time gap between Ptd-C synthesis in the worms and appearence of labelled ES-62 in the culture medium.

The time-course experiment undertaken earlier using radioactive choline had failed to produce radiolabelled PC-containing glycosphingolipids in A. viteae, hence an experiment including longer culture periods (up to 72 h) and continuous exposure to the radiolabel was carried out. Subsequent HPTLC analysis revealed a continuous increase with regard to incorporation of radioactivity into Ptd-C, whereas SM displayed a maximum at 48 h. Labelling of A. viteae glycolipids with [14C]choline

Results 32 could be detected after 24 hours reaching a maximum value also at 48 h (see Fig. 13). ES-62 radioactivity in the culture medium was detected within 8 hours. Thus, as predicted earlier, labelling of glycosphingolipids with [14C]choline takes significantly longer than that of ES-62. In addition to radiolabelled glycolipids, several additional bands of radioactivity could be revealed by HPTLC/autoradiography. Identification of these metabolites is in progress. The main portion of radioactivity present in the aqueous phase was found to be associated with a peak at 48 h. HPLC analysis revealed free choline and phosphorylcholine as the major radiolabelled compounds in this fraction, whereas only small amounts of radiolabelled CDP-choline were detected. In general, radiolabelling of metabolites tended to peak at 48 hours and then plateau or decrease. Exceptions to this were Ptd-C and ES-62, the latter in particular, both of which increased. Summarizing these data, we concluded that CDP-choline is obviously not the PC donor for ES-62, since PC-transfer to its N-glycans is assumed to occur in the Golgi lumen whereas CDP-choline is synthesized in the ER for phosphatidylcholine biosynthesis (see Fig. 14). Sphingomyelin synthesis is predominantly located in the – Golgi apparatus, but more specifically confined to the trans-Golgi network [190, 191], representing a later compartment than that suggested to contain a putative PC- transferase acting on ES-62, i.e. the medial Golgi [14]. Hence, it is perhaps not surprising that the parasite glycoprotein was radiolabelled earlier than SM. Ptd-C is synthesized via the Kennedy pathway in the ER and would be, therefore, expected to be radiolabelled before ES-62 in the time-course experiment, and this was indeed the case. However, phosphatidylserine synthase [192] and phosphatidylserine decarboxylase [193], enzymes involved in utilizing serine for phosphatidylcholine biosynthesis are generally associated with mitochondrial membranes. This might be the reason, that labelling of ES-62 with serine was not successful. Phosphatidylethanolamine methyltransferase which transfers methyl groups from SAM to produce Ptd-C is an ER-located enzyme [194, 195] and is, thus, present in the same local environment as CDP-choline: 1,2-diacylglycerol cholinephosphotransferase which synthesizes Ptd-C via the Kennedy pathway. Indeed, we could obtain labelled ES-62 by using SAM or ethanolamine. However, with respect to the SAM result, there was a delay of ~20 hours between Ptd-C and ES-62 radiolabelling. This strongly suggests that the radiolabelled PC, which is being detected on ES-62 is not acquired directly from Ptd-C. It may be that after beeing labelled by [3H]SAM, Ptd-C undergoes phospholipase D digestion with the cleaved choline being re-routed through the Kennedy pathway [196, 197] culminating in the synthesis of PC-bound ES-62. It is, therefore, concluded that neither sphingomyelin nor phosphatidylcholine act as PC donors for filarial nematode glycoproteins. In contrast to ES-62, Ptd-C and SM biosynthesis, choline radiolabelling of glycolipids could only be achieved after prolonged incubation times. This might be due to a slow turnover of these compounds. All these metabolic labelling studies were indicative of a more complex choline metabolism in nematodes than in higher vertebrates. A hypothesized pathway might diverge at the level of phosphorylcholine leading to the synthesis of PC-substituted (glyco)proteins and glycosphingolipids, probably via as yet unknown metabolites and the involvement of nematode-specific transferases, that are highly regulated during development (see Fig. 12).

Results 33

TGN SM

trans

Ptd-S PC-ES-62 medial Golgi GSLs Ptd-E MIT

cis

ER Ptd-C Ptd-C Cer DAG + CDP-choline SAM + Ptd-E

Fig. 14 Cellular localization of choline metabolism and glycosphingolipid biosynthesis. Sphingomyelin (SM) synthesis is located in the trans Golgi network (TGN). Modification of ES-62 with PC was found to take place in the medial Golgi. Phosphatidylserine (Ptd-S) and phosphatidylethanolamine (Ptd-E) are synthesized in the mitochondria (MIT). Formation of phosphatidylcholine (Ptd-C) from CDP-choline and diacylglycerol (DAG) as well as by methylation of Ptd-E using S-adenosylmethionine are located in the endoplasmic reticulum (ER). Ceramide is synthesized in the ER. Glucosylceramide is probably formed in the ER or cis Golgi, whilst the elongation of the carbohydrate chains happens in the Golgi.

