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

Karina Mondragon-Shem, Katherine Wongtrakul-Kish, Radoslaw P. Kozak, Shi Yan, Iain Wilson, Katharina Paschinger, Matthew E. Rogers, Daniel I. R. Spencer, Alvaro Acosta-Serrano

Preprint posted on June 04, 2020

More than just saliva: Insights into the N-glycome of sandfly saliva

Selected by Mariana De Niz

Categories: biochemistry, cell biology


Multiple infectious diseases are vector-borne, and are transmitted during an infected insect’s feed on blood. Leishmania is transmitted by sand flies, which inoculate parasites and saliva into the skin of  vertebrates. Saliva has anti-haemostatic and anti-inflammatory activities that facilitate bloodfeeding, while modulating the host’s immune responses. Among the molecules that make up sand fly saliva, most research has focused on proteins. In most eukaryotic cells, the addition of glycans to proteins is a conserved and diverse post-translational modification. In Drosophila melanogaster, various important biological functions have been attributed to glycans, including morphology, locomotion, cell-cell interactions, and signaling (1). This and other studies have suggested that protein-linked glycans in the saliva of blood feeding arthropods may have key relevance in the transmission of vector-borne diseases. In their work, Mondragon-Shem, Wongtrakul-Kish and colleagues characterised the profile of N-glycans from the salivary glycoproteins of Lutzomyia longipalpis, vector of visceral leishmaniasis in the Americas (2).

Figure 1. Characterisation of the profile of N-glycans from the salivary glycoproteins of Lutzomyia longipalpis, vector of visceral leishmaniasis in the Americas. (Adapted from figure 1, Ref2, generated by Karina Mondragon-Shem).

Key findings and developments

In silico analyses showed that approximately half of commonly known salivary proteins from Lutzomyia longipalpis contain conventional N-glycosylation sites (3,4). For further in-depth investigation, the authors dissected Lu. longipalpis salivary glands and released their saliva for analysis. Using a variety of methods, they first identified those that were glycosylated. Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) allowed identification of multiple candidates, from which the authors focused on 43 potentially N-glycosylated protein candidates, some of which were identified in the in silico analysis, and some which have been proposed as vaccine candidates in the past. Further computational analysis of the chosen candidates identified conserved protein domains, with family distributions showing candidates belonging to the actin, tubulin, 5’nucleotidase, peptidase M17, and the major royal jelly protein families. Further analysis on the “molecular function breakdown” suggested that about half of the candidate glycoproteins are involved in binding. Prediction of protein subcellular localization and solubility suggested that 85% of candidate glycoproteins are soluble, with 10 of them being extracellular too.

The authors then went on to determine the N-glycome modifying the salivary proteins of Lu. longipalpis, and identified 14 different structures. They conclude that most salivary glycoproteins from Lu. longipalpis are of the high mannose type (Man5GlcNAc2), while only a few hybrid-type species were identified. A more detailed analysis of the saliva using Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) of pyridylaminated glycans revealed the major oligomannosidic species, and suggested the existence of a series of sandfly salivary glycans containing unidentified modifications of 144 Da. This modification was mainly found in two isomeric glycans, however it seems to be located in different positions in the two structures. Altogether, the identity of these modified glycans remains to be confirmed. Finally, as the in silico analysis suggested that the majority of the identified proteins have putative O-glycosylation sites, the authors went on the explore this finding further, but did not detect any O-glycans in the samples. Together, this work is the first detailed structural analysis of sandfly salivary glycans.


What I like about this preprint

I like that the authors identified, and bridged a gap in scientific knowledge, and show the importance of understanding better the composition and glycan modifications of vector saliva in general, and sandfly saliva in particular. Hopefully this will transcend to other vectors of public health, and veterinary, and scientific relevance.


Open questions

  1. Congratulations on the very interesting work, which opens many exciting questions! On a general level, and assuming a naïve host who has never seen vector saliva, how do your overall findings relate to potential recognition by vertebrate hosts of saliva, upon injection? Or conversely, ‘hiding’?
  2. Following from the previous question, you mention in your work the potential of salivary proteins as vaccine candidates. You discuss also how people living in endemic areas get numerous bites by the vector, even when the vector is not infected. Have you or others studied if and how vector saliva – and particularly glycoproteins- elicit immunological memory? If so, how would the progress of infection with Leishmania differ if the mammalian host has ‘seen’ salivary glycoproteins before, versus if they have not.
  3. Thinking of transmission from vector to mammalian host, does presence of Leishmania alter in any way the modifications you found in your work?
  4. You discuss in your work, previous findings on the glycome of Drosophila melanogaster Do you think the N-glycome contributes to Leishmania dynamics in the sandfly midgut?
  5. You studied the presence of O-linked glycans, and did not detect these sugars in fly saliva. Can you expand further on the importance of these sugars, and why surprisingly you did not detect them?
  6. You discuss in your work that the candidates you identified belong to the actin, tubulin, 5’nucleotidase, peptidase M17, and the major royal jelly protein families. What is the relevance of these families to saliva, and potentially to pathogen transmission?
  7. You discuss in your work also, the relevance of saliva in multiple vectors responsible for transmission of infectious pathogens. How do your findings compare to research conducted on other vectors?


  1. Katoh, T. & Tiemeyer, M. The N’s and O’s of Drosophila glycoprotein glycobiology. Glycoconj J 30, 57-66, (2013).
  2. Mondragon-Shem, K., Wongtrakul-Kish K., et al. Insights into the salivary N-glycome of Lutzomyia longipalpis, vector of visceral leishmaniasis. bioRxiv (2020).
  3. Abdeladhim, M., et al. What’s behind a sand fly bite? The profound effect of sand fly saliva on host hemostasis, inflammation and immunity. Infect Genet Evol 28, 691-703, (2014).
  4. Valenzuela, J. G., et al. Identification of the most abundant secreted proteins from the salivary glands of the sand fly Lutzomyia longipalpis, vector of Leishmania chagasi. J Exp Biol 207, 3717-3729, (2004).




Posted on: 24th June 2020


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