Cysteine proprotease colocalizes with vitellogenin in compound granules of the cockroach fat body.

A cysteine proprotease has been identified in developing embryos of the cockroach Blattella germanica and found to be a maternally encoded gene product that is transferred endocytically to the oocyte. The present study aims at establishing how this maternally derived proprotease is synthesized, packaged, and secreted during vitellogenesis. To this end, proprotease was localized immunocytochemically in the fat body of postmating females and its localization compared with that of vitellogenin over the same developmental periods. Fat bodies in cockroaches are comprised of two different cell types: trophocytes and bacteriocytes. Data show that proprotease and vitellogenin come to colocalize in compound granules of the fat body trophocytes. While synthesis of vitellogenin can be traced back to granules resulting from the coalescence of Golgi-derived vesicles in the trophocyte cytoplasm, proprotease appears to be localized predominantly on the cytolysosomes of both trophocytes and bacteriocytes. When probed with an anti-proprotease antiserum, bacteria are also positively labeled, regardless of whether they are segregated inside the cytolysosomes or free in the bacteriocyte cytoplasm. Since vitellogenin and proprotease colocalize within the same cell organelle, it is assumed that Golgi-derived vesicles, which contain vitellogenin, may fuse with cytolysosomes bearing proprotease to yield compound secretory granules. To account for the present observations, the origin and role of proprotease are discussed in relation to the turnover of bacteria in the fat body and to the requirements of endosymbiosis.

Localization of the proenzyme form of the vitellin-processing protease in Blattella germanica by affinity-purified antibodies.

During Blattella germanica embryo development, the nutritive yolk protein vitellin is processed by a cysteine protease, which is activated proteolytically from a proprotease during acidification of yolk granules. A murine polyclonal antiserum was generated with the purified proprotease as the immunogen. The antiserum was made monospecific to proprotease by subtractive affinity chromatography using proprotease-free yolk proteins as ligand. The purified antibodies were employed to investigate the temporal and spatial expression of the proprotease during vitellogenesis and embryo development. Anti-proprotease-reactive peptides appeared in extracts of fat bodies and ovarian follicles of post-mating females, but not in fat bodies of males or the fat bodies or follicles of unmated females, suggesting that the proprotease is synthesized extraovarially. Use of the antibodies was extended to monitor the kinetics of proprotease disappearance during early embryo development.

The vitellin-processing protease of Blattella germanica is derived from a pro-protease of maternal origin.

Proteolytic processing of vitellin in Blattella germanica embryos is accomplished by activation of a yolk-borne cysteine protease (Mr 29 000) derived from a pro-protease precursor of Mr 40 000 (Liu et al., 1997). In the present study, fat body, ovaries and embryos of different developmental stages were examined immuno-cytochemically with purified murine anti-proprotease antibodies (Liu, 1995) to determine the intracellular location of the pro-protease. Proenzyme was detected in discrete secretory granules of the fat body and in large lysosome-like vesicles of both the follicle cell cytoplasm and the cortical ooplasm of previtellogenic ovarian follicles. In vitellogenic oocytes, coated pits and vesicles are scantily labelled for proprotease and no clear gold pattern could be discerned over the yolk granules. During embryonic development, pro-protease is associated with some, but not all, yolk granules. In newlyovulated eggs (day 0), pro-protease is either distributed over the entire granule or confined to some internal vesicles. As development proceeds, it becomes associated with almost every yolk granule and restricted to the superficial layer. By day 6, pro-protease is evident over all yolk granules but the intensity of reaction has greatly diminished, due probably to conversion of the pro-protease to the mature enzyme. Yolk granules are flanked along their margin by vesicles that are stained after zinc-osmium fixation. This observation suggests that the pro-protease may be transferred between yolk granules via vesicular shuttling. B. germanica embryos of different developmental stages were also exposed to [(3)H]-DAMP. Data show that autoradiographic grains are not evenly distributed among closely adjacent yolk granules within vitellophagic cells, a result consistent with the known slight temporal asynchrony of the acidification event.

A cysteine protease that processes insect vitellin. Purification and partial characterization of the enzyme and the proenzyme.

A cysteine protease that initiates degradation of vitellin (Vt) in the orthopteran Blattella germanica, and its proprotease precursor, were purified from yolk and partially characterized. The protease, purified 300-fold, contains three peptides of Mr 27,000, 29,000, and 31,000. A comparison of the purified enzyme's action pattern on Vt in vivo and in vitro confirmed its role in Vt processing. Protease-deficient yolk (day 0 postovulation) contained peptides of Mr 35,500, 37,000, 39,000, and 41,000, which were absent from yolk with protease activity. These were replaced by three peptides of approximately Mr 29,000, at days 2-3, the same time in development that protease expression and acidification of yolk granules occur (Nordin, J. H., Beaudoin, E. L., and Liu, X. (1991) Arch. Insect Biochem. Physiol. 18, 177-192). Acidification of purified proprotease converted it to three peptides of approximately Mr 29, 000 with cysteine protease activity. This conversion also required participation of a cysteine protease. Activated proprotease had the same pH activity profile, susceptibility to inhibitors, and cathepsin classification (L) as the protease. These results indicate that the Vt-processing protease is derived from a proprotease, which is activated in vivo by a developmentally regulated decrease in intragranular pH.

