Hemelipoglycoprotein from the ornate sheep tick, Dermacentor marginatus: structural and functional characterization

Abstract

Background: Tick carrier proteins are able to bind, transport, and store host-blood heme, and thus they function also as antioxidants. Nevertheless, the role of carrier proteins in ticks is not fully understood. Some of them are found also in tick males which do not feed on hosts to such an extent such as females (there are differences in male feeding in different tick species) and thus they are not dealing with such an excess of heme; some of the carrier proteins were found in salivary glands where the processing of blood and thus release of heme does not occur. Besides, the carrier proteins bind relatively low amounts of heme (in one case only two molecules of heme per protein) compared to their sizes (above 200 kDa). The main aim of this study is the biochemical characterization of a carrier protein from the ornate sheep tick Dermacentor marginatus, hemelipoglycoprotein, with emphasis on its size in native conditions, its glycosylation and identification of its modifying glycans, and examining its carbohydrate-binding specificity.

Results: Hemelipoglycoprotein from D. marginatus plasma was purified in native state by immunoprecipitation and denatured using electroelution from SDS-PAGE separated plasma. The protein (290 kDa) contains two subunits with molecular weights 100 and 95 kDa. It is glycosylated by high-mannose and complex N-glycans HexNAc(2)Hex(9), HexNAc(2)Hex(10), HexNAc(4)Hex(7), and HexNAc(4)Hex(8). The purified protein is able to agglutinate red blood cells and has galactose- and mannose-binding specificity. The protein is recognized by antibodies directed against plasma proteins with hemagglutination activity and against fibrinogen-related lectin Dorin M from the tick Ornithodoros moubata. It forms high-molecular weight complexes with putative fibrinogen-related proteins and other unknown proteins under native conditions in tick plasma. Feeding does not increase its amounts in male plasma. The hemelipoglycoprotein was detected also in hemocytes, salivary glands, and gut. In salivary glands, the protein was present in both glycosylated and nonglycosylated forms.

Conclusion: A 290 kDa hemelipoglycoprotein from the tick Dermacentor marginatus, was characterized. The protein has two subunits with 95 and 100 kDa, and bears high-mannose and complex N-linked glycans. In hemolymph, it is present in complexes with putative fibrinogen-related proteins. This, together with its carbohydrate-binding activity, suggests its possible involvement in tick innate immunity. In fed female salivary glands, it was found also in a form corresponding to the deglycosylated protein.

Figure 1

Identification of putative FReD proteins in D. marginatus plasma and detection of HLGP in tick tissues. Molecular weights of standard proteins are depicted. A) Immunoblotting of non-reduced electrophoretically separated D. marginatus plasma proteins. 1 - D. marginatus plasma proteins, stained with Coomassie Brilliant Blue, 2 - immunostaining of plasma proteins using antibodies against HA of D. marginatus hemolymph serum, 3 - immunostaining of plasma proteins by anti-Dorin M antibodies. B) Immunoblotting of HLGP in D. marginatus tissues using mouse polyclonal anti-HLGP serum. 1 - fed female plasma, 2 - unfed female plasma, 3 - fed male plasma, 4 - unfed male plasma, 5 - fed female hemocytes, 6 - unfed female hemocytes, 7 - fed male hemocytes, 8 - unfed male hemocytes, 9 - fed female gut, 10 - fed female salivary glands. * marks the position of native glycosylated HLGP, # marks the deglycosylated form of HLGP.

Figure 2

2 D and native electrophoreses of plasma proteins and HLGP. A) Blue native/SDS-PAGE 2 D electrophoretic separation of D. marginatus plasma proteins, stained by Coomassie Brilliant Blue (CBB). BN-PAGE separated plasma proteins are showed above the gel and acrylamide gradients are depicted above the gel (native electrophoresis) and on the right side (SDS-PAGE electrophoresis). HLGP is marked with an arrow. B) Blue native PAGE of D. marginatus plasma proteins, stained by CBB. 1 - native molecular weight standard, 2 - tick plasma. Complex of HLGP with other proteins is marked with an arrow.

Figure 3

HLGP is a glycosylated protein. Molecular weights of standard proteins are depicted. A) Schiff staining and lectin staining of electrophoretically separated and electroblotted purified HLGP. 1 - tick plasma proteins, stained with Coomassie Brilliant Blue, 2-5 electroblotted HLGP staining. 2 - Schiff staining, 3 - SNA lectin staining, 4 - GNA lectin staining, 5 - DSA lectin staining of purified HLGP. Positive reaction in Schiff staining confirms the presence of glycans on HLGP. Types of glycans identified by positive reactions in lectinoblotting are described in text. B) Immunodetection of deglycosylated purified HLGP using anti-HLGP serum. 1,3,4 - non-reduced electroeluted HLGP and 2,5,6 - reduced electroeluted HLGP stained with anti-HLGP serum. Samples in lanes 3 and 5 were deglycosylated using Endo H, samples in lanes 4 and 6 deglycosylated using PNGase A/F. * - native glycosylated HLGP, # - deglycosylated form of HLGP.

Figure 4

Mass spectra of N-glycans from the purified HLGP and from D. marginatus hemolymph. A) MALDI-TOF spectrum of enzymatically released N-glycans from HLGP. Four peaks corresponding to s HexNAc₂Hex₉ (m/z 2396.7), HexNAc₂Hex₁₀ (m/z 2600.8), HexNAc₄Hex₇ (m/z 2477.8), and HexNAc₄Hex₈ (m/z 2684) are marked. Three more peaks are visible corresponding to contaminating series of hexoses and O-glycans (not labeled). B) MALDI-TOF spectrum of enzymatically released N-glycans from D. marginatus hemolymph. A range of glycan structures was identified, namely high-mannose type glycans HexNAc₂Hex₃ to HexNAc₂Hex₁₀ (m/z 1171.3, 1375.4, 1783.6, 1987.7, 2191.8, 2395.9, 2599.9) and complex fucosylated and non-fucosylated glycans such as HexNac₃Hex₃ (m/z 1416.5), HexNAc₂Hex₄dHex₁ (m/z 1549.5), HexNAc₂Hex₅dHex₁ (m/z 1753.6). Again, contaminants were present in the sample - series of hexoses as well as O-glycans (not labeled).

Figure 5

HLGP is a carbohydrate-binding protein - HA and SPR. A) Hemagglutination activity of purified HLGP. C - negative control (no hemagglutination activity), H - serial two-fold dilution of electroeluted HLGP. Hemagglutination activity is visible in dilutions 2× to 16×. B) Carbohydrate-binding specificity of purified HLGP assessed by SPR on a chip with four channels arranged sequentially as galactose-fucose/mannose-mannose-fucose. Red line corresponds to signal from the channel with immobilized galactose, green to channel with immobilized fucose and mannose, blue to channel with immobilized mannose and purple to channel with immobilized fucose. The fucose/mannose channel contained half amount of the corresponding carbohydrates and serves as control. Lines correspond to SPR response in real-time (not normalized for the sequential flow through channels). Galactose showed the strongest binding by HLGP, some binding was observed also for mannose. Fucose was not recognized by the protein.