2.2.3 Inhibitors of the choline metabolism and glycosphingolipid biosynthesis have anthelminthic potential Chemotherapeutics for filariasis, such as, diethylcarbamazine [198] target only distinct larval stages and show severe side-affects, e.g., in the case of O. volvulus and L. loa infections. Benzimidazoles have, in part, teratogenic potential, thus disqualifying them for the treatment of pregnant women, who are at a high risk of neonatal abnormalities. Furthermore, reported resistances to anthelminthics like Ivermectin, a macrolide paralyzing nematodes by disrupting the GABA-mediated transmission of nerve signals, show the importance for the development of new drugs. In this context, nematode-specific biomolecules and the enzymes involved in their biosynthesis represent promising potential targets. Furthermore, the free-living worm C. elegans has been found to be a good model for the screening of anthelminthics [199, 200]. Since it can be assumed, that PC-conjugated molecules may play an important role in nematodes for their survival within the host and/or for their development and maturation, we investigated the effects of various inhibitors of

Results 34 choline metabolism and glycosphingolipid biosynthesis (see Fig. 15) on the model organism C. elegans in axenic culture, in terms of, viability and fertility [201]. Inhibitor concentrations were used in the range normally given in the literature for the corresponding cell culture systems. To investigate the importance and drug sensitivity of the PC metabolism via the classical Kennedy pathway, we used: amiloride [202] and tamoxifen [203] as inhibitors of choline uptake in cells; hemicholinium-3 as an inhibitor of choline kinase [204]; hexadecylphosphocholine [189] and N-acetyl-erythro-sphingosine-1- phosphocholine [205] as inhibitors of CTP-phosphocholine cytidylyl transferase; and farnesol and geranylgeranol as inhibitors of choline phosphotransferase [181]. The conversion of Ptd-E to Ptd-C by N-methylation was investigated with the inhibitors 3- deazaadenosine [206] and S-adenosyl-homocysteine [207]. The following inhibitors were used to investigate glycosphingolipid biosynthesis: L- cycloserine, an inhibitor of 3-ketodihydrosphingosine synthase [208]; fumonisin B1, which prevents N-acylation of sphinganine [209]; and 1-methylthiodihydroceramide which leads to depletion of sphinganine in cells [210]. Tunicamycin and 3´-azido-3´- deoxythymidine inhibit glycosphingolipid biosynthesis by interfering with the transport of sugar nucleotides across membrane vesicles [185, 211]. N-Oleoylethanolamine [212], DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride (PDMP) [213], N-butyldeoxynojirimycin (NB-DNJ) [214], N-nonyldeoxynojirimycin (NN-DNJ) [215], doxorubicin [216, 217] and mifepristone [218, 219] are known as inhibitors of glucosylceramide synthase.

OH OH CH3 HO N HO N CH HO HO 3 OH OH NN-DNJ NB-DNJ

HO OH

H3C + + CH3 N O O N H C CH 3 2 Br- 3 Hemicholinium-3 OH O OH O HO NH (CH ) CH(CH ) N O N 3 2 2 8-11 HO O N O O NH NHAc O OH C H 9 19 O OH Tunicamycin PDMP HO OH OH

Fig. 15 Compounds successfully used as inhibitors of choline metabolism and glycosphingolipid biosynthesis [201].

Results 35

From the inhibitors of choline metabolism tested, tamoxifen showed some effects in reducing the size of the nematodes and lowering the number of larvae produced, whilst hemicholinium-3 resulted at 50 µM concentration in the arrest of larval development at the L1/L2 stage and in lower mobility to some extent. No influence was observed on the viability of the worms. Other inhibitors of choline metabolism showed no significant effects on C. elegans.

embryos in pretzel multicellular four-cell stage

vulva 50 µm

gut

50 µm

multicellular stage

Fig. 16 Effect of inhibitors of glycosphingolipid biosynthesis on the development of C. elegans [201]. (A) without inhibitor; (B) Tunicamycin; (C) NB-DNJ

Similarly, only some inhibitors of glycosphingolipid biosynthesis were effective in C. elegans culture. Fumonisin B1 was found to be toxic to the larvae at concentrations between 10 and 50 µM. Tunicamycin at concentrations of 0.5 and 1 mM was found to be toxic to the larvae, whereas at lower concentrations significant effects on nematode development were observed. The larvae showed a significant reduction in

Results 36 size and motility, whilst the gonads remain undeveloped. The last group of inhibitors we tested were directed against the glycosylation of ceramide as the first step of glycosphingolipid biosynthesis. Of the inhibitors employed only PDMP, NB-DNJ and NN-DNJ showed anthelminthic effects. In contrast to tunicamycin, larvae developed to adult worms with slightly reduced size but fully developed organs. Embryonic development in these worms was, however, found to be impaired by an arrest of the embryos at the multicellular stage (see Fig. 16). No larvae were produced in the presence of these inhibitors.

A without inhibitor tunicamycin 50 µM L4/adults

100% 13%

100 B 90

40 L4 / adults(%) 30

0 Control Tunicamycin H-3 NB-DNJ NN-DNJ PDMP 50 µM 10 mM50 µM 50 µM 50 µM Fig. 17 Analysis of inhibitor effects on C. elegans development with the COPAS Biosort [201]. (A) size distribution of worm without and with tunicamycin (50 µM); (B) percentage of L4/adults after 14 days of cultivation without (100%) or with inhibitors.

Results 37

For the quantitative analysis of inhibitory effects, cultures were analyzed with the COPAS BIOSORT system in co-operation with UnionBiometrica (Geel, Belgium). This instrument allows the measurement of length and organ development of living C. elegans. After 14 days of culture, L4 / adult populations were gated and compared (Fig. 17). At this time the F1 generation reaches adulthood in axenic culture. The results indicated a 87% reduction in the case of tunicamycin and at least 60% reduction for the other effective inhibitors, thus corroborating the microscopic observations. The lower reduction for the inhibitiors of glucosylceramide synthase is due to the fact that the parental generation reached adulthood, but produced no offspring thereafter. Biochemical analysis of the treated worms revealed no differences in the chloroform phase, where Ptd-C and SM were found to be the dominant species. A dramatic reduction in zwitterionic glycosphingolipid synthesis was found, however, in the case of hemicolinium-3, tunicamycin, NB-DNJ, NN-DNJ and PDMP as indicated by the absence of TEPC-15-positive glycosphingolipids and incorporation of [3H]glucosamine (Fig. 18).

A B TEPC-15 Autoradiography TEPC-15

kDa

97.4 66.2 42.7

31.0 Zw GSLs 21.5 14.4 1 2 3 4 1 2 3 4 1 2 3 4

Fig 18 HPTLC (A) and Western blot (B) analysis of the glycosphingolipid fractions and proteins from axenic C. elegans cultures without inhibitor (lane 1), in the presence of tunicamycin (50 µM, lane 2), NB-DNJ (50 µM, lane 3) or H- 3 (10 mM, lane 4) [201]. For glycosphingolipid analysis worms were grown in the presence of [6-3H]- glucosamine (106 cpm/ml), harvested and the glycosphingolipids were obtained by phase-separation. After HPTLC chromatography, they were visualized either by immunostaining using the PC-specific antibody TEPC-15 or by autoradiography. Protein bound PC-epitopes were visulalized after SDS-PAGE by western blotting using TEPC-15. ZwGSLs, zwitterionic glycosphingolipids identified by co-migration with corresponding glycolipid standards from A. suum.

Results 38

To investigate the time during larval and embryonic development when the inhibitors mostly affected C. elegans ontogeny, the drugs were administered at different time points to the axenic culture (Fig. 19). Cultures were started at day 1 and inhibitors were added either simulataneously or after 24, 48, 72 or 96 hours. The effects were monitored at day 7. From the inhibitors tested, NB-DNJ and NN-DNJ showed effects even when applied 96 h after the onset of culture, resulting in adult worms with impaired embryonic development of eggs arrested at the multicellular stage. Tunicamycin was effective when applied until 48 h after onset of culture, whereas PDMP and H-3 had to be applied within the first 24 h or at the culture start, respectively.

Start of Appearance L1/L2 L2/L3 L3/L4 culture of larvae

Day 1 2 3 4 5 6

NB-DNJ

NN-DNJ

Tunicamycin

PDMP

Hemicholinium-3

Fig 19 Time dependence of inhibitor effects on C. elegans development [201]. Inhibitors were added either at the beginning of cultivation or after 24, 48, 72 and 96 hours. Nematode development was examined microscopically. Arrow heads indicate the developmental stage at which the inhibitors were found to be active.

Only a limited number of drugs reported to be effective on mammalian cells resulted in altered phenotypes in C. elegans development when applied at comparable concentrations. This might be due to differences in cell physiology or a higher resistance of the nematode´s enzymes towards these drugs. The observed effects of these inhibitors in this study could be divided into two groups: one represented by tunicamycin and hemicholinium-3 resulted in developmental arrest in early larval stages; and a second represented by inhibitors of glucosylceramide synthesis impaired the embryonic development of the nematodes. Biochemically we observed a dramatic reduction in the biosynthesis of phosphorylcholine-substituted glycosphingolipid, but no effect on protein bound PC-epitopes for the effective inhibitors. Nematodes appeared to be very sensitive towards interference with the transport of nucleotide-activated sugars (tunicamycin) or the formation of glucosylceramide

Results 39

(PDMP, NB-DNJ, NN-DNJ). It should be noted that tunicamycin acts further as an inhibitor of dolichol glycosylation [220], whilst deoxynojirimycins act as glucosidase inhibitors [221]. Therefore, additional effects on N-glycosylation resulting in the observed phenotypes could not be excluded. Nevertheless, all effective inhibitors have in common their interference with the biosynthesis of glycosphingolipid biosynthesis and choline metabolism. Together with the biochemical data obtained, we conclude, therefore, that the phenotypes observed resulted, at least in part, from the depletion of the zwitterionic glycosphingolipids. Whereas inhibitors like tunicamycin, due to their additional effects on N-glycosylation, cannot be used as anthelminthics, NB-DNJ has been found to be a highly efficient and well tolerated therapeutic in lysosomal storage diseases [222, 223, 224, 225]. The described anthelmintic effect might be a further field of application for this drug. The time- dependence of drug administration demonstrated different sensitivities towards the depletion of biosynthetic intermediates. The levels of nucleotide-activated sugars and PC seem to be important during early development, whereas the synthesis of glucosylceramide is critical during embryogenesis.

2.3 Biological implications of phosphorylcholine epitopes

2.3.1 Zwitterionic glycosphingolipids induce proinflammatory cytokines Little is known as to the biological activity of glycolipids, in general, and parasitic helminth-derived glycolipids, in particular, as regards their putative modulation of the host´s immune response via the cytokine network. Glycosphingolipids have been shown to be immunomodulatory molecules that suppress cells of the immune system, both in vivo and in vitro. Thus, gangliosides inhibit the in vitro proliferative response of various classes of activated immune cells, such as, T- and B-lymphocytes, macrophages and natural killer cells [226]. However, the molecular mechanism(s) underlying the immunosuppressive activity of glycosphingolipids are incompletely understood, but include the direct interaction of ganglioside micelles with IL-2 and IL-4 in the modulation of IL-2-/IL-4-dependent processes [227] and the interference of monocytes at the level of antigen presentation [228, 229], whilst neutral glycosphingolipids of the cestode, E. multilocularis, inhibited the production of IL-2 [230]. The immunomodulation of T- lymphocyte activation in vivo and in vitro, observed in the case of T. cruzi glycoinositolphospholipids, could be directly related to the molecule´s ceramide moiety [231]. A functional resemblance of LPS to glycosphingolipids has been proposed and reinforced by the former´s ability to mimic the second messenger ceramide in TNF-α- and IL-1-stimulated cells [232, 233]. Since there is no structural similarity between these two classes of lipid molecules, it may be assumed that this coincidence of biological activity is based on similar physico-chemical properties. Because of the physico-chemical similarity between glycosphingolipids and LPS of Gram-negative bacteria and the induction of bioactive protein mediators by the latter in the host, i.e., cytokines, responsible for the effects of endotoxaemia [234], a comparative study was performed by Krziwon et al. [235] on the ability of the former to stimulate the production of inflammation-associated cytokines. An atypical, zwitterionic glycosphingolipid (as regards the linkage of the glucuronic acid residue to the ceramide moiety and the presence of non-acetylated glucosamine) from the LPS- negative, Gram-negative bacterium, Sphingomonas paucimobilis, induced the synthesis and release of the human mononuclear cell-derived, inflammation-

