Distinctive cationic proteins of the human eosinophil granule: major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin

The human eosinophil granule contains a number of cationic proteins that have been identified and purified to homogeneity, including the major basic protein (MBP), the eosinophil cationic protein (ECP), and the eosinophil-derived neurotoxin (EDN). Because of confusion in the literature regarding the distinctiveness of MBP and ECP, we investigated the immunochemical and physicochemical properties of these purified proteins by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), by specific double antibody radioimmunoassays (RIA) for MBP and ECP, and by fractionation of acid-solubilized eosinophil granules on Sephadex G-50 columns. Analysis of a mixture of the three purified proteins by SDS-PAGE showed that they migrated as three distinct bands with differing m.w. Comparison by specific RIA for MBP and ECP did not demonstrate any appreciable immunochemical cross-reactivities among the three proteins. Sephadex G-50 column fractions of acid-solubilized eosinophil granules were analyzed by RIA and by SDS-PAGE analysis of individual column fractions. MBP, ECP, and EDN eluted at different volumes from Sephadex G-50 columns as determined by RIA and SDS-PAGE. Soluble extracts of eosinophil granules from patients with the hypereosinophilic syndrome contained between six and 64 times more MBP than ECP on a weight basis. These observations demonstrate that MBP, ECP, and EDN are distinctive cationic proteins of the human eosinophil granule and that eosinophil granules from patients with eosinophilia contain considerably greater quantities of MBP than ECP.     

Identification of granule-bound starch synthase in potato tubers

Starch granules isolated from potato (Solanum tuberosum L.) tubers were extracted with sodium dodecyl sulfate and the extract was analyzed. A major protein with a molecular weight of 60,000 daltons was detected. This protein was purified by preparative sodium dodecyl sulfate-gel electrophoresis and specific antibodies were prepared. The anti-60-kilodalton antibodies obtained (a) cross-reacted with the waxy proteins of both maize (Zea mays L.) and grain amaranth (Amaranthus hypochondriacus L.), and (b) inhibited starch synthase activity in partially digested starch granules of the grain amaranth. This evidence strongly suggests that the major 60-kilodalton protein present in potato starch granules represents the granule-bound starch synthase.     

Human eosinophil-derived neurotoxin and eosinophil protein X are likely the same protein

The human eosinophil granule contains a series of cationic proteins. Two of these, eosinophil-derived neurotoxin (EDN) and eosinophil protein X (EPX), are reported to have similar m.w. and both possess neurotoxic and helminthotoxic activities. Therefore, the properties of these molecules were analyzed to determine whether they differ. EDN was purified from eosinophils of patients with the hypereosinophilic syndrome and EPX from the buffy coat cells of normal individuals. By SDS-PAGE, both proteins showed a major band at 18.7 kDa and a minor band at 21.4 kDa. By two-dimensional non-equilibrium gel electrophoresis the proteins migrated identically. With radiolabeled proteins in reverse phase HPLC, both proteins eluted at the same concentration of acetonitrile and showed identical tryptic maps. Both proteins possessed comparable ribonuclease activity and both were comparably neurotoxic in the rabbit. By immunodiffusion the two proteins showed a reaction of identity; by RIA, with both polyclonal and monoclonal antibodies, the proteins had very similar inhibitory activities. These results indicate that EDN and EPX have virtually identical properties and are probably the same protein.     

ACID PHOSPHATASE LOCALIZATION IN RABBIT EOSINOPHILS

Eosinophil (and heterophil) leukocytes of glycogen-induced rabbit peritoneal exudates were fixed for 1½ min in 2% glutaraldehyde and examined for acid phosphatase activity both biochemically and cytochemically. Biochemical assays showed that enzymatic activity had been inhibited by only ~10% under these conditions. The cytochemical reaction in the eosinophil was confined to the granules in which the reaction product appeared in the matrix, not in the crystalline core (or in the core region after the latter's extraction). Granules wherein the matrix was disrupted and the crystalline core degraded or extracted showed the most intense deposition of reaction product, whereas well preserved granules with morphologically intact matrix and crystals were unreactive. Yet, not all disrupted granules gave a positive reaction, indicating that disruption was a necessary but not sufficient condition for reactivity. In many eosinophil leukocytes, most if not all granules were acid phosphatase-positive, provided they had become disrupted to a certain degree. Factors possibly involved in converting the granules from an unreactive to a reactive state are discussed.     