Processing of pro-vitellogenin in insect fat body: a role for high-mannose oligosaccharide.

Several discrete events were resolved in the processing of vitellogenin in Blattella germanica. Using tunicamycin to inhibit the synthesis of high-mannose oligosaccharide, a high molecular weight pro-vitellogenin peptide (apo-proVG, Mr 215,000) was identified in fat body. Dosages of tunicamycin which inhibited glycosylation of vitellogenin by 98% inhibited its synthesis by as much as 59%, yet led to an intracellular accumulation of apo-proVG. Reversibility and dose dependency of these effects on vitellogenin synthesis, glycosylation, proteolytic processing, and secretion were demonstrated. In control insects, glycosylation of apo-proVG yielded a Mr 240,000 pro-vitellogenin peptide (proVG). FITC-Concanavalin A bound to purified proVG but not to apo-proVG, thus confirming an absence of high-mannose oligosaccharide in the apo-protein. Following its glycosylation, proVG was processed rapidly in fat body to Mr 160,000 (VG160) and Mr 102,000 (VG102) peptides which subsequently were secreted into hemolymph. After uptake into developing oocytes, the VG160 peptide was processed further prior to chorionation, yielding subunits of Mr 95,000 and 50,000. Uniqueness of the peptides of mature vitellin (Mr 102,000, 95,000, and 50,000) was indicated by comparison of the CNBr fragments of each purified subunit. Staining of CNBr fragments with FITC-Concanavalin A also indicated that high-mannose oligosaccharides are attached at one or more sites within each vitellin subunit. Resolution of the substructure of this insect vitellin and identification of events involved in the processing and secretion of its fat body apo-protein provide a basis for further study of the assembly and transport of vitellogenin, its packaging in eggs, and utilization during embryogenesis.

Further studies on the surface saccharides in Trichomonas vaginalis strains by fluorescein-conjugated lectins.

Fluorescence emitted by individual cells of several Trichomonas vaginalis strains, nearly all of which were cloned, incubated with fluorescein-conjugated lectins in the absence (experimental) or presence (control) of inhibitory sugars, or else in phosphate-buffered saline alone (autofluorescence) was measured with a Leitz MPV Compact microfluorometer. Irrespective of whether the organisms were postfixed in formalin or glutaraldehyde, the relative fluorescence emitted by the cells was closely comparable, provided that appropriate neutral density filters were employed. However, autofluorescence was much higher for glutaraldehyde-fixed trichomonads. Therefore, although better preserved and more amenable to subsequent manipulations, such organisms were found unsuitable for use in "qualitative" titration of the fluorescence emitted by various strains. Provided that the necessary precautions were taken, comparable fluorescence readings were obtained with trichomonads affixed to glass slides by heat (41 degrees C, on a section spreader) or by a cytologic centrifuge (Cytospin 2). Large numbers of concanavalin A (Con A)-binding sites were present on organisms of all strains, irrespective of their virulence for human patients and as estimated by the subcutaneous mouse assay; these sites were shown with the aid of D-mannose to be mannose or mannose-related residues. More binding sites for soybean agglutinin (SBA) were found on the virulent than on avirulent strains. On the basis of inhibition experiments, the sugar residues mainly responsible for these differences appeared to be D-lactose residues. Similar differences were observed with Ricinus communis agglutinin Type I (RCA I), for which D-galactose was employed as the competing sugar. However, with two cloned strains the situation with regard to RCA I binding was reversed - more of the lectin bound to a mild than to a virulent strain. The results obtained with Ricinus communis Type II agglutinin (RCA II) were often similar to those noted for RCA I; however, in most instances the inhibition with N-acetyl-D-galactosamine (GalNAc) was lower. Furthermore, the results noted with the GalNAc-specific agglutinins from Dolichus biflorus and Helix pomatia suggested that only very few GalNAc residues were available for binding on the surfaces of all T. vaginalis strains examined in the course of this study. Statistical analyses of fluorescence emitted by four clones of each, Balt 42 (virulent) and JH31A (avirulent) T. vaginalis strain upon incubation with Con A and SBA revealed homogeneity of these strains with regard to the number of the specific surface saccharide residues, D-mannose and D-lactose.(ABSTRACT TRUNCATED AT 400 WORDS)

A comparative study of the size-heterogeneous high mannose oligosaccharides of some insect vitellins.