Results 40 associated cytokines tumor necrosis factor-α (TNF-α), IL-1 and IL-6, but with approximately 10,000-fold less activity than LPS, in this respect [235]. In collaboration with A. Ulmer (Research Center Borstel) we tested, therefore, whether this was also true for our zwitterionic glycosphingolipids from A. suum [112]. Since we considered A. suum merely as a model for the human parasitic nematode, Ascaris lumbricoides, all in vitro procedures were performed with human and not porcine peripheral blood mononuclear cells (PBMCs). The zwitterionic components A and C, and the component A-derived CPH (see Table 1), were assayed as to their biological activity in inducing the inflammatory monokines TNF-α, IL-1 and IL-6. Components A and C, but not ceramide pentasaccharide, were shown to be biologically active in terms of a dose-dependant response in the release of TNF-α, IL- 1 and IL-6, thus indicating a PC-dependent effect [112]. For IL-1 and IL-6, this dose- dependency of cytokine release was evident up to and including 1000 ng/ml of Component A, with the presumption that higher concentrations were inhibitory at the cellular level. Of the two zwitterionic glycolipids tested, component A was the more bioactive one in inducing the monokines TNF-α and IL-1; component A, and to a lesser extent component C, were also capable of inducing low levels of IL-6 activity. Interestingly, the zwitterionic glycosphingolipids of A. suum stimulated human PBMC production of the cytokines TNF-α and IL-6 in a concentration range similar to that of LPS, whereas these molecules were at least a factor 100-fold less potent than LPS in the stimulation of IL-1. The expression of the inflammatory response-cytokines TNF- α, IL-1 and IL-6 is usually considered to be concomitant [236]. There are, at least, two plausible explanations for the detection of the anomalous, non-concomitant levels of these cytokines induced by the A. suum-derived zwitterionic glycosphingolipids in this study. Firstly, kinetic studies of LPS- and zwitterionic glycosphingolipid-induced inflammatory cytokine expression demonstrated maximal activity in the temporal sequence of TNF-α and IL-1 at approximately the same time- point, and prior to that of IL-6 [235, 237]. The fixed-point determination of cytokine release in the assay used in this study at 8h of incubation introduces an experimental artefact, whereby TNF-α and IL-1 approach their maximal levels of activity whilst IL-6 induction is sub-optimal at this time-point. Secondly, it is known that activation of monocytes by LPS is a receptor-mediated process, which is transduced by the cell- surface molecule CD14 [238]. The mechanism by which the A. suum-derived zwitterionic glycosphingolipids induce cytokine production is, however, unknown. It may be postulated that they act in a direct way by replacing intracellular lipid second messengers, such as, ceramide. Therefore, a different pattern of cytokines released by activated monocytes may be due to different mechanisms of activation.

2.3.2 The modulation of the host´s innate and adaptive immune responses by zwitterionic glycosphingolipids can be dissected into PC-dependent and PC-independent effects In order to further elucidate the immunomodulatory capacities of zwitterionic glycosphingolipids we investigated, in collaboration with B. and M. Harnett (University of Strathclyde and University of Glasgow), their effects on immune cells from BALB/c mice and compared the results with those obtained with ES-62 from A. viteae [239]. For this model compound, it had been shown, that the immunomodulatory properties mainly depended on the PC-substituent [45, 80]. In our study, we have also used the HF-treated, i.e., PC-depleted and formerly zwitterionic glycosphingolipid fraction from A. suum to dissect PC-dependent and PC-independent effects.

