Pronase and proteinase K digestion of human immunoglobulin M

Human 19S IgM was digested with pronase and proteinase K. Proteolysis was relatively fast, producing Fab2 mu-like fragments (approx. mol. wt 114,000) and Fab mu-like fragments (approx. mol. wt 46,500) as major products. Immunochemical analysis indicated that the fragments produced by either enzyme are very similar and that they are produced by cleavage at the C mu 2-C mu 3 and C mu 1-C mu 2 domain junctions respectively. An intermediate species of mol. wt 74,300, immunologically identical to F(ab)2 mu, was also identified. This is thought to represent an F(ab)2 mu fragment with one Fab mu fragment removed. Fc mu-related fragments were not identified in the digestion mixture with either enzyme. Covalently linked and non-covalently linked 7S human IgM (IgMs and IgMr respectively) were digested with pronase and proteinase K. IgMs was degraded very rapidly by either enzyme, producing relatively stable F(ab)2 mu- and Fab mu-like fragments. These fragments were similar in mol. wt and immunochemical properties to those produced from 19S IgM. IgMr was also degraded rapidly by either enzyme, in this case producing Fab mu-like fragments with no detectable F(ab)2 mu-like fragments. The kinetics of digestion and nature of the products suggest that cleavage of 19S IgM by pronase or proteinase K proceeds via an initial attack at the C mu 2-C mu 3 junction, followed by further degradation at the Cmu 1-C mu 2 junction. The results obtained using 7S IgM show that the intersubunit disulphide bonds, and the associated pentameric structure, are responsible for the relative resistance of 19S IgM to proteolysis. The inter-heavy-chain disulphide bonds, in particular the bond at cys 337, are responsible for the limited susceptibility of F(ab)2 mu-like fragments to proteolysis.     

Two Plasmodium falciparum merozoite surface polypeptides share epitopes with a single Mr 185 000 parasite glycoprotein

The malarial parasite Plasmodium falciparum synthesizes a major glycoprotein (gp) of Mr 185 000 during its asexual blood cycle. Immunoprecipitation of [35S]methionine- or [3H]glucosamine-labeled schizont antigens indicated that two groups of polypeptides were distinguished with anti-gp 185 mouse monoclonal antibodies: group A was composed of glycosylated molecules of Mr 185 000, 120 000, 90 000, 88 000, 46 000, and 40 000 while group B contained, in addition to gp 185, polypeptides of Mr 152 000, 106 000 and 83 000. The latter polypeptides lacked detectable amounts of radiolabeled saccharide. The smaller Mr polypeptides were specifically immunoprecipitated and not merely coprecipitated with gp 185. Our results suggest that gp 185 contains at least two structurally distinct domains which may be processed independently into either the group A or group B polypeptides. Although gp 185 may not be a merozoite surface protein, representative group A and group B-specific monoclonal antibodies bound to surface antigens of the merozoite as demonstrated by immunolabeling followed by electron microscopy. Therefore, at least one group A antigen and one group B antigen appeared to be on the extracellular surface of the merozoite. The proteins found in immunoprecipitates after both (1) sonication in aqueous medium and ultracentrifugation and (2) solubilization and phase separation of parasite molecules with Triton X-114 suggested that the group A and group B polypeptides and glycoproteins are either soluble or peripheral membrane proteins. Some of these, therefore, may be components of the surface coat of the merozoite.     

A 50 kilodalton exoantigen specific to the merozoite release-reinvasion stage of Plasmodium falciparum

The immunoglobulins G of a human plasma inhibiting in vitro Plasmodium falciparum merozoite reinvasion have been purified and used to immunoprecipitate the antigens released into the culture medium by an [35S]methionine-labeled synchronous culture. Several of the major exoantigens identified were found throughout the entire life cycle; they were also immunoprecipitated from the labeled parasitized cells. Some antigens were found only after the reinvasion stage, and especially a major one of molecular mass 50 kDa and pI 5.5. The latter was not found in the parasitized cells but derived most likely from the processing of a major 126 kDa antigen which disappeared from the parasites during the reinvasion period and which was immunoprecipitated by an anti-50 kDa monoclonal antibody.     