Comparative studies of the carbohydrate component from vitellins of the cockroaches Blattella germanica, Blaberus discoidalis, Periplaneta americana and Simploce capitata and the locust Locusta migratoria have been conducted. Chemical, enzymatic and chromatographic analyses show that each vitellin contains variably processed high mannose type oligosaccharides. While all have a common size range they occur as two distinct classes based on the proportion of individual saccharides present. Oligosaccharide size distribution is not a characteristic of an individual animal but of the species. Because oligosaccharide heterogeneity also occurs in B. germanica vitellogenin (the hemolymph precursor of vitellin), it does not result from structural changes during or after its uptake by the egg.

Production and composition of an exocellular Nigeran-protein complex isolated from cultures of Aspergillus awamori.

Aspergillus awamori Nakazawa (QM873) hyphae maintained on a nitrogen-deficient medium produced an exocellular complex consisting of the wall alpha-glucan, nigeran (94%), water-soluble nigeran oligosacchrides (1 to 2%), protein (2 to 4%), and a small amount of beta-glycan (less than 1%). Nigeran was not covalently linked to protein. The complex appeared in the growth medium only under those temporal or nutritional conditions where the hyphal wall content to nigeran reached at least 30% of the cell dry weight. Rates of nigeran accumulation in the hypha cell wall, scanning electron microscopy of hyphae, and pulse-chase experiments with [14C]glucose suggested that the complex arises via displacement of a portion of the hyphal wall into the medium. The nigeran portion of the complex contained lamellar crystalline domains similar to those in the intact cell wall. Enzymic digestion of nigeran in the complex indicated that it has a degree of crystallinity lower than that observed with pure nigeran crystals grown in vitro. Therefore, polymerization in vivo resulted in somewhat less chain organization in the crystallite. Since this complex was isolated without any prior chemical or exogenous enzymatic treatment, it should be useful for studies of hyphal wall biogenesis and organization in this organism.

Production by Aspergillus awamori of homologous oligosaccharides, each with an uneven number of glucosyl units and a reducing terminal (alpha , 1 leads to 4) linkage.

Homologous oligosaccharides with degrees of polymerization of 7 to 15 have been isolated from the nigeran-protein complex of Aspergillus awamori Nakazawa (QM873). Each contains an uneven number of glucose units, differs from its nearest homolog by two residues, and contains the sequence Glc(alpha , 1 leads to 3)Glc- (alpha , 1 leads to 4)Glc at its reducing terminus.

Structural and Immunochemical Studies on the Phytotoxic Peptidorhamnomannan of Ceratocystis ulmi.

A wilt-inducing peptidorhamnomannan produced by Ceratocystis ulmi, the causative agent in Dutch Elm disease, has been subjected to additional chemical and physical characterization. Gel filtration, reductive beta elimination, hydrofluoric acid deglycosylation, and ultracentrifugation experiments provide evidence that the wilt-inducing polymer is polydisperse with a molecular weight range of approximately 105,000 to 120,000. The carbohydrate portion of each molecule is composed of small percentages of mannose, mannobiose, mannotriose, and a tetra- or pentasaccharide composed of mannose and rhamnose plus a major component consisting of two or three long rhamnomannan chains each with a molecular weight range of 32,000 to 34,000. All saccharide units are attached via O-glycosidic linkages to a protein with a molecular weight of approximately 35,000.Rabbit antibodies directed against both C. ulmi and the purified peptidorhamnomannan have been prepared. Their possible use in evaluating the role of the polymer in the disease is discussed.

Distribution and conformation of crystalline nigeran in hyphal walls of Aspergillus niger and Aspergillus awamori.

Hyphal walls of Aspergillus awamori containing increased amount of the alpha-glucan, nigeran, became correspondingly more opaque when viewed in the electron microscope as shadowed preparations. However, increased polymer deposition was not accompanied by any significant change in wall thickness. The nigeran of both A. awamori and Aspergillus niger occurred in situ in a crystalline conformation identical to that of single crystals prepared with pure polysaccharide. Furthermore, this polymer was the dominant crystalline material in the hyphae whether or not they were enriched in nigeran. Enzymic digestion of nigeran in A. niger and A. awamori revealed that the bulk of the polymer was exposed to the cell's exterior. However, a certain fraction was accessible to enzymic attack only after the wall was treated with boiling water. A third portion, detectable only by x-ray diffraction, was associated with other components and could not be extracted, even with prolonged boiling. It was removed by hot, dilute alkali and was associated in the wall with another glucan fraction. Dry heating of A. niger walls altered their susceptibility to enzymic digestion of nigeran in situ. It is proposed that this treatment introduces interstices in the crystal surface that facilitate attack.