Results 41

Proliferation of splenic B-cells induced either via F(ab’)2 fragments of anti-murine Ig (anti-Ig) or LPS was significantly reduced when the glycosphingolipids were present in the culture medium. Intriguingly, however, whereas the LPS-mediated effect was dependent on the PC moiety of the glycosphingolipids, whereas the result generated when using anti-Ig was not. Analysis of cell cycle-status and mitochondrial potential indicated that the combination of glycosphingolipid and anti-Ig reduced B cell proliferation was, at least in part, due to inducing apoptosis. In the case of ES-62, however, reduced proliferation was found to be independent of increased apoptosis (Wilson et al, submitted). As for ES-62, the reduced proliferation after activation by anti-Ig correlated with a reduction in Erk/MAPkinase phosphorylation. The glycosphingolipids were also investigated for their effect on LPS/IFNγ-induced inhibition of Th1/pro-inflammatory cytokine production by peritoneal macrophages. As had been shown for ES-62 [240], employed glycolipids revealed, in a PC-dependent manner, a reduction in IL-12p40 levels. Unlike ES-62 [97], this required a pre- incubation of the cells. No effects were observed for IL-6 and TNF-α. Moreover, again unlike ES-62 [97], the glycosphingolipids alone failed to induce the production of IL-12, IL-6 or TNF-α in macrophages [239]. These results indicated that the glycosphingolipids contain a non-PC immunomodulatory component and that ES-62 and the glycosphingolipids inhibit B- cell proliferation by different mechanisms. Nevertheless, the key mitogenic signalling target, Erk/MAPKinase, is affected in both cases, presumably reflecting the induction of growth arrest [241] by both agents. It is interesting to note that the two parasite molecules, in spite of both mediating inhibition of this pivotal transducer of cellular proliferation in B-cells, appear to ultimately induce distinct effects by subsequent differential modulation of putative downstream regulators of cell survival/apoptosis. Of the two non-PC components of the glycosphingolipids - carbohydrate and ceramide -, the latter is perhaps more likely to be the immunomodulatory moiety. This is because ceramide has been shown to be an apoptotic agent in many cell types including B-cells [242]. The finding that treatment with Ascaris glycosphingolipids on their own does not induce apoptosis but that it requires co-stimulation with anti-Ig, however, suggests that a molecular cross talk between signals elicited by the ceramide moiety and the B-cell receptor (BCR) are required for induction of cell death, presumably via an activation-induced cell death mechanism. The inhibitory effect on LPS-induced activation of B-cells on the other hand is suggested to result from either PC targeting the LPS receptor or from molecular cross talk following ligation of the LPS and PC receptors. BiaCore and Far Western analyses have indicated that ES-62 binds to lymphocyte and monocyte membrane fractions with receptor-like affinity in a PC-dependent manner (M. und B. Harnett, unpublished results). This binding could reflect the PC-receptor previously identified on B cells [243]. Alternatively, as PC has been conserved by pathogens throughout evolution and can be detected in a wide range of prokaryotic and eukaryotic pathogenic organisms [45], it is possible that PC-containing pathogen products are recognized via binding of PC to pattern recognition receptors, such as, the recently identified Toll-like receptors (TLRs). TLRs have been proposed to initiate and direct innate and adaptive immune responses by recognizing conserved “Danger signals” such as LPS on pathogens [244, 245, 246]. Consistent with the idea that TLRs may act as potential PC/ES-62 receptors, recent studies have shown that signalling via TLR2 suppresses LPS-stimulated generation of IL-12, IL-6 and TNF-α [247] in a manner reminiscent of ES-62-induced modulation of macrophage function. We could only mirror part of the ES-62-induced results with Ascaris glycosphingolipids, specifically,

Results 42 we found strong inhibition of LPS/IFN-γ – induced IL-12, but lesser effects on TNF- α and no suppression of IL-6 secretion. The failure to see any induction of cytokine production by the glycosphingolipids was also surprising, since we have previously shown them to cause secretion of pro-inflammatory cytokines from human PBMC, as determined by bioassay [112]. This presumably reflects differences in the host species and/or cell type analyzed or, perhaps, even the nature of assay utilized. Analogous experiments with synthetic PC-substituted arthro-series glycosphingolipids carrying different (host-like) ceramides or only short hydrophobic alkyl-chains [248] and synthetic PC-substituted, neogala-series glycosphingolipids [249] (provided by N. Hada, Kyoritsu College of Pharmacy, Tokyo) will help to identify the additional immunomodulatory component(s) in A. suum glycosphingolipids.

2.3.3 PC-substituted antigens can bind to the PAF-receptor and are internalized Relatively little is known about the signal transduction into cells to activate these abortive mechanisms. No cell-surface proteins have been identified so far as the PC- receptor(s). Furthermore, it´s still a matter of speculation, as to whether PC- containing molecules have to be internalized after binding to their receptor or not. The receptor for platelet-activating factor (PAF), present on numerous cell types [250], has been proposed to be a potential receptor for PC-substituents, as shown for bacterial pathogens [251, 252]. The alternate possibility of internalization of PC- substituted proteins, degradation of the receptor/protein complex and liberation of the PC-component, however, could not be excluded. Preliminary data from BiaCore experiments and Far Western studies suggested that ES-62 mediates its effects on B cells, in a PC-specific manner, by binding to proteins of 82 and 135 kDa. Western blotting studies gave the first evidence that the 82 kDa protein present in B- and T- cells as well as in macrophages might be TLR2 [95]. We, therefore, started investigations in collaboration with L. Filgueira (University of Zurich) to identify the receptor(s) and to study the uptake of PC-antigens by immune cells (Lochnit unpublished results). For the identification of cellular receptors we developed a photoaffinity labelling technique using a trifunctional crosslinker carrying a photolabile azido-group, a biotin label and a disulfide linkage (Fig. 20 A). After coupling of the crosslinker to the PC- antigen, different cell types (THP-1, U937 and PBMCs) were incubated with this conjugate in the dark. After photolabelling, cell extracts were separated by gel electrophoresis under reducing conditions and biotinylated proteins were detected with avidin. For all cell types, we detected two bands of approximately 40 kDa and 80 kDa (Fig. 20 B). Since the apparent molecular mass of the lower band corresponded with the mass of the PAF-receptor, photolabelling was repeated after incubation of the cells with PAF. Analysis of the photolabelling experiments revealed significant reduction of labelling of the 40 kDa band, whereas the 80 kDa band was unaffected. Isolation of the biotinylated proteins by avidin-affinity-chromatography and identification of the proteins by mass spectrometric peptide fingerprinting is currently in progress. To study the cellular uptake of PC-bearing molecules, we synthesized PC- substituted, FITC-labeled human serum albumin (HSA). The binding was tested for FC4, a B-lymphocyte clone, for Jurkat cells, a T-lymphocyte tumor cell line, and for in vitro generated human dendritic cells. HSA without PC was used as a control

Results 43 molecule. Both, PC-HSA and HSA were labeled with FITC and binding was measured with flow cytometry. All three types of immune cells tested bound PC-HSA and HSA, although at different intensities. The FC4 cells bound lower amounts of PC- HSA-FITC in comparison to HSA-FITC, whereas Jurkat cells and dendritic cells bound higher amounts of PC-HSA-FITC in contrast to HSA-FITC. Comparing the three cell types, Jurkat cells appeared to have the highest number of binding sites, whereas FC4 cells and dendritic cells reflected a low number of binding sites. Binding of PC-HSA-FITC, but not of HSA-FITC, could be decreased with increasing concentrations of platelet-activating-factor, but could not be blocked completely.

