Analyse der posttranslationalen Modifikationen von Proteinen in den Mikrofilarienscheiden von Litomosoides sigmodontis

Abstract

Lymphatic filariases are wide-spread diseases in humans and beasts in the tropics. According to the WHO about 400 million individuals are estimated to be infected [1]. Lymphatic diseases are caused by nematodes like Wuchereria bancrofti and Brugia species, employing arthropods as vectors.
The rodent filaria, Litomosoides sigmodontis, and its mammal host, Sigmodon hispidus, have been found to be a competent model system for laboratory studies on filariasis and its pathogenesis.
Despite of very complex immunological implications during the pathogenesis of an Litomosoides-infected host, a concomitant decrease of the immune response towards the larvae, i.e. microfilariae, is characteristic. These are ensheathed, and -still for unknown reasons- protected against immune attacks of the host. Yet, recently observed featured of the micro-filariae, such as molecular mimikry or chemically modified surfaces, are suggested to have modulating effects on the host's immune system. Therefore, the biochemical analysis of the modifications in the Litomosoides sigmodontis-microfilarial sheath is considerably important to help elucidate the molecular mechanisms involved during immune evasion.

Exclusively two surface polypeptides of the L. sigmodontis-microfilarial sheath, shp3 and shp3a, are posttranslationally modified by the biogenic amine, dimethylaminoethanol (DMAE), which is known for its antidepressive properties.
The structure of DMAE was extensively described by nuclear magnetic resonance as the Fmoc-derivative. As revealed by amino acid analysis and MALDI-TOF-mass spectrometry, the modifications of shp 3a accounted for 75% of its molecular mass, and 25% of DMAE, respectively [2]. Upon dephosphorylation with hydrofluoric acid (HF) the loss of mass of ca. 18kDa correlated with the amount of 110 phosphorylated DMAE-residues. The DMAE-homologues, choline and monomethylaminoethanol, were detected only in trace amounts. In contrast to other sheath polypeptides, shp3 and shp3a could be stained with the cationic dye, Stains-All, in SDS-PAGE, thus suggesting a poly-anionic structure [3].
Component analyses of carbohydrates yielded 6% (w/w) of N-acetylgalactosamine, 3% (w/w) of galactose and 0.2% (w/w) of uronic acids, suggesting the presence of O-glycans. By means of hydrazinolysis, reductive beta-elimination and mass spectrometry, the carbohydrate moiety was subjected to a detailed structural analysis. Mass spectrometric analyses yielded mono-meric N-acetylgalactosamine and the disaccharide, Gal1-3GalNAc.Unlike other O-glycans, these were not substituted by a sialic acid, a fucose nor a sulfate residue, thus resembling the so-called Tn- and the T-antigen, respectively.
The data based on combined MALDI-TOF-MS, methylation analysis (GC/MS) and HF-treatment revealed a trisaccharide with an unusual sequence, Gal1-3Gal1-3GalNAc. Seven OH-groups of the trisaccharide were substituted with phosphorylated DMAE. Four of which could be located on the terminal galactose, the remaining three residues were not exactly assigned.
Finally, the chemical synthesis of bovine insulin and serum albumin, both substituted with phosphorylated DMAE as a hapten, was successful. These synthetic products were purified via HPLC and could be employed to achieve antibodies for further histochemical experiments.

References

[1] WHO. Lymphatic Filariasis: The Disease and its Control WHO Technical Report Series 821, 1-71 (1992)

[2] M. Hintz, M. Kasper, B. Stahl, R. Geyer, H.-O. Kalinowski, M. Karas, S. Kühnhardt, H.-H. Schott, F. Conraths, H. Zahner, S. Stirm: Dimethylaminoethanol is a major component in the Litomosoides carinii microfilarial sheath. Mol. Biochem. Parasitol. 76, 325-328 (1996)

[3] M.R. Green, J.V. Pastevka, A.C. Peacock: Differential Staining of Phosphoproteins on Polyacrylamide Gels with a Cationic Dye. Anal. Biochem. 56, 43-51 (1973)

[4] M. Hintz: Die Proteine der Mikrofilarienscheide von Litomosoides carinii. Doctor's thesis 1994

[5] M. Kasper: Strukturanalyse der posttranslationalen Modifikationen in den Proteinen der Mikrofilarienscheide von Litomosoides sigmodontis. Doctor's thesis 2000, submitted