Insights into the salivaryN-glycome ofLutzomyia longipalpis, vector of visceral leishmaniasis

Abstract

24During Leishmania transmission sand flies inoculate parasites and saliva into the skin of 25 vertebrates. Saliva has anti-haemostatic and anti-inflammatory activities that evolved to facilitate 26 bloodfeeding, but also modulate the host's immune responses. Sand fly salivary proteins have 27 been extensively studied, but the nature and biological roles of protein-linked glycans remain 28 overlooked. Here, we characterised the profile of N-glycans from the salivary glycoproteins of 29 Lutzomyia longipalpis, vector of visceral leishmaniasis in the Americas. In silico predictions 30 suggest half of Lu. longipalpis salivary proteins may be N-glycosylated. SDS-PAGE coupled to LC-31 MS analysis of sand fly saliva, before and after enzymatic deglycosylation, revealed several 32 candidate glycoproteins. To determine the diversity of N-glycan structures in sand fly saliva, 33 enzymatically released sugars were fluorescently tagged and analysed by HPLC, combined with 34 highly sensitive LC-MS/MS, MALDI-TOF-MS, and exoglycosidase treatments. We found that the 35 N-glycan composition of Lu. longipalpis saliva mostly consists of oligomannose sugars, with 36Man5GlcNAc2 being the most abundant, and a few hybrid-type species. Interestingly, some 37 glycans appear modified with a group of 144 Da, whose identity has yet to be confirmed. Our 38 work presents the first detailed structural analysis of sand fly salivary glycans. 39 40 41 42 43 44 45 72There are some reports describing the presence of salivary glycoproteins in sand flies through in 73 silico and blotting analyses [13][14][15][16][17][18][19] ; however, to our knowledge no detailed structural studies have 74 been published to date. Therefore, we set out to identify the salivary glycoproteins in the sand fly 75 vector species Lutzomyia longipalpis, and structurally characterise their N-glycan conjugates. We 76 further discuss their implications for insect bloodfeeding as well as vector-host interactions. 77 78 Results 79 Identification of Lutzomyia longipalpis salivary glycoproteins.80 To determine the degree of N-glycosylation, an in silico analysis was carried out on 42 salivary 81 proteins previously reported in Lu. longipalpis 4,20 to predict protein N-glycosylation sites using the 82 NetNGlyc server (http://www.cbs.dtu.dk/services/NetNGlyc/). This revealed 48% of the 83 commonly known salivary proteins contain conventional N-glycosylation sites (Supplementary 84 those without recognizable glycosylation sequons (as determined by NetNGlyc), obtaining a final 109 list of 43 potentially N-glycosylated protein candidates (Supplementary Table S2). Fourteen of 110 these potential glycoproteins were also identified in our initial in silico analysis (Supplementary 111 Table S1), including LJM11, LJM111 and LJL143, which have been proposed as potential vaccine 112 components against Leishmania infection 4 . Using the InterProScan tool to identify conserved 113 protein domains, family distributions ( Supplementary Fig. S4) show five of the candidates 114 belonging ...

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

“…To evaluate the presence of O-linked glycans in tsetse salivary glycoproteins, we released the O-glycans by hydrazinolysis [24] and analyzed fluorescently labelled samples by HILIC-HPLC and MS. However, we did not detect O-glycans by either HILIC or MS (S6 Fig), which coincides with our recent findings in the saliva of Lutzomyia longipalpis sand flies [42]. This suggests that either these sugars are made in very low abundance by salivary gland cells of these insects, or they may be structurally different compared to mammalian O-glycans.…”

“…albopictus [40], and Phlebotomus duboscqi sandflies [41]. However, the most complete structural characterization of salivary glycoproteins in disease vectors to date has been that of Lutzomyia longipalpis, vector of visceral leishmaniasis in the Americas [42]. This sand fly species makes mostly oligomannose N-glycans, with Man 5 GlcNAc 2 being the most abundant structure.…”

Insights into the proteomic profile and gene expression of Lutzomyia longipalpis-derived Lulo cell line

“…longipalpis genome in VectorBase ( , Table II), as well as their importance in the literature. 42 , 43 In addition, identification with a score greater than 50 reflects the quality of the peptide sequences obtained and the consequent identification of the protein, in the studied condition. These three proteins had their corresponding nucleic acid sequences analysed for the design of specific RT-PCR primers ( Table ).…”