AB O N3 HN NH O H S PBMC S N S kDa O HN O 100 S 75 S 50 37 O O O N O 25

SO3Na

Fig. 20 Photolabeling of interaction partners of PC-modified antigens [253]. PBMCs were photolabelled with the trifunctional crosslinker (A), lysed and marked proteins were detected with avidin-conjugated alkaline phosphatase (B).

The influence of PC-HSA on human dendritic cells was tested in a mixed leukocyte reaction assay (Fig. 21). The immature in vitro generated dendritic cells were preincubated with PC-HSA or HSA for 1 hour, irradiated and used at different cell numbers to stimulate allogeneic non-adherent PBMC. Independent of the amount of dendritic cells, PC-HSA treatment completely blocked the proliferative response of the allogeneic non-adherent PBMC. With HSA-treatment, there was a significant proliferative response, even at lower numbers of dendritic cells used. These data clearly showed that PC-containing antigens can bind specifically to different cell types. This binding can be, in part, inhibited by PAF. Thus, at least two receptors might exist on the cells. Besides the PAF-receptor and TLR-2, the as yet unidentified 135 kDa band observed by Deehan et al. [95] might represent a third receptor. Recently, additional G-protein-coupled receptors for sphingosylphosphorylcholine and lysophosphatidylcholine have been identified [254, 255, 256, 257, 258, 259]. Thus, cells seem to possess a much larger repertoire of receptors for choline-bearing molecules than previously expected.

Results 44

Variations in the expression of these receptors coupled with different subsequent intracellular signaling pathways might, therefore, explain the multiple effects of PC- antigens on and in immune cells. Whether a subsequent internalization of the PC- containing antigens is necessary for their biological activity remains to be studied. Furthermore, the effects on dendritic cells support earlier results [98] showing an interference of PC-antigens with dendritic cell maturation.

16000 stddev 14000 average 12000 10000 8000 cpm 6000 4000 2000 0 PC-HSA HSA PC-HSA HSA PC-HSA HSA 1:20 1:20 1:40 1:40 1:80 1:80

Fig. 21 Influence of PC-HSA on dendritic cells in the mixed leukocyte reaction assay (MLR) [253]. Human immature in vitro generated dendritic cells were incubated with HSA or PC- HSA (5µg/ml) for 1 hour on ice, irradiated and used at different ratios (DC:PBMC; 1:20, 1:40 and 1:80) to stimulate allogeneic non-adherent PBMC (2x105/well, 96-well plate, quadruplate). Proliferative response was measured in a 5 days MLR assay using a 12 hours pulse with 6-[3H]thymidine as counts per minute (cpm). Representative of 3 independent experiments.

Summary 45

3 Summary Nematodes contain PC-substituted glycosphingolipids and (glyco)proteins. The former can be considered as nematode specific markers. Detailed structural analyses of the zwitterionic glycosphingolipids from A. suum have revealed a large number of complex carbohydrate structures, thus reflecting the high glycosylation potential of nematodes. These structures are based on the arthro-series oligosaccharide core and support close phylogenetic relationship of nematodes to the arthropods. Preliminary studies on the PC-substituted (glyco)proteins from C. elegans have given a first indication for the presence of PC-epitopes which are not bound via N-linked glycans. Data from axenic cultures and immunolocalization studies have shown the presence of these epitopes, not only on somatic structures of the nematode, but also on their surface and, additionally, on excretory-secretory products. This supports the hypothesis of an offensive use of these immunomodulatory compounds in host- parasite interactions. Although, C. elegans clearly demonstrated the developmentally regulated expression of PC-epitopes, little is known as to the choline metabolism of nematodes, especially, with regard to the biosynthesis of these epitopes. Neither the donor of PC, nor the involved transferases have been identified so far. Our studies have demonstrated for the first time the presence of the classical Kennedy pathway in nematodes. Metabolic labelling experiments gave the first indication for an additional choline pathway leading to the PC-epitopes on glycosphingolipids and (glyco)proteins, probably diverging at the level of PC from the Kennedy-pathway. CDP-choline, Ptd-C and SM could be excluded as direct donors for the PC group. Impairment of choline metabolism and glycosphingolipid biosynthesis revealed the physiological importance of the PC-modification during embryogenesis and larval- development. Inhibitors of these biosynthetic pathways, therefore, have anthelmintic potential. PC-antigens bind, at least, via two receptors to their target cells, possibly TLR2 and PAF-receptor and can be internalized by the cells. It remains unclear, whether this internalization is of biological significance. Of the immune system-target cells, these antigens can induce several cytokines, and inhibit cell proliferation and maturation, thus directing the immune system to an appropriate Th2-type immune response characterized by a reduced capacity for antigen presentation and the expression of non-protective immunoglobulins. The cellular signalling pathways targeted by PC-antigens and their mutual impairment remain to be elucidated. Summarizing, we have structurally elucidated highly complex PC-substituted antigens from nematodes that express a widespread cellular distribution. These antigens appear to have important functions in embryogenesis and larval development. Inhibitors of their biosynthesis, therefore, have anthelminthic potential. The nematodes can modulate, via these antigens, the immune response of their hosts thus allowing for a long persistence.