Digestion of legume starch granules by larvae of Zabrotes subfasciatus (Coleoptera: bruchidae) and the induction of alpha-amylases in response to different diets

Zabrotes subfasciatus larvae possess three alpha-amylase isoforms as determined by in gel assays following SDS-PAGE. The two minor isoforms present lower electrophoretic mobility than the major form, and seem to occur as a heterodimer. When developed inside Vigna unguiculata (cowpea) seeds, fourth instar larvae have minor quantities of the slow-migrating forms, but when reared on seeds of Phaseolus vulgaris (common bean) or Phaseolus lunatus, the two slow-migrating forms are expressed in higher amounts, while activity of the major form was independent of the host seed. Larvae developing inside cowpea seeds at the beginning of the fourth instar were fed on flour from cotyledons of cowpea or common bean. Larvae fed on the common bean flour started to express the dimer in higher amounts when compared with the control larvae fed on cowpea flour. In an attempt to correlate differences between starch granules and the induction of alpha-amylases, a detailed study on the digestive process of the granules was conducted. Incorporation of purified starch granules into artificial diets did not induce the two minor alpha-amylases. The in vitro hydrolysis rates of purified granules and the pattern of dextrins liberated by the different alpha-amylases were similar for the two legume species. The starch granules enter the midgut extensively damaged, which may facilitate the access to the more susceptible parts of the granules to enzymatic attack.     

Binding of the major phasin, PhaP1, from Ralstonia eutropha H16 to poly(3-hydroxybutyrate) granules

The surface of polyhydroxybutyrate (PHB) storage granules in bacteria is covered mainly by proteins referred to as phasins. The layer of phasins stabilizes the granules and prevents coalescence of separated granules in the cytoplasm and nonspecific binding of other proteins to the hydrophobic surfaces of the granules. Phasin PhaP1(Reu) is the major surface protein of PHB granules in Ralstonia eutropha H16 and occurs along with three homologues (PhaP2, PhaP3, and PhaP4) that have the capacity to bind to PHB granules but are present at minor levels. All four phasins lack a highly conserved domain but share homologous hydrophobic regions. To identify the region of PhaP1(Reu) which is responsible for the binding of the protein to the granules, N-terminal and C-terminal fusions of enhanced green fluorescent protein with PhaP1(Reu) or various regions of PhaP1(Reu) were generated by recombinant techniques. The fusions were localized in the cells of various recombinant strains by fluorescence microscopy, and their presence in different subcellular protein fractions was determined by immunodetection of blotted proteins. The fusions were also analyzed to determine their capacities to bind to isolated PHB granules in vitro. The results of these studies indicated that unlike the phasin of Rhodococcus ruber, there is no discrete binding motif; instead, several regions of PhaP1(Reu) contribute to the binding of this protein to the surface of the granules. The conclusions are supported by the results of a small-angle X-ray scattering analysis of purified PhaP1(Reu), which revealed that PhaP1(Reu) is a planar, triangular protein that occurs as trimer. This study provides new insights into the structure of the PHB granule surface, and the results should also have an impact on potential biotechnological applications of phasin fusion proteins and PHB granules in nanobiotechnology.     

Localization of the guinea pig eosinophil major basic protein to the core of the granule

The localization of the guinea pig eosinophil major basic protein (MBP) within the cell was investigated by the use of immunoelectron microscopy and by isolation of the granule crystalloids. First, by immunoperoxidase electron microscopy, we found that the MBP of eosinophil granules is contained within the crystalloid core of the granule. Specific staining of cores was present when rabbit antiserum to MBP was used as the first stage antibody in a double antibody staining procedure, whereas staining was not seen when normal rabbit serum was used as the first stage antibody. Second, crystalloids were isolated from eosinophil granules by disruption in 0.1% Triton X-100 and centrifugation through a cushion of 50% sucrose. Highly purified core preparations yielded essentially a single band when analyzed by electrophoresis on polyacrylamide gels containing 1% sodium dodecyl sulfate (SDS). The E1%1cm of the core protein was 26.8 +/- 1.0 (X +/- SEM); the E1%1cm for the MBP was 26.3. The core protein could not be distinguished from the MBP by radioimmunoassay (RIA) and essentially all of the protein in the core preparations could be accounted for as MBP. The results indicate that the MBP is contained in the core of the guinea pig eosinophil granule and that it is probably the only protein present in the core.     

Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. II. Cell surface localization of granule membrane and content proteins before and after degranulation

The compositional relationship between the cell surface of rabbit polymorphonuclear leukocytes (PMNs) and the membranes of PMN cytoplasmic granules has been investigated. Heterophilic PMNs obtained from peritoneal exudates contained 13 cell surface polypeptides ranging in molecular weight from 220,000 to 12,000 daltons as determined by lactoperoxidase-catalyzed protein iodination and gel electrophoresis. Of these, four polypeptides co-migrated with proteins identified as the major constituents of specific (SpG) and azurophilic (AzG) granule membranes. The most notable of these were cell surface proteins of 145,000 and 96,000 daltons that co-migrated with proteins identified as granule content proteins released from PMNs during exocytosis. Extensive washing did not remove these proteins from the cell surface. Iodination of PMNs after the release of SpG and AzG contents by calcium ionophore- induced exocytosis revealed that there was not a dramatic quantitative change in the proteins on the cell surface. Instead, there were large, quantitative increases in the relative amounts of (125)I that were incorporated into several pre-existing cell surface proteins; all of these cell surface proteins co-migrated as a set with those polypeptides identified as either granule membrane or content proteins. Although nearly all of the major polypeptides of SpG and AzG had counterparts on the cell surface of freshly isolated peritoneal exudates PMNs, there were several polypeptides that were unique to the cell surface. Thus, the PMN has at least three membrane compartments with strikingly different protein compositions.     

A comparative study of the polypeptides of three iridescent viruses by N-terminal analysis,amino acid analysis,and surface labeling

Polyacrylamide gel analysis of the structural proteins of three types of iridescent viruses (2, 6, and 9) demonstrated that the purified virions had one major and more than 20 minor polypeptides. Surface labeling procedures performed on pure intact virions, using ¹²⁵I in the presence of lactoperoxidase and chloramine T (at low iodine concentrations), demonstrated that the major and two or three minor polypeptides were located on the outside. The major structural polypeptide was isolated from each virus type by preparative polyacrylamide gel electrophoresis. Amino acid analysis indicated that this protein was very similar in the three iridescent viruses. The three polypeptides had an identical N terminal (proline). While the major polypeptide of each virus has a slightly different molecular weight as determined by polyacrylamide gel electrophoresis, the similarities in iodine labeling, N terminals, and amino acids suggests a common function for this protein.     

Characteristics of structural proteins of infectious flacherie virus from the silkworm, Bombyx mori

Structural proteins and the characteristics of infectious flacherie virus (IFV) purified from the silkworm, Bombyx mori, are described. The purified IFV had four major structural proteins, which were detected only in high concentration gels of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a few minor ones. Molecular weights of the major proteins were 35,200 (VP 1), 33,000 (VP 2), 31,200 (VP 3), and 11,600 (VP 4), and numbers per virion were 62, 57, 54, and 31, respectively. Amino acid compositions of VP 1, VP 2, and VP 3 were similar to each other but that of VP 4 was somewhat different. By isoelectric focusing and two-dimensional electrophoresis, high resolution of the structural proteins was obtained with silver staining. The isoelectric points of the four major proteins were determined as 7.7(VP 1), 6.7(VP 2), 4.8(VP 3), and 5.5(VP 4). This work is the first report on insect picornaviruses that presents some discriminative properties of each viral protein that was compared to those of mammalian picornaviruses.     

SEGREGATION AND PACKAGING OF GRANULE ENZYMES IN EOSINOPHILIC LEUKOCYTES

During their differentiation in the bone marrow, eosinophilic leukocytes synthesize a number of enzymes and package them into secretory granules. The pathway by which three enzymes (peroxidase, acid phosphatase, and arylsulfatase) are segregated and packaged into specific granules of eosinophils was investigated by cytochemistry and electron microscopy. During the myelocyte stage, peroxidase is present within (a) all rough ER cisternae, including transitional elements and the perinuclear cisterna; (b) clusters of smooth vesicles at the periphery of the Golgi complex; (c) all Golgi cisternae; and (d) all immature and mature specific granules. At later stages, after granule formation has ceased, peroxidase is not seen in ER or Golgi elements and is demonstrable only in granules. The distribution of acid phosphatase and arylsulfatase was similar, except that the reaction was more variable and fully condensed (mature) granules were not reactive. These results are in accord with the general pathway for intracellular transport of secretory proteins demonstrated in the pancreas exocrine cell by Palade and coworkers. The findings also demonstrate (a) that in the eosinophil the stacked Golgi cisternae participate in the segregation of secretory proteins and (b) that the entire rough ER and all the Golgi cisternae are involved in the simultaneous segregation and packaging of several proteins.     