Changes in cell surface proteins and glycoproteins during the encystation of Entamoeba invadens

Changes in cell surface components of axenically grown trophozoites of Entamoeba invadens which occur during encystation were followed. Protein patterns of trophozoites, immature and mature cyst forms, were analyzed by sodium dodecyl sulphate gel electrophoresis. Total protein profiles of trophozoites and cyst forms stained by Coomassie blue gave similar patterns. In contrast, a number of different bands were observed in gels stained with the carbohydrate-specific Schiff's reagent as well as when nitrocellulose blottings were treated with 125I-radiolabelled wheat germ or soybean agglutinins. The most notable differences were bands at 250 and 95-105 kDa present in the cyst forms and absent in the trophozoites, and two bands at 70 and 75 kDa present in the latter and missing in the cysts. Labelling of trophozoites and cyst cell surfaces by iodination with lactoperoxidase revealed a number of protein bands which were exposed on the trophozoite surface and missing in the cysts. Moreover, gel electrophoresis patterns of non-reduced or reduced samples also differed considerably, indicating that a number of proteins are linked by disulphide bonds. This study shows that specific glycoproteins are produced during cyst formation.     

Characteristic proteins of micronemes and dense granules from Sarcocystis tenella zoites (Protozoa, Coccidia)

A subcellular fractionation procedure has been developed, which allowed the recovery of purified fractions of micronemes, dense granules and pellicles from Sarcocystis tenella zoites. As expected, sodium dodecyl sulphate polyacrylamide gels electrophoresis of the pellicles showed a fairly heterogenous protein content. In contrast, only one major omponent (42 000) daltons) was found in the dense granules, whereas two major bands (20 000 and 22 000 daltons) were observed for the micromemes. These characteristic proteins were also major components of the whole zoite and might have important functions in the physiology of the organism.     

Cell free synthesis of Toxoplasma gondii antigens

Functionally active poly(A)-containing mRNA was isolated from tachyzoites of the RH strain of Toxoplasma gondii. The T. gondii mRNA was capable of directing the synthesis of proteins in a wheat germ in vitro translation system, but not in a cell free system derived from rabbit reticulocyte lysate. Efficient translation in the wheat germ system required spermine and exogenous tRNA. Amino acid incorporation was maximal at 110 mM K+ and 1.8 mM Mg2+. Tachyzoite antigens synthesized in vitro were immunoprecipitated with T. gondii antibodies from rabbits, mice and humans. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated polypeptides yielded patterns that differed according to antibody source, but all T. gondii antibody preparations reacted with a translation product with an apparent molecular weight of 24 000.     

Isolation of a Plasmodium falciparum rhoptry protein

A monoclonal antibody raised against the malaria parasite Plasmodium falciparum recognised a protein of 140000 molecular weight which was synthesized during schizogony. The protein has been purified by monoclonal antibody affinity chromatography from extracts of parasitized red cells. Antibodies against the protein have been used to determine its subcellular location. The protein is not expressed on the merozoite surface and has been located in the rhoptries, the apical organelles of the merozoite.     

Evidence for major differences in ribosomal subunit proteins from Plasmodium berghei and rat liver

Purified polysomes were isolated in high yield from the erythrocytic stages of the rodent malaria parasite, Plasmodium berghei, and from rat liver. Proteins extracted from the ribosomal subunits derived from these polysomes were fractionated and their number and molecular weights were estimated by two-dimensional polyacrylamide gel electrophoresis. Plasmodial small ribosomal subunits contained 30 proteins ranging in apparent molecular size from 11.7 to 40.7 kDa, while large subunits contained 35-36 proteins ranging from 12.1 to 42.6 kDa. None of these parasite proteins was shared by the two subunits nor altered in electrophoretic mobility by radioiodination. Rat liver 40 S ribosomal subunit proteins numbered 30 and ranged from 9.2 to 37.5 kDa, while liver 60 S subunits contained 41-43 proteins with apparent molecular sizes of 10.3-45.2 kDa. Coelectrophoresis of trace amounts of radioiodinated P. berghei ribosomal subunit proteins and stainable quantities of liver proteins demonstrated that most of these 139 parasite and host ribosomal proteins possessed different two-dimensional electrophoretic mobilities under the conditions of this study. Based upon a comparative analysis of P. berghei and rodent ribosomal RNA and these data, it was concluded that parasite and host ribosomes contain distinct ribosomal RNAs and ribosomal proteins.     