Perspectives 46

4 Perspectives The structural analysis of glycosphingolipids from A. suum is nearly completed. The established methods allow a rapid characterization of these biomolecules. It will, therefore, be easily possible to investigate further nematodes and related phyla for the presence of similar or related PC-containing epitopes to obtain a complete view on the phylogenetic distribution of these antigens. Structural analyses in the near future will focus on a structural elucidation of PC-epitopes on (glyco)proteins. So far, structural studies on the location of the PC-moieties on the N-glycan structures are rare. Since there is evidence for further PC-epitopes, probably on O-glycans and/or directly linked to the protein backbone, (glyco)protein-based antigens might reflect a much higher structural diversity than glycosphingolipids. Additional transferases might be involved in their biosynthesis. Identification of the exact location within the amino acid sequence and context will probably help to identify potential signal sequences required by the respective transferases. To elucidate the choline metabolism radioactively labelled metabolites obtained during metabolic studies will have to be identified. This will help to identify the corresponding biosynthetic intermediates and transferases. RNAi-designed experiments will provide excellent tools for the identification of the enzymes involved in the biosynthesis of the PC- epitopes and the nematode-specific glycan structures. A further field of investigation will be the identification of additional, more specific inhibitors of choline metabolism. Axenic culture in combination with the COPAS system will allow a high-throughput screening. These inhibitors might form the basis for the development of a new class of nematicides with fewer side-effects and higher specificity. The next step will then be to elucidate the exact function of PC-conjugated molecures during embryogenesis and larval development for the nematodes themselves. To further elucidate the biological implications of PC, investigations on its interference with cytokine induction, signal transduction and cell maturation have to be continued. This will also include the identification of the cellular receptors for PC and the subsequent signalling pathways as well as their molecular crosstalk with other cellular metabolic pathways.

Literature 47

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6 Supplement

6.1 Complete list of own publications 1. Lochnit, G. & Geyer, R. (1995) Carbohydrate structure analysis of batroxobin, a thrombin-like serine protease from Bothrops moojeni venom. Eur. J. Biochem. 228, 805-816. 2.*2 Dennis, R.D., Baumeister, S., Smuda, C., Lochnit, G., Waider, T. & Geyer, E. (1995) Initiation of chemical studies on the immunoreactive glycolipids of adult Ascaris suum. Parasitology 110, 611-623. 3.* Lochnit, G., Dennis, R.D., Zähringer, U. & Geyer, R. (1997) Structural analysis of neutral glycosphingolipids from Ascaris suum adults (Nematoda: Ascaridida). Glycoconj. J. 14, 389-399. 4.* Gerdt, S., Lochnit, G., Dennis, R.D. & Geyer, R. (1997) Isolation and structural analysis of three neutral glycosphingolipids from a mixed population of Caenorhabditis elegans (Nematoda: Rhabditida). Glycobiology 7, 265-275. 5.* Lochnit, G., Dennis, R.D., Ulmer, A.J. & Geyer, R. (1998) Structural elucidation and monokine-inducing activity of two biologically active zwitterionic glycosphingolipids derived from the porcine parasitic nematode Ascaris suum. J. Biol. Chem. 278, 466-474. 6.* Dennis, R.D., Lochnit, G. & Geyer, R. (1998) Strategies for preliminary characterization of novel amphoteric glycosphingolipids. Methods Mol. Biol. 76, 197-212. 7.* Lochnit, G., Nispel, S., Dennis, R.D. & Geyer, R. (1998) Structural analysis and immunohistochemical localization of two acidic glycosphingolipids from the porcine, parasitic nematode, Ascaris suum. Glycobiology 8, 891-899. 8. Müthing, J., Duvar, S., Heitmann, D., Hanisch, F.G., Neumann, U., Lochnit, G., Geyer, R. & Peter-Katalinic, J. (1999) Isolation and structural characterization of glycosphingolipids of in vitro propagated human umbilical vein endothelial cells. Glycobiology 9, 459-468. 9. Wuhrer, M., Dennis, R.D., Doenhoff, M.J., Bickle, Q., Lochnit, G. & Geyer, R. (1999) Immunochemical characterisation of Schistosoma mansoni glycolipid antigens. Mol. Biochem. Parasitol. 103, 155-169. 10. Wuhrer, M., Dennis, R.D., Doenhoff, M.J., Lochnit, G. & Geyer, R. (2000) Schistosoma mansoni cercarial glycolipids are dominated by Lewis X and pseudo-Lewis Y structures. Glycobiology 10, 89-101. 11. Beermann, C., Lochnit, G., Geyer, R., Groscurth, P. & Filgueira, L. (2000) The lipid component of lipoproteins from Borrelia burgdorferi: structural analysis, antigenicity, and presentation via human dendritic cells. Biochem. Biophys. Res. Commun. 267, 897-905.

