Toshiya Osada

Structural changes in alpha-2- and ovomacroglobulins studied by gel chromatography and electron microscopy.

The structural change that occurs in alpha-2-macroglobulin upon its interaction with methylamine or chymotrypsin was studied by high-performance gel chromatography and electron microscopy. The result enabled us to estimate the Stokes radius of the protein as 8.8 nm and 7.9 nm before and after binding with the proteinase, respectively. The methylamine-treated protein also had the Stokes radius of 7.9 nm. Similar studies on the chicken and crocodilian ovomacroglobulins showed that these homologues of alpha 2-macroglobulin had Stokes radii of 9.2-9.3 nm and 8.5-8.7 nm before and after binding with chymotrypsin. Their Stokes radii did not change as a result of the methylamine treatment. Electron micrographs of the native and altered forms of the three proteins are presented. This study introduces a simple and quantitative method to study the structural change of alpha 2-macroglobulin and its homologues.

Antibodies against viral proteins can be produced effectively in response to the increased uptake of alpha2-macroglobulin: Viral protein conjugate by macrophages☆

We have previously shown that foreign antigens conjugated to alpha 2-macroglobulin (alpha 2M) were effectively taken up by macrophages, which was followed by a remarkable increase in the proliferation of immune T cells. We present in this report that the production of antibodies against viral proteins can also be effectively achieved when viral proteins were conjugated to alpha 2M and fed to the in vitro immune system. Proteins derived from Kirstein murine sarcoma virus (Ki-MSV) were partially purified by gel chromatography and conjugated to alpha 2M by the action of proteinases. Viral proteins conjugated to alpha 2M were taken up by thioglycolate-induced murine peritoneal exudate cells (PEC) more effectively than the free viral proteins. Murine spleen cells were then added to PECs fed with free viral proteins or with alpha 2M: viral protein conjugates, and the cell mixtures were incubated for five days. Each culture medium was then assayed for a specific antibody production by enzyme-linked immunosorbent assay (ELISA). Figures based on such assays revealed that the production of antibodies against viral proteins was ten times higher when the proteins were fed to macrophages in conjugated forms with alpha 2M.

Murine T cell proliferation can be specifically augmented by macrophages fed with specific antigen: α-2-macroglobulin conjugate

Foreign antigens conjugated to alpha-2-Macroglobulin (alpha-2-M) were effectively taken up by murine macrophages via alpha-2-M receptors. Such effective internalization of alpha-2-M:antigen conjugate by macrophages resulted in a remarkable increase in its ability to activate murine immune T cells under the following conditions. After macrophages were incubated with alpha-2-M:antigen conjugate or unconjugated antigen, they were cultured with immune T cells and antigen-stimulated tritiated thymidine incorporation by T cells was measured. The stimulation of T cell proliferative response by macrophages fed with the conjugate was sixteen times higher than what was observed with macrophages pretreated in the same concentration of unconjugated antigen. These findings suggest a physiological function of alpha-2-M and give us a new technique of immunization.

Open quaternary structure of the hagfish proteinase inhibitor with similar properties to human α-2-macroglobulin

A homologous protein to human plasma alpha-2-macroglobulin (alpha-2-M) was purified from the blood plasma of hagfish (Eptatretus buergeri) and its structure and function were studied. The hagfish protein inhibited several proteinases and its inhibitory activity was blocked with methylamine as in the case of human alpha-2-M. The molecular weight and sedimentation coefficient of the hagfish inhibitor were 390,000 +/- 20,000 and 11.0 S, respectively, as determined by sedimentation studies. The frictional ratio calculated from these parameters was 1.75. The Stokes radius estimated from HPLC gel chromatography was 8.8-8.9 nm, which was similar to that of human alpha-2-M despite the fact that the hagfish inhibitor was only one-half as large as human alpha-2-M in molecular weight. The hagfish inhibitor was expected to be more asymmetric and/or more hydrated than the human inhibitor. The electron micrographs of the negatively stained hagfish inhibitor showed that it had an open, rectangular quaternary structure of 15 +/- 1.5 X 19 +/- 2 nm in which two semiglobular units were located at the two shorter sides with a gap of 8 +/- 1 nm in width. Each semiglobular unit had an approximate width of 5 +/- 0.5 nm. The thickness of the unit was estimated to be 3 to 3.5 nm from the result of fixed-angle shadowing experiments. Although the two semiglobular units must be connected by some structure, very little material could be seen between them. Such an open quaternary structure may explain the high frictional ratio and large Stokes radius of this protein. The structural change of the inhibitor after reaction with proteinases or methylamine could be detected by electron microscopy and gel chromatography.

