Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes

Abstract

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins. Tsetse salivary N-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online with positive-ion ESI-LC-MS/MS. We found that the N-glycan profiles of T. brucei-infected and naïve tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type N-glycans. In overlay assays, these sugars were differentially recognized by the mannose receptor and DC-SIGN C-type lectins. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host.

Fig 1. Analysis of G. morsitans salivary glycoproteins.

10 μg of G. morsitans salivary proteins (lanes 1 and 2) and 10 μg of egg albumin (lanes 3 and 4) were incubated overnight with (2 and 4) and without (1 and 3) PNGase F. After digestion, proteins were resolved by SDS-PAGE and Coomassie blue-stained. There was a notable shift in migration in 4 bands following PNGase F treatment. After in-gel trypsinization and MALDI-TOF MS analysis these bands were identified as 5’ Nucleotidase (1), TSGF 2/Adenosine deaminase (2), TSGF 1 (3), and Tsal 1/2 (4). *, PNGase F enzyme.

Fig 2. Tsetse fly salivary glycoproteins are composed mainly of paucimannose and oligomannose N-glycans.

(A) Profile of salivary N-glycans from teneral (young, unfed) flies, before and after digestion with exoglycosidases. Aliquots of the total PNGase F-released 2-AB-labeled N-glycan pool were either undigested (i) or incubated with a range of exoglycosidases (ii-iv). (i) Undig, before digestion; (ii) GUH, Streptococcus pneumoniae in E. coli β-N-acetylglucosaminidase; (iii) JBM, Jack bean α-Mannosidase; (iv) bkF, Bovine kidney α-fucosidase. Following digestion, the products were analyzed by HILIC-UHPLC. Peaks labelled A correspond to the product of complete digestion with JBM; those labelled with an asterisk refer to buffer contaminants. The percent areas and structures of the different N-glycans are listed in Table 1. (B) Positive-ion ESI-MS spectrum of procainamide-labelled N-glycans from teneral tsetse fly saliva. Numbers refer to the structures in Table 1. The dagger symbol (‡) refers to m/z 1130.55 as [M+2H]²⁺ ion; the appearance of the Man₃GlcNAc₂-Proc as singly and doubly charged ion in positive mode, reflects on its high relative abundancy (~54%) in this sample. Green circle, mannose; blue square, N-Acetylglucosamine; red triangle, fucose; Proc, procainamide. GU, glucose homopolymer ladder. [22].

Fig 3. Analysis of tsetse salivary N-linked glycans in teneral, naïve and trypanosome-infected flies.

(A) Comparison of HILIC-UHPLC profiles of salivary N-glycans released by PNGase F. Analysis of 2AB-labelled glycans from (i) teneral, (ii) naïve, and (iii) trypanosome-infected saliva. Relative abundances are indicated in Table 2. Tbb, Trypanosoma brucei brucei. (B) Positive-ion ESI-MS analysis of procainamide labelled N-glycans from adult naïve and trypanosome-infected saliva. Spectra are shown for naïve (top) and trypanosome-infected (bottom) saliva. Numbers refer to the structures shown in Table 1. Green circle, mannose; blue square, N-Acetylglucosamine; red triangle, fucose; Proc, procainamide. Peaks labelled with an asterisk refer to buffer contaminants.

Fig 4. Analysis of the effects of infection on immunogenicity of tsetse fly saliva.

(A) 2 μg of G. m. morsitans salivary proteins were treated (+) or untreated (-) with PNGase F, fractionated by SDS-PAGE, transferred onto a PVDF membrane, and probed with an anti-G. m. morsitans saliva antibody. (B) Uniform protein loading for Western blot was confirmed by nigrosine staining of proteins transferred to PVDF membrane. (C) Con A blotting analysis of tsetse salivary glycoproteins from naïve and trypanosome-infected flies. OVA, egg albumin positive control. Asterisk indicates PNGase F enzyme band.

Fig 5. Analysis of the binding of salivary proteins to metacyclic trypanosomes.

Silver stained protein profiles and Western blot analysis to detect the presence of salivary proteins on tsetse salivary gland-derived trypanosomes. Two subsequent washes (W1 and W2) of metacyclic parasites (MF, equivalent of 3×10⁴ parasites loaded on gel) isolated from infected tsetse fly salivary glands and a corresponding sample of trypanosomes purified from mouse blood (BSF, equivalent of 3×10⁴ parasites loaded). Protein bands were revealed with purified rabbit anti-G. morsitans saliva IgGs and pre-immune IgGs as a control, and development with peroxidase-coupled goat anti-rabbit IgG. Asterisk indicates salivary proteins in the metacyclic trypanosome lysate.

Fig 6. Tsetse salivary N-glycans are recognized by C-type lectins Mannose Receptor and DC-SIGN.

2 μg of Glossina morsitans saliva (Gmm) were untreated (-) or treated (+) with PNGase F and then processed for overlay assays using either recombinant CTLD4-7-Fc (A) or DC-SIGN (B). MWM, lanes 1 and 7; Gmm saliva, lanes 2, 3, 8 and 9; OVA, egg albumin positive control (lanes 4, 5, 10 and 11); BSA, bovine serum albumin negative control (lanes 6 and 12). Nigrosine-stained membranes (B, D) are shown as loading controls for (A) and (B), respectively. Asterisk indicates PNGase F enzyme.