Question: Glycosylation of proteins is one of the most important post-translational modifications and important for protein stability, secretion and localization. In the case of parasitic nematodes, N-glycans frequently terminated with the zwitterion phosphorycholine can be identified. This specific modification seems to be biologically relevant as it is required for immunomodulatory effects; however, specific methods to test interactions with natural glycans need to be developed. Methods: To better define the biological function of these parasitic sugar modifications, N-glycans from the dog parasite Dirofilaria immits were prepared, derivatized with a bivalent fluorescent tag, analyzed by mass spectrometry and HPLC methods and finally used to generate a defined carbohydrate microarray. The array was probed with sera from non-infected as well infected dogs, various glycan- binding proteins, pentraxins and antibodies. Results: Using this approach, structures containing non-mammalian elements such as phosphorylcholine and multiple fucosylation on the antennae could be identified. These elements could be immunogenic to the dog host; however, on the glycan microarray only a weak binding signal of carbohydrate-binding IgM could be determined. Also, mannose-binding lectin failed to recognize the natural parasitic sugar molecules. Only after the chemically removal of the parasite-type modifications was binding to the glycans detected. In contrast, the pentraxin CRP was found to be present in the dog sera and could bind natural glycans in combination with the complement factor C1q. Conclusion: Taken together, the results show that the N-glycans of D. immitis contain a number of features, which may mediate immunomodulation in the host or confer the ability to avoid immune surveillance.

Understanding the biological role of parasitic N-glycans using microarrays

256th National Meeting (2018)

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