Characterization of surface proteins and glycoproteins on red blood cells from mice infected with haemosporidia:Plasmodium bergheiinfections of BALB/c mice

Parasitology ◽  
1980 ◽  
Vol 81 (2) ◽  
pp. 273-298 ◽  
Author(s):  
R. J. Howard ◽  
Patricia M. Smith ◽  
G. F. Mitchell
Keyword(s):  
Sialic Acid ◽  
Periodate Oxidation ◽  
Surface Protein ◽  
Surface Proteins ◽  
Red Cells ◽  
Galactose Oxidase ◽  
Infected Blood ◽  
Molecular Weights ◽  
Infected Cells

SUMMARYThe surface proteins and glycoproteins of red cells fromPlasmodium berghei-infected blood have been radio-isotope labelled and compared with those of normal mouse erythrocytes using the following protein labelling probes: lactoperoxidase-catalysed radio-iodination of tyrosyl residues, periodate oxidation and NaB3H4reduction of sialic acid and oxidation of galactosyl/N-acetylgalactosaminyl residues by galactose oxidase with subsequent NaB3H4reduction. DuringP.bergheiinfection, new tyrosyl-labelled proteins with apparent molecular weights (Mr) of 60000, 54000, 40000 and 27500 appeared on the surface of most, if not all, red cells in the blood. Purified multinucleate cells (mostly reticulocytes) differed only in that they also had a surface protein withMrof 83000. However, this molecule is thought to be specific to mouse reticulocytes rather than derived from parasites. In contrast to the relatively minor changes detected with radio-iodination, striking changes inglycoproteinradio-isotope labelling resulted from infection. All of the red cells in infected blood of greater than 20% parasitaemia lost their periodate-sensitive glycoprotein sialic acid. With some samples there was little change in glycoprotein labelling by the galactose oxidase method, provided neuraminidase was also added. Modification of the exocyclic hydroxyls of sialic acid is postulated to account for this. Other blood samples exhibited a dramatic loss of galactose oxidase-dependent labelling. It is suggested that these observations may relate to the excessive red cell destruction of uninfected as well as infected cells which has been inferred in many haemosporidial infections, including malaria.

Characterization of surface proteins and glycoproteins on red blood cells from mice infected with haemosporidia:Plasmodium yoeliiinfections of BALB/c mice

Parasitology ◽  
1980 ◽  
Vol 81 (2) ◽  
pp. 299-314 ◽  
Author(s):  
R. J. Howard ◽  
Patricia M. Smith ◽  
G. F. Mitchell
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Plasmodium Yoelii ◽  
Surface Proteins ◽  
Red Cells ◽  
Galactose Oxidase ◽  
Membrane Glycoproteins ◽  
Red Cell Membrane ◽  
Neuraminidase Treatment ◽  
Infected Blood ◽  
Inoculation Dose

SUMMARYLactoperoxidase-catalysed radio-iodination was used to compare the surface proteins on red cells fromPlasmodium yoelii-infected and normal BALB/c mice. The profile of radio-iodinated proteins separated by SDS-polyacrylamide gel electrophoresis was different for infected blood of similar parasitaemia from mice inoculated with different doses of the parasite. Inoculation with the lower dose resulted in the appearance of a major radio-iodinated protein of apparent molecular weight (Mr) 76000 which was labelled to a similar extent on uninfected red cells from infected blood and purified multinucleate infected cells. Several minor radio-iodinated bands, with identical mobilities to the minor bands on normal BALB/c erythrocytes, were also present on red cells from this infected blood. In contrast, the higher inoculation dose produced changes in the minor labelled bands, and the band withMrof 76000 was absent. In this case, the minor radio-iodinated proteins of the normal BALB/c erythrocyte (with Mr of 65000, 57000, 48000, 38000 and 32000) were replaced by a series of bands withMrof 60000, 50000, 43000 and 28000 on both uninfected and infected red cells. These differences with inoculation dose may be related to the different duration of these infections, the development of anaemia and the extent of pathological changes at the erythrocyte surface.P. yoeliiinfection caused a marked loss in periodate-dependent labelling of sialoglycoproteins on most, if not all, red cells in infected blood. There was also a large decrease in galactose oxidase-dependent glycoprotein labelling with or without neuraminidase treatment. These changes in the carbohydrate groups on red cell membrane glycoproteins may be linked to the excessive loss of both uninfected and infected red cells during some malaria infections.

