Glycosylation and antigenic properties of Phlebotomus perniciosus and P. orientalis salivary proteins

Cílem této práce bylo zmapovat glykosylaci a antigenní vlastnosti slinných proteinů dvou druhů flebotomů, Phlebotomus perniciosus a P. orientalis, kteří jsou významnými vektory viscerální leishmaniózy. Pomocí afinoblotu se značenými lektiny jsme prokázali, že řada slinných proteinů obou studovaných druhů flebotomů je N-glykosylována, přítomnost O-glykosylací nebylo možné potvrdit. Úroveň N-glykosylace většiny těchto proteinů je poměrně nízká, větší množství potenciálních N-glykosylačních míst bylo nalezeno pouze v aminokyselinových sekvencích hyaluronidázy P. orientalis a endonukleáz obou testovaných druhů. Pro expresi v bakteriálním expresním systému byly vybrány čtyři slinné antigenní proteiny P. perniciosus, z nichž dva proteiny (PpeSP01 a PpeSP01B) nebyly glykosylovány a úroveň glykosylace zbylých dvou proteinů (PpeSP03B a PpeSP07) byla nízká. Antigenní vlastnosti čtyř vybraných rekombinantních proteinů P. perniciosus byly následně testovány prostřednictvím imunoblotu a ELISA testů. Během počátečních pokusů se séry experimentálně exponovaných psů se dva proteiny (rSP07 a rSP01B) ukázaly jako nevhodné a byly tudíž z dalších experimentů vyřazeny. Rekombinantní proteiny rSP03B a rSP01 byly rozeznávány stejnými IgG protilátkami jako nativní formy těchto proteinů ze slinných žláz P. perniciosus....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...Department of ParasitologyKatedra parazitologiePřírodovědecká fakultaFaculty of Scienc

Salivary glycoproteins of bloodsucking arthropods

During obtaining their blood meal, bloodsucking arthropods salivate into their host. Bloodsucking arthropods' saliva contains wide array of bioactive macromolecules. Host organism develops antibody response against many of these molecules. Due to interspecies variability in salivary protein composition, detection of antibody response may serve as a marker of the exposure to individual species of bloodsucking arthropods. Host antibody response is mostly elicited by proteins or glycoproteins. Glycoproteins contain one or more oligosaccharide chains attached to the protein. Glycoprotein's antigenicity could be caused by either both parts, or by only the protein, or the sugar part. This fact has to be taken into consideration for choice of the expression system for recombinant glycoprotein synthesis. This work summarizes current knowledge about structure, function and features of salivary glycoproteins in various species of bloodsucking arthropods

Salivary glycoproteins of bloodsucking arthropods

Krevsající členovci injikují během sání krve obsah svých slinných žláz do hostitele. Sliny krevsajících členovců obsahují celé spektrum bioaktivních makromolekul, jejichž hlavní funkcí je umožnění sání krve. Proti řadě těchto molekul je ovšem namířena protilátková odpověď hostitelského imunitního systému. Detekce této protilátkové odpovědi může, díky mezidruhovým odlišnostem v proteinovém složení slin, sloužit jako měřítko expozice hostitele jednotlivým druhům krevsajících členovců. Molekulami vyvolávajícími protilátkovou odpověď mohou být proteiny nebo glykoproteiny. Glykoproteiny jsou proteiny, na něž jsou glykosidicky vázány oligosacharidové řetězce. Za antigenní vlastnosti slinných glykoproteinů mohou být zodpovědné obě jejich složky, či pouze cukerná nebo proteinová část. Tuto skutečnost je třeba zohlednit při výběru expresního systému pro syntézu rekombinantních glykoproteinů. Tato práce shrnuje poznatky o struktuře, funkci a vlastnostech slinných glykoproteinů různých druhů krevsajících členovců.During obtaining their blood meal, bloodsucking arthropods salivate into their host. Bloodsucking arthropods' saliva contains wide array of bioactive macromolecules. Host organism develops antibody response against many of these molecules. Due to interspecies variability in salivary protein composition, detection of antibody response may serve as a marker of the exposure to individual species of bloodsucking arthropods. Host antibody response is mostly elicited by proteins or glycoproteins. Glycoproteins contain one or more oligosaccharide chains attached to the protein. Glycoprotein's antigenicity could be caused by either both parts, or by only the protein, or the sugar part. This fact has to be taken into consideration for choice of the expression system for recombinant glycoprotein synthesis. This work summarizes current knowledge about structure, function and features of salivary glycoproteins in various species of bloodsucking arthropods.Katedra parazitologieDepartment of ParasitologyPřírodovědecká fakultaFaculty of Scienc

Salivary glycoproteins of bloodsucking arthropods

During obtaining their blood meal, bloodsucking arthropods salivate into their host. Bloodsucking arthropods' saliva contains wide array of bioactive macromolecules. Host organism develops antibody response against many of these molecules. Due to interspecies variability in salivary protein composition, detection of antibody response may serve as a marker of the exposure to individual species of bloodsucking arthropods. Host antibody response is mostly elicited by proteins or glycoproteins. Glycoproteins contain one or more oligosaccharide chains attached to the protein. Glycoprotein's antigenicity could be caused by either both parts, or by only the protein, or the sugar part. This fact has to be taken into consideration for choice of the expression system for recombinant glycoprotein synthesis. This work summarizes current knowledge about structure, function and features of salivary glycoproteins in various species of bloodsucking arthropods

Glycosylation and antigenic properties of Phlebotomus perniciosus and P. orientalis salivary proteins

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..