A comparison of knobby (K+) and knobless (K-) parasites from two strains of Plasmodium falciparum

Erythrocytes infected with Plasmodium falciparum develop knob-like protrusions on their membranes. Knobby (K+) parasites of the FCR-3 (Gambian) strain have been shown to possess a histidine-labelled protein of apparent molecular weight 80 000 which is absent from knobless (K-) variants of the same strain. Here we report similar findings with K+ and K- parasites of another strain, the Malayan Camp strain, and also with cloned K+ and K- parasites of the FCR-3 strain. A histidine-labelled protein unique to the two K+ parasites was identified as a broad band with an apparent molecular weight of 89 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of this protein in both K+ Malayan Camp parasites and K+ FCR-3 (Gambian) parasites and its absence from K- parasites of both strains is consistent with this protein being a major component of knobs.     

Selective acquisition of plasma proteins by Trichomonas vaginalis and human lipoproteins as a growth requirement for this species

Trichomonas vaginalis avidly bound numerous host macromolecules which were not removed by repeated washing in phosphate buffered saline. The use of radioiodinated Cohn plasma fractions in binding studies allowed the identification of plasminogen, fibrinogen, immunoglobulin G, lipoproteins A and B, transferrin, α₁-antitrypsin, and albumin on intact organisms. The binding of immunoglobulin G, albumin, transferrin, and lipoproteins to intact, motile trichomonads was further demonstrated using ¹²⁵I-labeled plasma that was chromatographically depleted of these proteins. Kinetic studies indicated that ¹²⁵I-labeled lipoproteins bind to T. vaginalis in a receptor-ligand-like manner. The surface localization and uptake of bound lipoproteins was shown by treatment of intact organisms with pronase at various times after incubation with lipoproteins. Purified lipoproteins could be substituted for plasma or serum as a growth supplement in a complex medium of trypticase/yeast extract/maltose and supported growth and multiplication rates equal to those in the same medium with plasma.     

Isolation and characterization of surface antigens from Schistosoma mansoni schistosomula

Surface antigens of Schistosoma mansoni schistosomula were isolated using antibodies produced in rat and human schistosomiasis. Three immunoreactive surface proteins of 40 000, 37 000 and 32 000 daltons were identified by SDS—PAGE analysis of immune complexes formed by incubation of a detergent extract of surface labelled schistosomula with infected rat sera. Surface antigens of similar molecular weight were also isolated when using sera of patients with schistosomiasis. Binding of schistosomula surface antigens to specific antibodies was substantially inhibited by components released by adult worms. The results suggest that these schistosomula surface antigens could be involved in the immune response against challenge infection but their protective role in immunity still remains to be determined.     

Purification of Plasmodium lophurae cathepsin D and its effects on erythrocyte membrane proteins

The cathepsin D of Plasmodium lophurae was purified using a combination of CM-Sephadex, pepstatin-agarose and Sephadex G-100 chromatography. The plasmodial enzyme was distinct from that of the host red cell and bovine spleen in its low isoelectric point (pI 4.3). The cathepsin D of P. lophurae, as well as plasmodial extracts demonstrating such proteinase activity, were able to digest the membrane proteins of duckling and human red cells at pH 7.4; proteolysis was not inhibited in phosphate-buffered saline by 100 microM pepstatin. Membrane proteins most susceptible to proteolysis were those of the cytoskeleton, notably bands 1 and 2 (spectrin), bands 2.1-2.6 (spectrin-binding proteins) and band 3. Membrane protein degradation by crude plasmodial extracts was partially inhibited by a combination of 10 mM FeCl3, and 10 mM phenylmethylsulfonyl fluoride in phosphate-buffered saline. The changes induced in erythrocyte membrane proteins by exposure to plasmodial cathepsin D parallel the alterations observed in red cell membranes obtained from malaria infected cells. Since the action of the plasmodial protease was confined to the inner surface of the red cell membrane, it is possible that protease-induced modifications in the red cell cytoskeleton could lead to merozoite release.     