Characterization of surface proteins and glycoproteins on red blood cells from mice infected with haemosporidia: Babesia rodhaini infections of BALB/c mice

Parasitology ◽  
1980 ◽  
Vol 81 (2) ◽  
pp. 251-271 ◽  
Author(s):  
R. J. Howard ◽  
Patricia M. Smith ◽  
G. F. Mitchell
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Surface Proteins ◽  
Red Cells ◽  
Galactose Oxidase ◽  
Intact Cells ◽  
Infected Blood ◽  
High Parasitaemia ◽  
Surface Glycoproteins

SUMMARYInfection of intact (nu/+) or hypothymic (nu/nu) BALB/c mice with the lethal intra-erythrocytic parasite, Babesia rodhaini, induced several changes in the surface proteins of red cells from infected blood. Lactoperoxidase-catalysed radio-iodination was used to compare the surface proteins on normal mouse erythrocytes and the total red cells from infected blood at different levels of parasitaemia. At very low parasitaemia, when only 2·5% of the red cells contained parasites, we observed significant changes in the profile of radio-iodinated proteins separated by SDS-polyacrylamide gel electrophoresis. These changes included the appearance of a group of high molecular weight proteins, and a protein with an apparent molecular weight (Mr) of 60 000, both of which were absent from normal erythrocytes. The major labelled band on the erythrocyte surface (Mr 92 000) also appeared to be less heavily labelled during infection. The magnitude of these differences in surface proteins increased as the parasitaemia rose, until the new bands dominated the radioactivity profile with blood of greater than 50% parasitaemia. Several control experiments established that the radio-iodinated proteins were surface molecules on intact cells and that artifactual proteolysis did not contribute to the observed differences. The results suggest that changes in the surface proteins occur on all red cells in the blood of infected mice. The results of labelling the surface glycoproteins by oxidation with periodate or galactose oxidase, followed by NaB3H4 reduction, have varied with the isolate of B. rodhaini. With the isolate currently in use, no significant differences were observed in the labelled surface glycoproteins of normal erythrocytes and red cells from infected blood of high parasitaemia, whereas an earlier isolate exhibited a marked decrease of glycoprotein labelling of both infected and uninfected red cells.

Electrophoretic and Chemical Characterization of the Charged Groups at the Surface of Murine CL3 Ascites Leukaemia Cells

1969 ◽  
Vol 4 (2) ◽  
pp. 289-298
Author(s):  
P. D. WARD ◽  
E. J. AMBROSE
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Chemical Characterization ◽  
Surface Protein ◽  
Subsequent Treatment ◽  
Surface Proteins ◽  
Carboxyl Groups ◽  
Neuraminidase Treatment ◽  
Ionic Nature ◽  
Ph Range

The electrophoretic characteristics of the murine CL3 ascites tumour were investigated. Treatment of the cells with formaldehyde raised the electrophoretic mobility (E.P.M.) from - 1.06 to - 1.28 µ/sec/V/cm; subsequent treatment with diazomethane reduced their mobility to zero. The E.P.M. of the diazomethane-treated cells did not alter over the pH range 3.0-8.0. This proved that the only ionic groups at this cell surface were amino and carboxyl groups. The absence of phosphate groups, another possibility, was confirmed by the lack of calcium-ion binding from 10 mM Ca2+ solutions. Neuraminidase treatment reduced the E.P.M. from -1.06 to -0.55 µ/sec/V/cm and free sialic acid was identified in the enzyme supernatant. Subsequent treatment of the cells with formaldehyde raised the mobility to -1.22 µ/sec/V/cm indicating that the change in E.P.M. on neuraminidase treatment was not due solely to the removal of the carboxyl groups of sialic acid but also to a change in the ionic nature of the surface. This change is ascribed to a change in the conformation of the surface protein. The reason for this change and a suggestion for the possible role of sialic acid at the cell surface are mentioned. Treatment of the cells with trypsin did not affect the viable cells in any way, suggesting that the surface proteins lack the basic amino acids lysine and arginine. Pronase treatment served only to show that much of the sialic acid was bound to protein; the total amount was not determined.

scholarly journals The P25 Ookinete Surface Proteins: Homology Modeling and Phylogenetic Relationships

