Uptake and incorporation of sialic acid by the tick Ixodes ricinus Marie Vancova a,b,⇑,1 , Jan Sterba b,1 , Jarmila Dupejova a,b , Zuzana Simonova b , Jana Nebesarova a , Milos V. Novotny c , Libor Grubhoffer a,b a Institute of Parasitology, Biology Centre of the ASCR, C ˇ eské Bude ˇjovice, Czech Republic b Faculty of Science, University of South Bohemia, C ˇ eské Bude ˇjovice, Czech Republic c National Center for Glycomics and Glycoproteomics, Department of Chemistry, Indiana University, Bloomington, IN 47405, USA article info Article history: Received 13 January 2012 Received in revised form 27 June 2012 Accepted 29 June 2012 Available online 7 July 2012 Keywords: Tick Ixodes ricinus Sialic acid Basement membrane Mass spectrometry Electron microscopy abstract We describe the detection of sialylated N-linked glycans in partially fed Ixodes ricinus tick females using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Sialylated glycans were detected in salivary glands as well as in tick guts and we propose the host origin of these structures. In addition, we mapped the transport of sialylated structures from the blood meal through the gut to the salivary glands using electron microscopy. Specific localization of sialylated glycans to base- ment membranes of salivary glands was observed. Finally, the influence of the sample preparation meth- ods for electron microscopy on ultrastructure and immunogold labeling was evaluated. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Ticks Ixodes ricinus are obligatory blood-feeding parasites, which transmit medically important pathogens such as the tick- borne encephalitis virus or the Lyme disease spirochetes Borrelia burgdorferi. When feeding on hosts, some tick tissues, especially the midgut and salivary glands (SGs), undergo substantial changes in their structure, size, and expression of proteins as well. SGs se- crete pharmacologically active molecules that allow feeding on hosts for a long time and facilitate the transmission of pathogens while gut produces predominantly the digestive enzymes. Poten- tial N-linked glycosylation sites were predicted in medically inter- esting molecules (such as Isac, Ixolaris, or Salp20 proteins) secreted into the host (Francischetti et al., 2002; Ribeiro et al., 2006; Tyson et al., 2007). However, the glycan structures of such glycoproteins are still completely unknown. The current improvements in ana- lytical techniques have now made it possible to analyze small amounts of glycoproteins obtained from tick organ homogenates and saliva either directly or after preparative chromatography iso- lations (Dupejova et al., 2011; Man et al., 2008). The exact structures of tick glycans have been identified only in few cases. Lectin Dorin M from the hemolymph of the soft tick Ornithodoros moubata was modified solely by the high-mannose- type of N-glycans with nine mannose residues and core-fucosylat- ed paucimannosidic glycans with four to five mannoses (Man et al., 2008). Recently, high-mannose and complex N-linked glycans without core-fucosylation have been described in a study of Heme- lipoglycoprotein, the protein responsible for binding, transporta- tion, and storage of heme, in Dermacentor marginatus (Dupejova et al., 2011). Analysis of glycans from electrophoretically separated proteins from the I. scapularis nymph SGs showed high-mannose glycans without core-fucosylation and core-fucosylated pauciman- nosidic (two to four mannoses) and complex (one or two N-acetyl- glucosamine residues) glycans (Pedra et al., 2010). The complex type glycans are the predominant structures in mammals, whereas the most dominant arthropod glycans are the high-mannose struc- tures as well as truncated (paucimannosidic type) N-glycans usu- ally modified by core-fucosylation (Aoki et al., 2007; Wilson et al., 2009). a1,3-Linked core-fucose was shown to trigger an im- mune reaction in vertebrates including humans (Altmann, 2007). In ticks, this modification is important for gut colonization by the obligate intracellular tick-borne pathogen Anaplasma phagocyto- philum (Pedra et al., 2010). Earlier, we detected the presence of sialic acid (Sia) residues in the secreting granules of SGs isolated from partially fed I. ricinus fe- males using indirect methods such as enzymatic deglycosylation and affinity labeling with Maackia amurensis agglutinin (MAA II) and Sambucus nigra agglutinin (SNA) (Vancova et al., 2006). SNA binds preferentially to Sia attached to terminal galactose in a2, 0022-1910/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinsphys.2012.06.016 ⇑ Corresponding author at: Institute of Parasitology, Biology Centre of the ASCR, Branisovska 31, CZ-37005 Ceske Budejovice, Czech Republic. Tel.: +420 38 7775403; fax: +420 38 5310388. E-mail address: vancova@paru.cas.cz (M. Vancova). 1 Both authors equally contributed to this work. Journal of Insect Physiology 58 (2012) 1277–1287 Contents lists available at SciVerse ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys