Hatisaburo Masuda

Haem detoxification by an insect

Haem is involved in many biological reactions, including oxygen transport, respiration and photosynthesis. In the free state, however, haem can generate reactive oxygen species that can damage biological molecules. It can also disrupt the phospholipid bilayer of cell membranes. In Plasmodium parasites, which are the aetiological agents of malaria disease, up to 80% of host-cell haemoglobin is digested, leaving the free haem group to be detoxified in the parasites food vacuole by polymerizing it into a harmless dark-brown crystalline structure called malaria pigment or haemozoin. Haem detoxification is also a challenge for blood-sucking insects, which digest several times their own weight of vertebrate blood during a blood meal. Here we show that haem polymerization into haemozoin is not exclusive to Plasmodium: it also occurs in the midgut of the blood-sucking insect Rhodnius prolixus(Hemiptera), an important vector of Trypanosoma cruzi, the causative agent of Chagas’ disease.

A missing metabolic pathway in the cattle tick Boophilus microplus

Heme proteins are involved in a wide variety of biological reactions, including respiration, oxygen transport and oxygen metabolism [1]. The heme prosthetic group is synthesized in almost all living organisms except for a few pathogenic bacteria and trypanosomatids that use blood as food [2] [3]. There is a general belief that all nucleated animal cells synthesize heme [1] [4]. However, blood-feeding arthropods ingest enormous amounts of vertebrate blood in a single meal and the heme pathway has not been studied in these animals. We have examined heme synthesis in two hematophagous arthropods - the blood-sucking bug Rhodnius prolixus and the cattle tick Boophilus microplus. We show that R. prolixus makes heme and has a fully operative heme biosynthetic pathway, while B. microplus does not. To our knowledge, this is the first report of an animal that does not synthesize its own heme and relies solely on the recovery of heme present in the diet. Because of the inability of Boophilus to synthesize heme and its ability to deal efficiently with large amounts of free heme, we propose this organism as a good model for studying heme transport and reutilization in animal cells.

Isolation of an aspartic proteinase precursor from the egg of a hard tick, Boophilus microplus

An aspartic proteinase precursor, herein named BYC (Boophilus Yolk pro-Cathepsin) was isolated from eggs of the hard tick, Boophilus microplus. As judged by electrophoresis on sodium dodecyl sulfate polyacrylamide slab gel (SDS-PAGE), purified BYC presented 2 bands of 54 and 49 kDa, bearing the same NH2-terminal amino acid sequence. By Western blot analysis, BYC was also found in the haemolymph, indicating an extraovarian site of synthesis. Several organs were incubated in culture medium with [35S]methionine, and only the gut and fat body showed synthesis of BYC polypeptides. Protein sequencing of both the NH2-terminal and an internal sequence obtained after cyanogen bromide (CNBr) cleavage of BYC revealed homology with several aspartic proteinase precursors. Incubation at pH 3.5 resulted in autoproteolysis of BYC, which produced the mature form of the enzyme, that displayed pepstatin-sensitive hydrolytic activity against haemoglobin. Western blot analysis using anti-BYC monoclonal antibodies showed proteolytic processing of BYC during embryogenesis and suggested activation of the enzyme during development. A role of BYC in degradation of vitellin, the major yolk protein of tick eggs, is discussed.

IMMUNIZATION OF BOVINES WITH AN ASPARTIC PROTEINASE PRECURSOR ISOLATED FROM BOOPHILUS MICROPLUS EGGS

The capacity of the Boophilus Yolk pro-Cathepsin (BYC) to induce a protective immune response in cattle against Boophilus microplus infestation was tested by vaccination experiments and by inoculation of monoclonal antibody (MAb) against BYC into fully engorged tick females. In immunization experiments the measurement of various biological parameters demonstrated a partial protection against B. microplus. A continuous decrease in the levels of specific antibodies was observed over 11 months when six bovines were maintained in field conditions. The inoculation of the MAb into tick females produced a dose-dependent decrease in oviposition and survival of the ectoparasite compared to the control.

Uptake of yolk proteins in Rhodnius prolixus

The uptake of yolk protein in Rhodnius prolixus was studied in vivo and in vitro using a metabolically labelled [32P]yolk protein purified on a potassium bromide gradient. The [32P]vitellin is readily removed from haemolymph and specifically accumulates in the ovary. The ability of oocytes to take up yolk protein at different stages of development increases with their size up to the time of chorion formation. This increase in uptake capacity of oocytes correlates with the opening of intercellular space in the follicular epithelium. The rate of uptake is dependent on the external concentration of yolk protein and temperature, and can be saturated. Separate analysis of binding shows a similar dependence on yolk protein concentration. Soon after a meal, the vitellogenin concentration increases in the haemolymph until it reaches a steady-state concentration at which the rate of vitellogenin uptake matches the rate of vitellogenin synthesis. Most ovarian growth occurs while the concentration of vitellogenin in the haemolymph is at this steady-state level, which is in turn very close to its ovarian binding constant of 12 mg/ml determined in vitro. Thus it is suggested that the uptake system normally works undersaturated and below its maximal rate of uptake.