Localization of eosinophil cationic protein, major basic protein, and eosinophil peroxidase in human eosinophils by immunoelectron microscopic technique

An immunoelectron microscopic technique using protein A-gold as a specific marker was used for precise intracellular localization of eosinophil granule proteins. Eosinophils from healthy individuals were isolated in metrizamide gradients. Eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) were clearly located in the matrix of the large crystalloid-containing granules. In addition, ECP was probably present in the small granules of eosinophils. Major basic protein (MBP) was present in the crystalloid structure of specific granules. This method can be applied in studies of eosinophil degranulation to trace the release of biological effector molecules.     

The composition of Staufen-containing RNA granules from human cells indicates their role in the regulated transport and translation of messenger RNAs

hStaufen is the human homolog of dmStaufen, a double-stranded (ds)RNA-binding protein involved in early development of the fly. hStaufen-containing complexes were purified by affinity chromatography from human cells transfected with a TAP-tagged hStaufen gene. These complexes showed a size >10 MDa. Untagged complexes with similar size were identified from differentiated human neuroblasts. The identity of proteins present in purified hStaufen complexes was determined by mass spectrometry and the presence of these proteins and other functionally related ones was verified by western blot. Ribosomes and proteins involved in the control of protein synthesis (PABP1 and FMRP) were present in purified hStaufen complexes, as well as elements of the cytoskeleton (tubulins, tau, actin and internexin), cytoskeleton control proteins (IQGAP1, cdc42 and rac1) and motor proteins (dynein, kinesin and myosin). In addition, proteins normally found in the nucleus, like nucleolin and RNA helicase A, were also found associated with cytosolic hStaufen complexes. The co-localization of these components with hStaufen granules in the dendrites of differentiated neuroblasts, determined by confocal immunofluorescence, validated their association in living cells. These results support the notion that the hStaufen-containing granules are structures essential in the localization and regulated translation of human mRNAs in vivo.     

Isolation of a protein from the plasma membrane of adrenal medulla which binds to secretory vesicles.

Solubilized proteins of the plasma membrane of bovine adrenal medulla were fractionated on the basis of their affinity for secretory vesicles. The isolation procedure included preparation of a highly purified fraction of plasma membranes, its solubilization in detergent, and application to a column prepared from glutaraldehyde-fixed chromaffin granules. Using this technique, one major polypeptide (80% of the material bound) was isolated. This protein has been shown to originate from the plasma membrane and has no affinity for fixed bovine adrenal medullary mitochondria or lysosomes. It is eluted most effectively by low pH (3.0) and can be rebound and re-eluted from fixed secretory granules. In sodium dodecyl sulfate and beta-mercaptoethanol it has an apparent molecular weight of 51,000. In addition, two minor components, comprising about 20% of the material bound were detected having apparent molecular weights in sodium dodecyl sulfate of 14,000 and 62,000. It is suggested that such a molecule could function as a plasma membrane-located receptor for chromaffin granules during the secretory process.     

Chemical and immunological similarities between the phloem proteins of three genera of the Cucurbitaceae

Phloem exudates from Cucurbita, Cucumis, and Citrullus were gelled by oxidative formation of disulphide bridges between the phloem filaments. Gellation could be inhibited by dithiothreitol or iodoacetamide and did not require the presence of the phloem lectin. Each exudate contained a dimeric lectin of similar relative molecular mass and purified specific activity; these were all specific for oligomers of N-acetyl-glucosamine, and shared antigenic determinants. The similarity of the phloem proteins between Cucurbita, Cucumis, and Citrullus implied that they served the same function in each genus. This is postulated to be the sealing of wounded sieve-tubes, with the lectin on the filaments binding and preventing the entry of micro-organisms. The phloem lectin and the filament-forming protein from Cucurbita shared sequence homologies as judged by amino-acid-composition comparisons, but antibodies raised against each showed no cross-reactivity with the other protein. The exudates from Cucurbita and Cucumis may contain a high concentration of phloem proteins because the large diameter of their sieve-pores does not allow rapid blocking by callose synthesis on wounding, and a chemical mechanism of gellation is required.     