Isolation and characterization of mouse C1q

C1q was isolated from mouse serum and ascites fluid by absorption onto human IgG-coated latex beads followed by seperation on 3–10% exponential gradient sodium dodecyl sulfate (SDS) polyacrylamide gels. Mouse C1q was also purified by low ionic strength precipitation of mouse serum. The purified C1q was heat-labile (56°C, 30 min) both structurally and functionally, contained 4.3% hydroxyproline, 1.38% hydroxylsine, and 18.5% glycine, had an apparent molecular weight of 380,000 daltons, and reconstituted the hemolytic complement activity of C1q-depleted mouse serum. The negatively stained ultrastructural appearance of this purified material consisted of 6 globular units connected by strands. These data demonstrate that mouse C1q structurally and functionally is similar to human and rabbit C1q. A portion of polyacrylamide gel containing mouse C1q was injected into rabbits resulting in the production of monospecific antisera against mouse C1q. Thus, this procedure is a new, rapid and simple method to obtain monospecific antisera against mouse C1q.     

Identification of parasite proteins in a membrane preparation enriched for the surface membrane of erythrocytes infected with Plasmodium knowlesi

A subcellular fraction enriched in erythrocyte membranes has been isolated from rhesus monkey erythrocytes infected with Plasmodium knowlesi. Infected cells were lysed by centrifugation through a zone of hypotonic buffer and membranes isolated by equilibrium density gradient centrifugation in the same tube. The purified membrane fraction was shown to include the erythrocyte surface membrane by several methods: electron microscopy, identification of Coomassie Blue stained erythrocyte membrane proteins, identification of band 3 with a monoclonal antibody, and identification of radioiodinated cell surface proteins. The resulting ghosts were shown to be specifically reactive with monkey sera against the variant surface antigens of P. knowlesi by indirect immunofluorescence and membrane agglutination. No reactivity was seen with a monoclonal antibody (13C11) against the intracellular schizont surface. A number of metabolically labelled parasite proteins were enriched in this membrane function, including peptides of 277, 208, 173, 153, 134, 109, 80, 60 and 48 kDa and the variant surface antigens of variable molecular mass (180-207 kDa). These proteins were distinct from the major parasite proteins of total infected erythrocytes and isolated merozoites. The major glucosamine labelled glycoprotein of the internal schizont (230 kDa) was not found in this fraction. Moreover, no fragment of this parasite glycoprotein was found in this membrane fraction, indicating that no part of this molecule is transported to the erythrocyte surface. In contrast, the variant antigen of P. knowlesi, known to be on the erythrocyte surface, could be readily identified as peptides unique to specific cloned parasite lines. We propose that the other nine parasite proteins found within this membrane fraction represent a starting point for the identification of other parasite proteins transported to the surface membrane of the infected erythrocyte.     

Evidence that the 32, 38 and 20 kilodalton surface antigens of schistosomula and schistosomes are a family of conserved proteins

The structures of the 38, 32 and 20 kDa surface antigens isolated from schistosomula and adult worms of Schistosoma mansoni were compared by two-dimensional peptide mapping, by immunological analysis and by one- and two-dimensional electrophoresis. Peptide mapping showed a high degree of similarity between the isolated antigens from both parasite stages. The NIMP/M47 monoclonal antibody showed cross-reactivity between the 32 and the 20 kDa antigens under denaturating and non-denaturating conditions, as demonstrated by immunoprecipitation and Western blotting. It is concluded that these antigens constitute a homologous family of surface antigens.     

The Fab fragments of monoclonal IgG to a merozoite surface antigen inhibit Plasmodium knowlesi invasion of erythrocytes