2 Publications directly related to the habilitation thesis.

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12.* Friedl, C.H., Lochnit, G., Geyer, R., Karas, M. & Bahr, U. (2000) Structural elucidation of zwitterionic sugar cores from glycosphingolipids by nanoelectrospray ionization-ion-trap mass spectrometry. Anal. Biochem. 284, 279-287. 13.* Wuhrer, M., Rickhoff, S., Dennis, R.D., Lochnit, G., Soboslay, P.T., Baumeister, S. & Geyer, R. (2000) Phosphocholine-containing, zwitterionic glycosphingolipids of adult Onchocerca volvulus as highly conserved, antigenic structures of parasitic nematodes. Biochem. J. 348, 417-423. 14.* Lochnit, G., Dennis, R.D. & Geyer, R. (2000) Phosphorylcholine substituents in nematodes: structures, occurrence and biological implications. Biol. Chem. 381, 839-847. 15.* Lochnit, G., Dennis, R.D., Müntefehr, H., Nispel, S. & Geyer, R. (2001) Immunohistochemical localization and differentiation of phosphocholine- containing antigens of the porcine, parasitic nematode, Ascaris suum. Parasitology 122, 359-370. 16. Hossain, H., Wellensiek, H., Geyer, R. & Lochnit, G. (2001) Structural analysis of glycolipids from Borrelia burgdorferi. Biochimie 83, 683-692. 17.* Lochnit, G., Geyer, R., Heinz, E., Holst, O., Rietschel, E.T., Zähringer, U. & Müthing, J. (2001) Glycolipids. Chemical biology and biomedicine in Glycoscience: Chemistry and Biological Chemistry (Fraser-Reid, B., Tatsuta, K. & Thiem, J., eds) pp. 2183-2252, Springer, Heidelberg. 18. Wuhrer, M., Kantelhardt, S.R., Dennis, R.D., Doenhoff, M.J., Lochnit, G. & Geyer, R. (2002) Characterization of glycosphingolipids from Schistosoma mansoni eggs carrying Fuc(α1-3)GalNAc-, GalNAc(ß1-4)[Fuc(α1-3)GlcNAc- and Gal(ß1-4)[Fuc(α1-3)]GlcNAc- (Lewis X) terminal structures. Eur. J. Biochem. 269, 481-493. 19.* Houston, K., Lochnit, G., Geyer, R. & Harnett, W. (2002) Investigation of the nature of potential phosphorylcholine donors for filarial nematode glycoconjugates. Mol. Biochem. Parasitol. 123, 55-66. 20. Kurokawa, T., Wuhrer, M., Lochnit, G., Geyer, H., Markl, J. & Geyer, R. (2002) Hemocyanin from the keyhole limpet Megathura crenulata (KLH) carries a novel type of N-glycans with Gal(ß1-6)Man-motifs. Eur. J. Biochem. 269, 5459-5473. 21.* Deehan, M.R., Goodridge, H.S., Blair, D., Lochnit, G., Dennis, R.D., Geyer, R., Harnett, M.M. & Harnett, W. (2002) Immunomodulatory properties of Ascaris suum glycosphingolipids - phosphorylcholine and non- phosphorycholine - dependent effects. Parasite Immunol. 24, 463-469. 22.* Friedl, C.H., Lochnit, G., Zahringer, U., Bahr, U. & Geyer, R. (2003) Structural elucidation of zwitterionic carbohydrates derived from glycosphingolipids of the porcine parasitic nematode Ascaris suum. Biochem. J. 369, 89-102.

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6.2 Submitted publications 1.* Lochnit, G., Bongaarts, R. & Geyer, R. (2003) A new class of anthelminthics: inhibitors of the glycosphingolipid biosynthesis and phosphorylcholine metabolism interfere with nematode development. 2.* Lochnit, G. & Geyer, R. (2003) Evidence for the presence of the Kennedy and Bremer-Greenberg pathways in Caenorhabditis elegans. 3.* Lochnit, G., Grabitzki, J., Henkel, B. & Geyer, R. (2003) Identification and characterization of an aspartyl protease-bound phosphorylcholine epitope in Caenorhabditis elegans.

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6.3 Acknowledgements First, I wish to thank my scientific teacher Rudolf Geyer for his excellent support, permanent commitment, continuous encouragement of this work and the wonderful atmosphere in his group for more than one decade. I wish to express my gratitude to Roger Dennis for the introduction in the fields of glycosphingolipids, nematodes and immunology, his helpful discussions and for the critical reading of many manuscripts. Many members of the lab contributed to this thesis: Claudia Friedl (A. suum zwitterionic glycosphingolipid structural analyses), Stefan Gerdt (C. elegans glycosphingolipid analyses), Hildegard Geyer (helpful discussions), Julia Grabitzki and Björn Henkel (C. elegans aspartyl protease project), Heike Müntefehr (A. suum zwitterionic glycosphingolipid localization), Sonja Nispel (A. suum acidic glycosphingolipid localization), Sandra Rickhoff (O. volvulus zwitterionic glycosphingolipid analyses) and Manfred Wuhrer (S. mansoni glycosphingolipid analyses). I thank all members of the lab for their patience, support, helpful discussions and for all the fun we have had during this time. I gratefully acknowledge Klaus Preissner, Stefan Stirm and all the other members of the Institute of Biochemistry for their support, fruitful discussions and the inspiring atmosphere. Many thanks also to my collaboration partners Bill and Maggie Harnett (Glasgow) for the support in immunological and biosynthetic aspects, Ulrich Zähringer (Borstel) for NMR-measurements, Jochen Ulmer (Borstel) for supporting the cytokine work, Louis Filgueira (Zurich) for investigations on the receptors and internalization of PC- antigens, Jacques Vanfleteren (Gent) for supporting the establishment of the axenic C. elegans culture, Einhardt Schierenberg (Cologne) for helpful discussions on the development of C. elegans, Horst Zahner (Giessen) for immunohistochemical cooperation, and Rico Bongarts and Angela Comas from UnionBiometrica (Geel) for the COPAS measurements. Special thanks are due for the excellent technical assistance of F. Busch, P. Kaese, S. Kühnhardt, W. Mink, M. Schwinn and D. Zimmer, without whose experience, carefulness, patience and support most of this work would not have been possible. The work was supported by the German Research Council (SFB 272 and 535 and Graduiertenkolleg “Molecular Biology and Pharmacology”) and the Hessian Ministry for Science and Arts.