Identification of a Tsetse Fly Salivary Protein with Dual Inhibitory Action on Human Platelet Aggregation

Background: Tsetse flies (Glossina sp.), the African trypanosome vectors, rely on anti-hemostatic compounds for efficient blood feeding. Despite their medical importance, very few salivary proteins have been characterized and functionally annotated. Methodology/Principal Findings: Here we report on...

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Bibliographic Details
Published in:PloS one 2010-03, Vol.5 (3), p.e9671-e9671
Main Authors: Caljon, Guy, Ridder, Karin De, Baetselier, Patrick De, Coosemans, Marc, Abbeele, Jan Van Den
Format: Article
Language:English
Subjects:
Adenosine
Adenosine diphosphate
Adenosine triphosphatase
adenosinetriphosphatase
Agglomeration
Amino Acid Sequence
Amino acids
Animals
Apyrase
Arthropoda
Arthropods
ATPases
Biochemistry
Blood
Blood platelets
Blood Platelets - cytology
Blood Platelets - metabolism
complementary DNA
Dendritic cells
Disaggregation
E coli
Enzymes
Feeding
Fibrin
Fibrinogen
Fusion protein
Gene expression
Gene Library
Genes
Genetics and Genomics/Functional Genomics
Genomics
Glossina morsitans
Glossina morsitans morsitans
Glycosylation
Hematophagy
Hemostasis
Humans
Immunology
Infectious Diseases/Neglected Tropical Diseases
Inflammation
Insect Proteins - genetics
Insect Proteins - physiology
Insects
Kinetics
Medical importance
Medicine
Molecular Sequence Data
Neutrophils
Nuc protein
Nucleotidase
Nucleotidases
Physiology
Platelet Aggregation
Platelets
Proteins
Recombinant proteins
Ribonucleic acid
RNA
RNA Interference
RNA-mediated interference
Saliva
Saliva - metabolism
Salivary gland
salivary glands
Salivary Proteins and Peptides - genetics
Salivary Proteins and Peptides - metabolism
Salivary Proteins and Peptides - physiology
Sequence Homology, Amino Acid
Trypanosome
Tsetse Flies
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Summary:Background: Tsetse flies (Glossina sp.), the African trypanosome vectors, rely on anti-hemostatic compounds for efficient blood feeding. Despite their medical importance, very few salivary proteins have been characterized and functionally annotated. Methodology/Principal Findings: Here we report on the functional characterisation of a 5′nucleotidase-related (5′Nuc) saliva protein of the tsetse fly Glossina morsitans morsitans. This protein is encoded by a 1668 bp cDNA corresponding at the genomic level with a single-copy 4 kb gene that is exclusively transcribed in the tsetse salivary gland tissue. The encoded 5′Nuc protein is a soluble 65 kDa glycosylated compound of tsetse saliva with a dual anti-hemostatic action that relies on its combined apyrase activity and fibrinogen receptor (GPIIb/IIIa) antagonistic properties. Experimental evidence is based on the biochemical and functional characterization of recombinant protein and on the successful silencing of the 5′nuc translation in the salivary gland by RNA interference (RNAi). Refolding of a 5′Nuc/SUMO-fusion protein yielded an active apyrase enzyme with K(m) and V(max) values of 43±4 µM and 684±49 nmol Pi/min×mg for ATPase and 49±11 µM and 177±37 nmol Pi/min×mg for the ADPase activity. In addition, recombinant 5′Nuc was found to bind to GPIIb/IIIa with an apparent K(D) of 92±25 nM. Consistent with these features, 5′Nuc potently inhibited ADP-induced thrombocyte aggregation and even caused disaggregation of ADP-triggered human platelets. The importance of 5′Nuc for the tsetse fly hematophagy was further illustrated by specific RNAi that reduced the anti-thrombotic activities in saliva by approximately 50% resulting in a disturbed blood feeding process. Conclusions/Significance: These data show that this 5′nucleotidase-related apyrase exhibits GPIIb/IIIa antagonistic properties and represents a key thromboregulatory compound of tsetse fly saliva.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0009671