Insights into the salivary N-glycome of Lutzomyia longipalpis, vector of visceral leishmaniasis

Abstract

During Leishmania transmission sand flies inoculate parasites and saliva into the skin of vertebrates. Saliva has anti-haemostatic and anti-inflammatory activities that evolved to facilitate bloodfeeding, but also modulate the host's immune responses. Sand fly salivary proteins have been extensively studied, but the nature and biological roles of protein-linked glycans remain overlooked. Here, we characterised the profile of N-glycans from the salivary glycoproteins of Lutzomyia longipalpis, vector of visceral leishmaniasis in the Americas. in silico predictions suggest half of Lu. longipalpis salivary proteins may be N-glycosylated. SDS-PAGE coupled to LC-MS analysis of sand fly saliva, before and after enzymatic deglycosylation, revealed several candidate glycoproteins. to determine the diversity of N-glycan structures in sand fly saliva, enzymatically released sugars were fluorescently tagged and analysed by HPLC, combined with highly sensitive LC-MS/MS, MALDI-TOF-MS, and exoglycosidase treatments. We found that the N-glycan composition of Lu. longipalpis saliva mostly consists of oligomannose sugars, with Man 5 GlcnAc 2 being the most abundant, and a few hybrid-type species. Interestingly, some glycans appear modified with a group of 144 Da, whose identity has yet to be confirmed. Our work presents the first detailed structural analysis of sand fly salivary glycans. Sand flies are small insects that can transmit bacteria and viruses 1,2 , but are known mainly as vectors of leishmaniasis, a disease that threatens 350 million people worldwide 3. When female sand flies feed, they inject a saliva comprised of molecules that facilitate the ingestion of blood, and modulate the host immune system and pathogen transmission 4-6. These effects have led researchers to explore the potential of insect salivary molecules as markers of biting exposure 5,7 (to determine risk of disease), or even as components of vaccines against leishmaniasis 8. Of the many types of molecules that make up saliva, most research has focused on the proteins; here, we have investigated the glycans that modify these proteins. In most eukaryotic cells, the addition of glycans to proteins is a highly conserved and diverse post-translational modification. The most common types of protein-linked glycans are N-linked (attached to asparagine residues in the sequon Asn-X-Thr/Ser), and O-linked (attached to serine or threonine residues). Glycoconjugates display a wide range of biological roles, from organism development to immune system functions against pathogens 9. One study has addressed the types and roles of glycans in insects using the model fruit fly, Drosophila melanogaster. In this species, biological functions have been attributed to different glycan classes, such as morphology and locomotion (N-linked glycans), or cell interaction and signalling (O-linked glycans) 10 .

Lectin Microarray Analysis of Salivary Gland Glycoproteins from the Arboviral VectorAedes aegyptiand the Malaria VectorAnopheles stephensi

“…O-glycans, terminal glucuronic acids and sulphate residues could not be detected in Glossina morsitans morsitans salivary proteins in this MS study [19]. Terminal αGal was not detected in protein glycoconjugates in the saliva of L. longipalpis [18] or G. morsitans morsitans [19]. This suggests that the presence of α-gal epitopes in the glycoproteins of arachnid tick vectors may not be a characteristic of insect mosquito vectors.…”

“…albopictus C6/36 cells [11] may be due to the acquisition of α-gal containing glycoconjugates from foetal bovine serum used in the culture medium and requires further investigation. Analysis of N-glycans in saliva of the sandfly vector of visceral leishmaniasis, Lutzomyia longipalpis, by MS also showed a preponderance of oligomannose, fucosylation of the core GlcNAc, and terminal HexNAc residues, but O-glycans, terminal glucuronic acids and sulphate residues were not detected [18]. N-glycans in saliva from the vector of African trypmanosomiasis Glossina morsitans morsitans showed a preponderance of pauci-mannose and oligomannose, α-linked fucose in the core GlcNAc, and terminal GlcNAc in a study by MS [19].…”

An analytical study on the identification of N-linked glycosylation sites using machine learning model

“…7 . It is observed, there are 13 studies including ( Chien et al, 2020 ; Taherzadeh et al, 2019 ; Liu et al, 2019 ; Li et al, 2019 ; Thomès, Burkholz & Bojar, 2021 ; Pitti et al, 2019 ; Adolf-Bryfogle et al, 2021 ; Park et al, 2019 ; He, Wei & Zou, 2019 ; Audagnotto & Dal Peraro, 2017 ; Mondragon-Shem et al, 2020 ; Dimeglio et al, 2020 ; Ruiz-Blanco et al, 2017 ) which have developed the tool specific to the N-Linked site identifications, few studied developed tool for glycosylation sites identification irrespective of the specific type including ( Bojar et al, 2021b ; Carpenter et al, 2022 ; Lundstrøm et al, 2022 ; Burkholz, Quackenbush & Bojar, 2021 ; Coff et al, 2020 ; Shek, Kotidis & Betenbaugh, 2021 ) and some authors ( Le, Sandag & Ou, 2018 ; Liu et al, 2021 ; Yang et al, 2019 ; Campbell, 2017 ) develop tool without mentioning the type of PTM. These all tools have list down in the Table 9 .…”

A sand fly salivary protein acts as a neutrophil chemoattractant

“…Of note, the amount of protein in a pair of salivary glands varies by sand fly species 16,17 , and a sand fly delivers 70-90% of its salivary protein content while feeding [18][19][20] . For P. duboscqi and L. longipalpis, a pair of salivary glands contains between 0.78 to 1 μg of protein 17,20 and 0.5 to 1 μg of protein 21,22 , respectively. Further, transcriptomic analysis indicates that the relative abundance of yellow proteins in the salivary glands of L. longipalpis and P. duboscqi is 16.0% 23 and 7.5% 24 , respectively.…”