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Glycosylation and antigenic properties of Phlebotomus perniciosus and P. orientalis salivary proteins
Sumová, Petra ; Volf, Petr (advisor) ; Grubhoffer, Libor (referee)
The goal of this study was to map the glycosylation pattern and antigenic properties of the salivary proteins of two closely related sand fly species, Phlebotomus perniciosus and P. orientalis. Affinity blotting with commercially available lectins revealed that many salivary proteins of these species are N-glycosylated, while the presence of O-glycosylation could not be confirmed. The level of N-glycosylation of most of these proteins is quite low, a larger number of potential N-glycosylation sites were found only in the amino acid sequences of P. orientalis hyaluronidase and endonucleases of both species tested. Four antigens from P. perniciosus salivary glands were selected for expression in a bacterial expression system; two of these proteins (PpeSP01 and PpeSP01B) were not glycosylated and the glycosylation level of the remaining two (PpeSP03B and PpeSP07) was low. The antigenic properties of the four chosen recombinant proteins were subsequently tested using immunoblot and ELISA. During the initial experiments with the sera of dogs experimentally bitten by P. perniciosus, two proteins (rSP07 and rSP01B) were proven unsuitable and they were excluded from further experiments. Recombinant proteins rSP03B and rSP01 were recognized by the same IgG antibodies as the native forms of these proteins...
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Characterization and antigenic properties of salivary yellow-related proteins in phlebotomine sand flies
Cikrtová, Petra
2 ABSTRACT Yellow-related proteins (YRPs) form an abundant protein family, whose members were found in salivary gland transcriptomes of all sand fly species studied up to date. This protein family belongs to the group of MRJP/Yellow proteins occurring in insects and some other organisms. Though sharing similar folding as a six-bladed β-propeller comprising central tunnel, MRJP/Yellow proteins adopted different functions. The structure and biogenic amine-binding property described for the sand fly salivary YRPs was based on a single study conducted on Lutzomyia longipalpis. In the present work, we have modelled the structures of 32 salivary YRPs belonging to 13 different sand fly species. We have shown the general structural similarity of these proteins along with both inter- and intra-specific differences in surface charge, tunnel parameters and in amino acids composition of the amine-binding motif. These modifications indicated divergence in function of individual YRPs, which was experimentally verified in the second project focused on identification of the amine-binding properties of YRPs in two important vectors of Leishmania; Phlebotomus perniciosus and P. orientalis. In each species, two YRPs differ in affinities for biogenic amines serotonin, histamine and catecholamines. However, in both sand fly...
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Characterization and antigenic properties of salivary yellow-related proteins in phlebotomine sand flies
Cikrtová, Petra
2 ABSTRACT Yellow-related proteins (YRPs) form an abundant protein family, whose members were found in salivary gland transcriptomes of all sand fly species studied up to date. This protein family belongs to the group of MRJP/Yellow proteins occurring in insects and some other organisms. Though sharing similar folding as a six-bladed β-propeller comprising central tunnel, MRJP/Yellow proteins adopted different functions. The structure and biogenic amine-binding property described for the sand fly salivary YRPs was based on a single study conducted on Lutzomyia longipalpis. In the present work, we have modelled the structures of 32 salivary YRPs belonging to 13 different sand fly species. We have shown the general structural similarity of these proteins along with both inter- and intra-specific differences in surface charge, tunnel parameters and in amino acids composition of the amine-binding motif. These modifications indicated divergence in function of individual YRPs, which was experimentally verified in the second project focused on identification of the amine-binding properties of YRPs in two important vectors of Leishmania; Phlebotomus perniciosus and P. orientalis. In each species, two YRPs differ in affinities for biogenic amines serotonin, histamine and catecholamines. However, in both sand fly...
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Glycosylation and antigenic properties of Phlebotomus perniciosus and P. orientalis salivary proteins
Sumová, Petra ; Volf, Petr (advisor) ; Grubhoffer, Libor (referee)
The goal of this study was to map the glycosylation pattern and antigenic properties of the salivary proteins of two closely related sand fly species, Phlebotomus perniciosus and P. orientalis. Affinity blotting with commercially available lectins revealed that many salivary proteins of these species are N-glycosylated, while the presence of O-glycosylation could not be confirmed. The level of N-glycosylation of most of these proteins is quite low, a larger number of potential N-glycosylation sites were found only in the amino acid sequences of P. orientalis hyaluronidase and endonucleases of both species tested. Four antigens from P. perniciosus salivary glands were selected for expression in a bacterial expression system; two of these proteins (PpeSP01 and PpeSP01B) were not glycosylated and the glycosylation level of the remaining two (PpeSP03B and PpeSP07) was low. The antigenic properties of the four chosen recombinant proteins were subsequently tested using immunoblot and ELISA. During the initial experiments with the sera of dogs experimentally bitten by P. perniciosus, two proteins (rSP07 and rSP01B) were proven unsuitable and they were excluded from further experiments. Recombinant proteins rSP03B and rSP01 were recognized by the same IgG antibodies as the native forms of these proteins...
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