2013 ◽  
Vol 2013 ◽  
pp. 1-17
Author(s):  
Babita Sharma ◽  
Manoj Kumar Jaiswal
Keyword(s):  
Phylogenetic Relationships ◽  
Surface Protein ◽  
Surface Proteins ◽  
Parasite Development ◽  
Transmission Blocking ◽  
Malaria Burden ◽  
Mosquito Midgut ◽  
Egf Domain ◽  
Domain Iii

Sexual stages of Plasmodium such as zygote, ookinete, and young oocysts express 25 kDa surface protein P25, which along with P28 proteins protect the parasite from harmful environment inside mosquito midgut. Vaccines against these proteins induce antibodies in vertebrate host capable to inhibit parasite development in mosquito midgut and thus preventing the transmission of parasite from mosquito to other human host. Transmission-blocking vaccines help reduce malaria burden. The purpose of this study was in silico structural characterization of P25 family proteins and to predict their phylogenetic relationships with other proteins. Results indicate that members of P25 family have four EGF domains arranged in triangular fashion with major variations lying in the loop regions. All 22 cysteines are conserved forming 11 disulphide bonds. The C-loop of EGF domain IV in P25 proteins is smaller in comparison to P28 proteins. B loop of EGF domain II showed maximum RMSD variations followed by loops of EGF domain III. P25 proteins are tile-like triangular flat proteins that protect the parasite inside mosquito midgut. Obtained structures will help in understanding the biology of the parasite inside the mosquito midgut. These structures may also help in designing transmission-blocking vaccine against malaria in absence of experimentally determined structures.

scholarly journals Development of a Multivalent ISCOM Vaccine against Anaplasmosis

1993 ◽  
Author(s):  
Guy H. Palmer ◽  
Eugene Pipano ◽  
Terry F. McElwain ◽  
Varda Shkap ◽  
Donald P. Knowles, Jr.
Keyword(s):  
T Lymphocytes ◽  
Outer Membrane ◽  
Membrane Surface ◽  
Surface Protein ◽  
The United States ◽  
Surface Proteins ◽  
Efficient Production ◽  
Major Surface ◽  
Homologous Challenge

Anaplasmosis is an arthropod+borne disease of cattle caused by the rickettsia Anaplasma marginale and an impediment to efficient production of healthy livestock in both Israel and the United States. Our research focuses on development of a recombinant membrane surface protein (MSP) immunogen to replace current vaccines derived from the blood of infected cattle. The risk of widespread transmission of both known and newly emergent pathogens has prevented licensure of live blood-based vaccines in the U.S. and is a major concern for their continued use in Israel. Briefly, we accomplished the following in our BARD supported research: i) characterization of the intramolecular and intermolecular relationships of the native Major Surface Proteins (MSP) in the outer membrane; ii) expression, purification, and epitope characterization of the recombinant MSP-2, MSP-3, MSP-4, and MSP-5 proteins required to construct the recombinant ISCOM; iii) demonstration that the outer membrane-Quil A induces CD4+ T lymphocytes specific for the outer membrane polypeptides; iv) identification of CD4+ T lymphocytes that recognize outer membrane polypeptide epitopes conserved among other wise antigenically distinct strains; v) determination that immunization with the outer membrane-Quil A construct does not affect the ability of ticks to acquire or transmit A. marginale; and vi) demonstration that the outer membrane-Quil A construct induces complete protection against rickettsemia upon homologous challenge and significant protection against challenge with antigenically distinct strains, including tick transmission. Importantly, the level of protection against homologous challenge in the MSP vaccinates was comparable to that induced by live blood-based vaccines and demonstrates that development of a new generation of vaccines is feasible.

scholarly journals The structure of a glycopeptide purified from porcine thyroglobulin

1971 ◽  
Vol 123 (3) ◽  
pp. 415-420 ◽  
Author(s):  
Minoru Fukuda ◽  
Fujio Egami
Keyword(s):  
Sialic Acid ◽  
Sodium Borohydride ◽  
Periodate Oxidation ◽  
Galactose Oxidase ◽  
Borohydride Reduction ◽  
Enzymic Hydrolysis ◽  
End Groups ◽  
Sodium Borohydride Reduction