Membranes of chromaffin granules. Isolation and partial characterization of two proteins

Membranes of chromaffin granules were isolated from the adrenal glands of four different species. The solubilized membrane proteins could be resolved into several bands by polyacrylamide-gel electrophoresis (alkaline and acid gel systems). Two major protein components appeared to be common to the chromaffin granule membranes of ox, horse, pig and man. The various membrane proteins of bovine chromaffin granules were separated by filtration on Sephadex G-200 in the presence of sodium dodecyl sulphate. Two major membrane proteins (A and B) were obtained in purified form. Treatment of protein A with 2-mercaptoethanol before electrophoresis resulted in two more rapidly migrating subunits, whereas protein B was unaffected by mercaptoethanol treatment. The amino acid compositions of the two purified proteins were determined. They are very similar to that of the total membrane proteins but significantly different from that of the chromogranins, the soluble proteins of chromaffin granules.     

Fluorescence of eosinophil leucocyte granules induced by 1-hydroxy-3,6,8-pyrenetrisulfonate. Visualization of differences in protein isoelectric points

After treatment of horse, rat and human blood smears with alkaline solutions of 1-hydroxy-3,6,8-pyrenetrisulfonate (HPTS), eosinophil leucocyte granules were the unique cell components which showed a bright green fluorescence. When stained with HPTS at pH 10, the whole granule of horse eosinophils showed high emission which strongly diminished after washing or staining in salt solutions or by using blocking methods for amino groups. Using HPTS at pH 12, the fluorescence reaction of house granules was specifically located in the peripheral region, appearing as fluorescent rings. These microscopic observations, which indicate differences in the isoelectric point of proteins within the eosinophil granule, were also confirmed by HPTS staining of protein blots as model substrates. Spectral analysis of HPTS at pH 10 and 12 showed practically identical absorption and emission spectra with peaks at 450 nm and 510 nm, respectively. Our results indicate that mainly ionic binding occurs between cationic proteins and HPTS in alkaline solution, and that the most cationic proteins (with isoelectric points at pH higher than 12) are located in the peripheral annular region of horse eosinophil granules.     

Preliminary analysis of root exudates of in vitro-micropropagated Alnus glutinosa clones

Biochemical analysis at Alnus glutinosa (L.) Gaertn. root exudates was carried out in order to investigate the probable role of these exudates in the establishment of the Alnus-Frankia symbiosis. Sufficient amounts of sterile root exudates were collected from plants originating from five in vitro-micropropagated A. glutinosa clones (AG1, AG2, AG3, AG4, AG8) grown under aerobic conditions in nutrient solution with Ballotini glass beads. Cytological controls showed good preservation of roots at the end of the collecting period. The total protein and sugar contents of crude root exudates were determined and related to the dry root weight. These results showed a provenance effect. Soluble high molecular-weight fractions (i.e. the mucilage) were isolated following rigorous purification of the crude root exudates and within this fraction proteins were characterized by isoelectric focusing. The protein pattern of the five clones tested showed major bands in common and some minor specific ones. These experimental data will serve as a useful basis for future physiological investigations.     

Identification of a stress-induced protein in stem exudates of soybean seedlings root-infected with Fusarium solani f. sp. glycines

Sudden death syndrome of soybean (Glycine max) is caused by the soilborne fungus, Fusarium solani f. sp. glycines, that infects soybean roots. Besides root necrosis, symptoms include interveinal leaf chlorosis, necrosis and premature defoliation. It is proposed that a fungal toxin is produced in soybean roots and translocated to foliage. In this study, we isolated compounds from soybean stem exudates from plants that were either inoculated or not inoculated with F. solani f. sp. glycines. A protein with an estimated molecular mass of 17 kDa and designated as FISP 17 for F. solani f. sp. glycines-induced stress protein was identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein occurred only in F. solani f. sp. glycines-infected soybean stem exudates. The N-terminal amino acid sequence of the purified protein had 100 % identity with a starvation-associated message 22 protein, and 80 and 78 % identity with purified bean pathogenesis-related proteins, PvPR1 and PvPR2, respectively. To determine if the protein was of plant or fungal origin, a synthetic peptide was designed based on the N-terminal sequence and used to raise a polyclonal antibody from rabbit. Western blot analysis showed that the antibody only reacted with a 17-kDa protein in F. solani f. sp. glycines-infected plant exudates, but no reaction occurred with healthy plant exudates or with culture filtrates of F. solani f. sp. glycines. This is the first report of the presence of a stress-induced protein in stem exudates of soybean seedlings root-infected with F. solani f. sp. glycines.