Two rat monoclonal antibodies (both IgG2a isotype and having closely related specificities) and a pool of rhesus immune IgG, all of which inhibit Plasmodium knowlesi merozoite invasion of rhesus erythrocytes, have been studied before and after proteolytic digestion. The F(ab')2 and Fab fragments of both rat monoclonal antibodies show considerably enhanced inhibition of merozoite invasion as compared with the intact IgG. Inhibition by monovalent fragments indicates that these antibodies are not dependent upon merozoite agglutination and may act by blocking merozoite attachment to the specific red cell receptor. The fact that the inhibitory activities of F(ab')2 and Fab are equally enhanced on a weight basis, as compared with IgG, suggests that the removal of Fc may reduce electrostatic repulsion between antibody and merozoite surface, both of which are negatively charged at neutral pH. By contrast, papain digestion of polyclonal IgG derived from an immunised rhesus pool markedly reduces its inhibitory activity. This suggests that much of the inhibition mediated by polyclonal IgG results from merozoite agglutination and that the specificity of the rat inhibitory monoclonal antibodies is poorly represented in the immune pool. The P. knowlesi antigen reactive with the inhibitory monoclonal antibodies is known to be synthesized as a minor 66 kDa polypeptide during the last 1.5 h. of schizont development and is processed to smaller products (44 and 42 kDa) present on the merozoite surface. The present results suggest that this antigen may have particular interest as a vaccine against P. knowlesi malaria.     

Antigenic diversity of the circumsporozoite proteins in the Plasmodium cynomolgi complex

Antigenic diversity was observed in the circumsporozoite (CS) proteins of five of the six Plasmodium cynomolgi isolates (NIH, Mulligan, London, Gombak, Ceylon, RO) that we examined. Monoclonal antibodies were produced against salivary gland sporozoites of three of the isolates. Interaction of these monoclonal antibodies with the sporozoites was isolate specific, the exception being the anti-NIH monoclonals which also reacted with Mulligan strain sporozoites. Inhibition of binding between the different monoclonal antibodies indicated that for each of the NIH, London, and Gombak strains, the homologous monoclonals were recognizing the same or a topographically close immunodominant epitope on the respective CS protein. Also the binding of a polyvalent anti-NIH rhesus serum to the homologous antigen could only be inhibited by anti-NIH monoclonal antibody. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of sporozoite extracts demonstrated clear differences in the apparent molecular weights of the CS proteins of four of the six isolates. This is the first study which provides evidence of antigenic diversity in the CS proteins of different isolates of a primate plasmodial species.     

A mild procedure for separating polypeptide chains prior to immunoprecipitation and western blotting analysis

Conventional cleavage of linked polypeptide chains by heating in SDS can so alter molecular structure as to interfere with antibody binding, on which both immunoprecipitation and 'western blotting' depend. As an alternative, gentle treatment with acid at room temperature or at 0 degrees C was effective in separating the alpha and beta chains of human MHC Class II glycoprotein dimers and proved superior in terms of preservation of at least one labile epitope on the beta chain.     

A major change in surface antigens during the maturation of newborn larvae of Trichinella spiralis

Newborn larvae of Trichinella spiralis were collected for 30 min from female worms in culture, incubated in vitro for various times up to 18 h, and surface-labelled with iodine. The detergent-solubilised products were examined by SDS-polyacrylamide gel electrophoresis. At time periods up to 6 h these larvae expressed only one M_r 64 000 iodine-labelled surface protein. Some time between 6 h and 18 h a further three components (apparent M_r 58 000, 34 000 and 32 000) became accessible to surface labelling. All four of these components are antigenic in that they can be immunoprecipitated with T. spiralis immune sera. Tryptic peptide analysis revealed that the 32 and 34 kDa antigens were structurally very similar, but the 58 and 64 kDa proteins differed from each other and the 32–34 kDa pair. Thus T. spiralis not only undergoes a total change in surface antigens between moults, but also major changes in surface antigen expression within one stage.     

Evidence that the major surface proteins of three Leishmania species are structurally related

Promastigotes of Leishmania major LRC-L137, L. donovani LEM 75, and L. tropica LRC-L32 were surface radioiodinated. The proteins of the parasites were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and labeled molecules were revealed by fluorography. A single major iodinated protein of Mr 63 000 (p63) was identified in each of the three species. These proteins were partially purified by phase separation in Triton X-114 solution, demonstrating that the p63 of each of the three species is the most abundant integral membrane protein in the promastigote. Peptide maps were obtained by partial proteolysis with N-chlorosuccinimide or Staphylococcus V8 protease followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The maps of L. major and L. donovani were identical, but only partially homologous to the maps of L. tropica p63. Finally, immunological crossreactivity among the three p63s was demonstrated with the serum of a mouse immunized with purified L. major p63, and the serum of a dog naturally infected with L. donovani. The data show that the major surface proteins found on promastigotes of three Old World Leishmania species are structurally related.