1. The structure of a purified glycopeptide isolated from porcine thyroglobulin was studied by sequential hydrolysis with specific glycosidases, by periodate oxidation and by treatment with galactose oxidase. 2. Sequential hydrolysis with several combinations of neuraminidase, α-l-fucosidase, β-d-galactosidase, β-N-acetyl-d-glucosaminidase and α-d-mannosidase presented the evidence for the following structure. 3. The monosaccharide sequence of the peripheral moiety of the heteropolysaccharide chain was sialic acid→galactose→N-acetylglucosamine. Some of the galactose residues were non-reducing end-groups with the sequence galactose→N-acetylglucosamine. 4. After removal of the peripheral moiety composed of sialic acid, fucose, galactose and N-acetylglucosamine, α-mannosidase released 1.4mol of mannose/mol of glycopeptide, indicating that two of the three mannose residues were located between peripheral N-acetylglucosamine and internal N-acetylglucosamine or mannose. 5. Periodate oxidation and sodium borohydride reduction confirmed the results obtained by enzymic degradation and gave information concerning the position of substitution. 6. Based on the results obtained by enzymic hydrolysis and periodate oxidation together with the treatment with galactose oxidase, a structure is proposed for the glycopeptide.

scholarly journals The glycoprotein of measles virus. External radioactive labelling of its carbohydrate and partial characterization of the glycopeptide

1980 ◽  
Vol 185 (1) ◽  
pp. 189-194 ◽  
Author(s):  
O Anttonen ◽  
M Jokinen ◽  
A Salmi ◽  
R Vainionpää ◽  
C G Gahmberg
Keyword(s):  
Sialic Acid ◽  
Measles Virus ◽  
Vero Cells ◽  
Galactose Oxidase ◽  
Culture Supernatants ◽  
Surface Carbohydrates ◽  
Specific Labelling

Measles virus was propagated in VERO cells and purified from the culture supernatants by two successive tartrate-density-gradient centrifugations. Surface carbohydrates were labelled both in vitro and in vivo with 3H after treatment with galactose oxidase/NaB3H4 or with [3H]glucosamine. The major labelled glycoprotein in measles virions had a mol.wt. of 79 000. After labelling with periodate/NaB3H4, which would result in specific labelling of sialic acid residues, the 79 000-mol.wt. glycoprotein was very weakly labelled. This suggests that there is no or a very low amount of sialic acid in the virions. Further analysis of the glycoprotein showed that galactose is the terminal carbohydrate unit in the oligosaccharide, and the molecular weight of the glycopeptide obtained after Pronase digestion is about 3000. The oligosaccharide is attached to the polypeptide through an alkali-stable bond, indicating a N-glycosidic asparagine linkage.

scholarly journals The Streptococcus gordonii Surface Proteins GspB and Hsa Mediate Binding to Sialylated Carbohydrate Epitopes on the Platelet Membrane Glycoprotein Ibα

2004 ◽  
Vol 72 (11) ◽  
pp. 6528-6537 ◽  
Author(s):  
Barbara A. Bensing ◽  
José A. López ◽  
Paul M. Sullam
Keyword(s):  
Sialic Acid ◽  
Surface Protein ◽  
Large Cell ◽  
Platelet Membrane ◽  
Surface Proteins ◽  
Membrane Glycoprotein ◽  
Streptococcus Gordonii ◽  
Platelet Membrane Glycoprotein ◽  
Carbohydrate Epitopes ◽  
Sialylated Glycoproteins

ABSTRACT Platelet binding by Streptococcus gordonii strain M99 is dependent on expression of the cell wall-anchored glycoprotein GspB. This large cell surface protein is exported from the M99 cytoplasm via a dedicated transport system that includes SecA2 and SecY2. GspB is highly similar to Hsa, a protein expressed by S. gordonii Challis that has been characterized as a sialic acid binding hemagglutinin. In this study, we compared the contribution of GspB and Hsa to the adherence of S. gordonii to selected glycoproteins. Our results indicate that GspB can mediate binding to a variety of sialylated glycoproteins. GspB facilitates binding to carbohydrates bearing sialic acid in either α(2-3) or α(2-6) linkages, with a slight preference for α(2-3) linkages. Furthermore, GspB readily mediates binding to sialic acid residues on immobilized glycocalicin, the extracellular portion of the platelet membrane glycoprotein (GP) Ibα (the ligand binding subunit of the platelet von Willebrand factor receptor complex GPIb-IX-V). Although Hsa is required for the binding of S. gordonii Challis to sialic acid, most of the Hsa expressed by Challis is retained in the cytoplasm. The deficiency in export is due, at least in part, to a nonsense mutation in secA2. Hsa export can be enhanced by complementation with secA2 from M99, which also results in significantly greater binding to sialylated glycoproteins, including glycocalicin. The combined results indicate that GspB and Hsa contribute similar binding capabilities to M99 and Challis, respectively, but there may be subtle differences in the preferred epitopes to which these adhesins bind.