Chemical structure of the galactomannan from the cell wall of Aspergillus niger.

The galactomannan of surface grown Aspergillus niger has been isolated by alkaline extraction of hyphal walls and characterized structurally. Its elution profile, from a column of Bio-Gel P-150, reveals a broad range of molecular sizes grouped into two fractions. Gas chromatographic and colorimetric analyses indicate that each fraction is composed of approximately equimolar quantities of galactose and mannose plus 12 to 14% glucose. Both have similar low optical rotations and contain acid labile galactose. Methylation, Smith degradation, acetolysis, reactivity with concanavalin A and beta-D-galactofuranosidase, plus digestionof galactose with D-galactose oxidase, were techniques employed to determine the polysaccharide's covalent structure. Results of these studies indicate that it is composed of a series of chains, 5 to 9 hexose units in length, connected by alpha1 leads to 6 bonds between mannopyranosyl moieties. The external portion of each chain consists of a galactose tri- or tetrasaccharide of the general structure Galf beta1 leads to 4 Galp(1-2) 1 leads to 4 Galp1 leads to. This segment is connected via a (1 leads to 2) linkage to the internal portion which is a di- to pentasaccharide of mannopyranosyl units joined in alpha1 leads to 2 glycoside linkage. Combination mild acid hydrolysis and methylation experiments indicate galactofuranosyl terminal units are attached only to galactose. The organization of glucose into the overall structure of the polymer has not been determined. Structural relationships of this polysaccharide to both fungal and yeast galactomannans are discussed.

Galactosaminogalactan from cell walls of Aspergillus niger.

A new heteropolysaccharide has been isolated by alkaline extraction of hyphal walls of Aspergillus niger NRRL 326 grown in surface culture. Its composition by weight, as determined by paper and gas chromatography and colorimetric analyses, is 70% galactose, 20% galactosamine, 6% glucose, and 1% acetyl. Two independent experiments have been used to ascertain copolymer structure: permeation chromatography in 6 M guanidinium hydrochloride, with controlled-pore glass columns of two fractionation ranges, and nitrous acid deaminative cleavage of galactosaminogalactan followed by reduction of fragments with [3H]borohydride and gel filtration chromatography. One of the tritiated fragments is tentatively identified as the disaccharide derivative galactopyranosyl 2,5-anhydrotalitol, on the basis of chromatographic properties and by kinetics of its acid hydrolysis. Smith degradation, methylation, deamination, and optical rotation studies indicate that the galactosaminogalactan consists of a linear array of hexopyranosyl units joined almost exclusively by alpha-(1 leads to 4) linkages. Hexosaminyl moieties are distributed randomly along the chains, which have an average degree of polymerization of about 100. The possible significance of this macromolecule in hyphal structure is considered.

Enzymes that hydrolyze fungal cell wall polysaccharides. The carbonhydrate constitution of mycodextranse, an endo-alpha (1 yields 4)-D-glucanase from Pencillium melinii.

The chemical constitution of the carbohydrate portion of mycodextranase, an exocellular endo-alpha(1 yields 4) D-glucanase of Penicillium melinii, has been investigated. At least 80% of the carbohydrate, consisting exclusively of mannose and glucose, is released from protein by treatment of the enzyme with 0.05 M potassium hydroxide plus 1 M sodium borohydride or 0.5 M sodium hydroxide at 50 degrees. There is concomitant destruction of 60% of the threonine and 15% of the serine of the treated enzyme and an increase in absorption, at 241 nm, of the treated protein's spectrum, indicative of an O-glycosidic beta-hydroxyamino acyl linkage between untreated protein and its associated carbohydrate. Mannose is the monosaccharide involved in this linkage. Smith degradation, methylation, and glycosidase digestions of the carbohydrate indicate that it is present in mycodextranase as side chains of mannose, glucosyl alpha(1 yields 2)-mannose, and mannosyl alpha(1 yields 2)-glucosyl alpha(1 yields 2)-mannose units with each enzyme molecule bearing a calculated average of 25 side chains. Separation of pronase glycopeptides by gel filtration on Sephadex G-25 revealed that 96% of the carbohydrate is present in the highest molecular weight fraction which contains 60% of the threonine of mycodextranase but only 3.5% of the aromatic acids judged by its absorbance at 280 nm. Further fractionation of this glycopeptide component on Sephadex G-75 indicates carbohydrate is restricted to two fractions, one containing 71% by weight of the threonine and serine of mycodextranase and 56% of its carbohydrate. These results suggest carbohydrate chains of mycodextranase are clustered in a few threonin-rich regions along the polypeptide chain rather than being separated from each other by nonglycosylated areas.