Haemozoin formation in the midgut of the blood-sucking insect Rhodnius prolixus

Malaria parasites digest haemoglobin and detoxify the free haem by its sequestration into an insoluble dark‐brown pigment known as haemozoin (Hz). Until recently, this pigment could be found only in Plasmodium parasites. However, we have shown that Hz is also present in the midgut of the blood‐sucking insect Rhodnius prolixus [Oliveira et al. (1999) Nature 400, 517–518]. Here we show that Hz synthesis in the midgut of this insect is promoted by a particulate fraction from intestine lumen. Haem aggregation activity is heat‐labile and is inhibited in vitro by chloroquine (CLQ). Inhibition of Hz formation in vivo by feeding insects with CLQ leads to increased levels of haem in the haemolymph of the insect, which resulted in increased lipid peroxidation. Taken together, these results indicate that a factor capable of promoting Hz crystallisation is present in R. prolixus midgut and that this activity represents an important physiological defence of this insect against haem toxicity.

Characterization of vitellin and vitellogenin from Rhodnius prolixus

Abstract The protein, phospholipid and carbohydrate components of vitellin and vitellogenin from Rhodnius prolixus were analyzed after being labelled by feeding the insects with blood enriched with 32 P i . The carbohydrate moiety is comprised mainly of mannose residue (90%), but also contains glucose and traces of galactose and glucosamine. Both vitellin and vitellogenin are present in two different aggregational states that differ in molecular weight. Each of the individual aggregates releases, under denaturing conditions, the same four polypeptides with approximate molecular weights of 180, 158, 44 and 38 K daltons with no consistent stoichiometries. Analysis of the phosphorylated yolk protein after specific precipitation with serum against vitellin showed 62% of the total radioactivity in the phospholipid fraction and 38% in the glycoprotein fraction. Upon further analysis by thin layer chromatography, the following phospholipids were found: phosphatidylcholine (64.2%), phosphatidylethanolamine (30%), phosphatidic acid (4,4%), sphingomyelin (0.7%), phosphatidylserine (0.6%) and cardiolipin (0.2%). Analysis of the glycoprotein fraction showed that all four subunits were phosphorylated, part of the radioactivity being found as [ 32 P]serine and [ 32 P]mannose.

Binding and storage of heme by vitellin from the cattle tick, Boophilus microplus

We have previously shown (, Curr. Biol. 9, 703-706) that the cattle tick Boophilus microplus does not synthesize heme, relying solely on the recovery of the heme from the diet to make all its hemeproteins. Here we present evidence that Vitellin (VN(1)), the main tick yolk protein, is a reservoir of heme for embryo development. VN was isolated from eggs at different days throughout embryogenesis. Immediately after oviposition, Boophilus VN contains approximately one mol of heme/mol of protein. During embryo development about one third of egg VN is degraded. The remaining VN molecules bind part of the heme released. These results suggest that VN functions as a heme reservoir, binding any free heme that exceeds the amount needed for development. In vitro measurement of the binding of heme to VN showed that each VN molecule binds up to 31 heme molecules. The association of heme with VN strongly inhibits heme-induced lipid peroxidation, suggesting that binding of heme is an important antioxidant mechanism to protect embryo cells from oxidative damage. This mechanism allows this hematophagous arthropod to safely store heme obtained from a blood meal inside their eggs for future use. Taken together our data suggest that, besides its known roles, VN also plays additional functions as a heme deposit and an antioxidant protective molecule.

Lipophorin and oögenesis in Rhodnius prolixus: Transfer of phospholipids

The lipophorin of Rhodnius prolixus was metabolically labelled with 32P exclusively in the phospholipid moiety and purified on a potassium bromide gradient. After injection into a vitellogenic female the radioactivity from [32P]lipophorin was readily removed from the haemolymph and accumulated in several organs, including the ovary. The rate at which ovaries incorporated radioactivity from injected [32P]lipophorin varied during the days following the meal. The ability of oocytes to take up radioactivity also varied, increasing with their sizes up to the time of chorion formation. The incorporation of radioactivity from [32P]lipophorin was several times higher than the sequestration of [14C]inulin, indicating that the process may be specific. The kinetics of incorporation were linear and the process was impaired at low temperature. Lipophorin delivered phospholipids to the growing oocytes but its apoproteins were not accumulated.

Vitellin processing and degradation during embryogenesis in Rhodnius prolixus

Abstract The fate of vitellin (VT), the major yolk protein in Rhodnius prolixus , was studied during embryogenesis by electrophoretic and immunological techniques. Analysis of the protein content of the eggs by polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions shows a continuous alteration in the VT molecule following oviposition. Two distinct phases of VT processing can be observed: (1) when limited proteolysis is dominant; and (2) when an extensive degradation of VT occurs inside the digestive system of the embryo. In the first phase (days 0–10 after oviposition), as embryogenesis proceeds, the two principal bands of VT give rise to several bands that migrate progressively further into the gel. Analysis of the same samples by SDS-PAGE reveals a continuous proteolysis of the subunits of the VT molecule. When the VT is analysed by rocket immunoelectrophoresis very little alteration in the total VT content per egg is observed from day 0 to day 10, suggesting that the antibodies used also recognize polypeptides produced by partial proteolysis. Western blot analysis demonstrated that some of the new polypeptides (112, 103, 64 and 56 kDa) generated during embryonic development are in fact derived from VT by limited proteolysis. After cleavage, the polypeptides remain in the same lipoprotein macromolecular complex. In the second phase of VT degradation, an extensive degradation of VT takes place, such that by day 14 after oviposition (hatching), VT content has declined to about half of the initial value. The VT remaining in the first instar is located entirely in the crop and disappears within 5 days after hatching.