scholarly journals Changes in neutrophil surface protein composition accompany phagocytosis

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 60-68 ◽  
Author(s):  
KM Skubitz ◽  
TK Kinkead
Keyword(s):  
Cell Surface ◽  
Defense Mechanism ◽  
Protein Composition ◽  
Surface Protein ◽  
Surface Proteins ◽  
Galactose Oxidase ◽  
Human Neutrophils ◽  
Host Defense Mechanism ◽  
Target Particle ◽  
Neutrophil Surface

Phagocytosis is a critical host defense mechanism of neutrophils. In this study, membrane protein changes occurring during phagocytosis were studied in human neutrophils using surface radiolabeling before or after phagocytosis of various target particles. Cells were labeled at the cell surface using lactoperoxidase-catalyzed iodination or neuraminidase-galactose oxidase-NaB3H4, galactose oxidase-NaB3H4, or periodate-NaB3H4 techniques. Such studies are complicated by the fact that these techniques identify many surface proteins on the phagocyte, and labeling after phagocytosis occurs often results in radiolabeling proteins of the target particle, thus making changes in cell-surface proteins more difficult to detect. Immunoprecipitation with monoclonal antibody AHN-1, which reacts with a carbohydrate present on several human neutrophil surface proteins and inhibits phagocytosis, eliminated interference caused by radiolabeled proteins of the target particle and simplified analysis by restricting the study to a limited number of proteins. AHN-1 immunoprecipitated less radiolabeled protein from neutrophils labeled after phagocytosis of particles opsonized with IgG or complement than from cells labeled before phagocytosis. Isolation of phagocytic vesicles containing opsonized emulsified paraffin oil demonstrated that three proteins of mol wt 105,000, 140,000, and 170,000 recognized by AHN-1 were internalized in the phagocytic vesicle during phagocytosis.

scholarly journals Changes in neutrophil surface protein composition accompany phagocytosis

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 60-68
Author(s):  
KM Skubitz ◽  
TK Kinkead
Keyword(s):  
Cell Surface ◽  
Defense Mechanism ◽  
Protein Composition ◽  
Surface Protein ◽  
Surface Proteins ◽  
Galactose Oxidase ◽  
Human Neutrophils ◽  
Host Defense Mechanism ◽  
Target Particle ◽  
Neutrophil Surface

Abstract Phagocytosis is a critical host defense mechanism of neutrophils. In this study, membrane protein changes occurring during phagocytosis were studied in human neutrophils using surface radiolabeling before or after phagocytosis of various target particles. Cells were labeled at the cell surface using lactoperoxidase-catalyzed iodination or neuraminidase-galactose oxidase-NaB3H4, galactose oxidase-NaB3H4, or periodate-NaB3H4 techniques. Such studies are complicated by the fact that these techniques identify many surface proteins on the phagocyte, and labeling after phagocytosis occurs often results in radiolabeling proteins of the target particle, thus making changes in cell-surface proteins more difficult to detect. Immunoprecipitation with monoclonal antibody AHN-1, which reacts with a carbohydrate present on several human neutrophil surface proteins and inhibits phagocytosis, eliminated interference caused by radiolabeled proteins of the target particle and simplified analysis by restricting the study to a limited number of proteins. AHN-1 immunoprecipitated less radiolabeled protein from neutrophils labeled after phagocytosis of particles opsonized with IgG or complement than from cells labeled before phagocytosis. Isolation of phagocytic vesicles containing opsonized emulsified paraffin oil demonstrated that three proteins of mol wt 105,000, 140,000, and 170,000 recognized by AHN-1 were internalized in the phagocytic vesicle during phagocytosis.

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