Endocytotic internalization of α-2-macroglobulin: α-galactosidase conjugate by cultured fibroblasts derived from Fabry hemizygote

Abstract Endocytotic internalization of α-galactosidase by cultured fibroblasts derived from a patient with Fabrys disease was achieved via receptor-mediated endocytosis of α-2-macroglobulin (α-2-M). α-galactosidase of coffee beans was conjugated to α-2-M when the latter was treated with trypsin. Internalization of the conjugate resulted in an increase of α-galactosidase activity in the crude cell extracts. The observed internalization was blocked by the presence of bacitracin, an inhibitor of binding between α-2-M and its receptor on the cell surface. When the cells were incubated at 4°C with the conjugate, internalization was also inhibited. The α-galactosidase activity in the cells was saturated when the concentration of the conjugate in the medium was 40 μg/ml. Since non-conjugated α-galactosidase was not effectively internalized, the observed internalization of the conjugate was mediated by recognition of α-2-M by its receptor. The effective internalization of α-galactosidase described in this paper has a potential use in the enzyme replacement therapy of Fabrys disease.

Structural changes of alpha2- and ovomacroglobulins

The plasma α2-macroglobulin and its egg white homologue ovomacroglobulin were purified from several different species and their structure before and after the reaction with proteinases studied by electron microscopy. The negatively stained specimens showed either a ringlike structure or a flowerlike one before the reaction with proteinses, but their structures changed into open rectangular ones after the reaction. The translational frictional ratio f/f0 of human α2-macroglobulin and crocodilian ovomacroglobulin given in the literature is between 1.5 and 1.6 before and after the reaction with proteinases. The value reflects asymmetry due not to a high axial ratio, but rather to an openness of the structure resulting in a partially free draining character of the molecules. The computational method developed by Bloomfield and his co-workers based on the formalism of Kirkwood is used to calculate the frictional ratio of several models constructed from small spheres. The overall shape of the models is derived from electron micrographs. Although the degree of hydration is an unknown parameter in the calculation, reasonable agreement is obtained between the experimental values of f/f0 and the calculated ones. Combination of electron microscopic and hydrodynamic methods would be fruitful in the structural study of giant proteins such as α2-macroglobulin.

The fate of internalized α-2-macroglobulin: α-galactosidase conjugate in fibroblasts from Fabry's hemizygote

In our previous report we described the endocytotic incorporation of coffee bean alpha-galactosidase conjugated to human alpha-2-macroglobulin (alpha-2-M) into cultured fibroblasts derived from a patient with Fabrys disease (1). The fate of internalized alpha-galactosidase according to the method described in the above report is now studied. Measurement of the enzyme activity of subcellular fractions showed that it was concentrated in the lysosomal-mitochondrial fraction. The half-life of internalized alpha-galactosidase was determined to be 2 h.

Polymerization of turtle α-macroglobulin through newly exposed sulfhydryls reveals the location of ex-thiolester bonds☆

Green turtle alpha-macroglobulin, which has previously been shown to contain thiolester bonds, formed linear polymers after being treated with proteinases. Biochemical analyses showed that the polymerization proceeded through disulfide-bond formation between monomers. The only sulfhydryl groups available for such polymerization after proteinase treatment were those created as the product of thiolester hydrolysis. Electron micrographs of polymers revealed H-shaped monomeric units aligned lengthwise in linear polymers. The average length per monomeric unit in the polymer estimated from the discrete distribution of polymer lengths was approximately 80% of the average length of free monomers, indicating that monomers overlapped each other within a region of about 4 nm. From such observations we concluded that the newly produced sulfhydryl groups were located on the four arms of the H-shaped molecule. The location of sulfhydryls can be taken as the site of the exposure of thiolesters which were originally sequestered in the hydrophobic interior of the molecule. Since the structure of turtle alpha-macroglobulin is very similar to that of human serum alpha 2-macroglobulin the results predict a similar location of sulfhydryls in human alpha 2-macroglobulin after proteinase treatment. The observed polymerization property is unique to sea turtle alpha-macroglobulin and has not been observed with human alpha 2-macroglobulin or other homologous proteins.