John H. Nordin

Cold-induced glycerol accumulation by Ostrinia nubilalis larvae is developmentally regulated

Abstract Ostrinia nubilalis larvae reared under both nondiapause and diapause-inducing conditions were chilled at 5°C for various periods and their haemolymph glycerol concentrations were measured enzymatically. The ability of fifth (final) instars to accumulate glycerol was dependent upon cold stress but not the diapause state. Furthermore this response was independent of any cold-induced release of cephalic or thoracic hormones. The capacity of O. nubilalis larvae to express cold-induced glycerol accumulation was found to require ecdysis from the fourth to fifth instar. Eggs as well as second, third and fourth instars were completely incompetent. These results indicate that, at the biochemical level, a specific developmental programme or sequence is required for O. nubilalis to demonstrate this response to cold stress.

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.

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 enzymes 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.

Studies on the low temperature induced biogenesis of glycerol by adult Protophormia terranovae

Physiological and biochemical changes accompanying cold stress in the diapausing adult arctic blowfly, Protophormia terranovae, have been observed. In the laboratory, this insect survives prolonged periods at temperatures in the range of −1°C to +4°C. Concentrations of free glycerol in excess of 10% of fresh body weight have been measured and the rate of its synthesis is greater at +1°C to +4°C than at −1°C to 0°C. Under these conditions Protophormia also undergoes significant weight loss (up to 58% over 39 days) presumably in part due to dehydration. Its respiration rate decreases as expected when first shifted from 20°C to 0°C but the rate declines an additional 70% after exposure to 0°C for 24 hr. This lowest rate, which is then maintained, when considered with the initial faster one suggests positive thermal modulation is coupled to inverse thermal compensation during cold stress. This was not observed with nondiapausing Protophormia. Increments in free glycerol are accompanied by decreases in the insects total glycogen reserves but upon rewarming, they return to pre-cold stress levels. While pre-stress glycogen stores are insufficient to provide for most of the free glycerol which accumulates, ingested carbohydrate present in the crop provides sufficient quantities. Studies with [14C] glucose indicate it is also metabolically active at low temperature. Neutral glyceride glycerol cannot contribute to net synthesis of free glycerol in significant amounts since the steady state concentrations present in pre-cold stressed insects decrease only slightly during cold stress. Furthermore, the specific radioactivity of acyl glyceride glycerol labelled in vivo with 2-[3H] glycerol before cold stress, remains unchanged during hibernation indicating that acyl glycerides are not turning over glycerol units produced by catabolism of hexose. The results of these studies argue that carbohydrate and not lipid glycerol is the source of the free glycerol which accumulates in Protophormia at low temperatures. The relationship of the above results to possible mechanisms which should permit glycerol accumulation under aerobic conditions are discussed.

Subunit composition of vitellin, and concentration profiles of vitellogenin, and vitellin in Phormia regina (Meig.) following a protein meal

1. 1. The vitellin (Vt) of Phormia regina eggs was isolated by extraction at low ionic strength and purified by TEAE-cellulose chromatography. SDS polyacrylamide gel electrophoresis and staining with horseradish peroxidase-concanavalin A showed that the Vt contains four subunits of Mr 42,000, 43,000, 44,000 and 45,500, all of which are glycosylated. 2. 2. Murine polyclonal antibodies to the Vt, employed in conjunction with radioimmunoblotting, recognized all four subunits, and revealed that Vt and/or vitellogenin (Vg) are present exclusively in the hemolymph, fat bodies and ovaries of liver-fed females but not those fed sugar. 3. 3. Using these antibodies in an ELISA, concentrations of Vg in the hemolymph and Vt in the ovaries were determined periodically following liver feeding. At 28°C, the hemolyph Vg titer reaches a maximum 28 hr post-feeding and then decreases rapidly as ovaries begin to sequester Vt, whose concentration in that organ peaks about 48 hr post-feeding.

Studies on Ligand Recognition by Vitellogenin Receptors in Follicle Membrane Preparations of the German-Cockroach, Blattella-Germanica

Abstract An in vitro binding assay was used to analyze the interaction between vitellin and its receptor in follicle membrane preparations of the cockroach, Blattella germanica . Binding was found to be optimal at pH 6.8. Specific binding was saturable, and Scatchard analysis yielded a K d -value of 79 nM and a total receptor concentration of 51 fmol/follicle. Glycopeptides and oligosaccharides prepared from vitellin competed for vitellin binding, whereas yeast mannan did not. Glycopeptides digested with α-mannosidase stimulated vitellin binding. Vitellins of the cockroach species Simploce capitata and Nauphoeta cinerea competed for binding of B. germanica vitellin, whereas vitellin of another cockroach, Leucophaea maderae , stimulated binding. Analysis of the oligosaccharide structure of the vitellin in these species revealed differences in the proportions of individual oligosaccharides in the total carbohydrate, however, these differences do not correlate with the ability of the vitellins to compete for binding. The results suggest that the high mannose oligosaccharides of vitellin are necessary but not sufficient for binding to the vitellogenin receptor.

Activation of the hexose monophosphate shunt during cold-induced glycerol accumulation by Protophormia terranovae

Abstract To evaluate the relative contributions made by glycolysis and the hexose monophosphate shunt during cold stress, glucose catabolism by adult Protophormia terranovae (which accumulate glycerol) and Musca domestica (which do not) was examined at 22°C and 2°C. Following injection of [1- 14 C]-glucose, [6- 14 C]-glucose and [3,4- 14 C]-glucose, rates of 14 CO 2 evolution were determined. In addition, the incorporation of label from both [1- 14 C]-glucose and [6- 14 C]-glucose into free glycerol and lipids was monitored. Respirometric studies show that both species increase their rates of glycolysis relative to the TCA-cycle at the lower temperature with P. terranovae exhibiting a slightly greater increment. The rate of the hexose monophosphate shunt relative to the TCA-cycle remains unchanged when M. domestica are shifted from 22°C to 2°C. However, in P. terranovae the rate of this pathway relative to the TCA cycle is doubled by the same temperature shift. The labelling patterns in free glycerol and lipids of P. terranovae support the respirometric data, indicating a significantly increased role for the hexose monophosphate shunt in this species during cold stress. The possible significance of this observation in relation to glycerol accumulation by P. terranovae is discussed.

Concanavalin A reactivity and carbohydrate structure of Blattella germanica vitellin.

Abstract The carbohydrate moiety of Blattella germanica vitellin has been investigated. A glycopeptide fraction obtained by protease digestion and gel filtration has a Man: GlcNAc ratio of approximately five and is enriched in aspargine and phosphate. Further structural characterization indicates only a single oligosaccharide species is present. It contains ten to eleven mannosyl units and is linked through a chitobiosyl-asparagine linkage to peptide. There are approximately twelve to sixteen of these oligosaccharide chains per mole of vitellin and they are located in the 100,000 mol. wt subunit. A mixture of glycopeptides derived from radiolabelled vitellogenin and unlabelled vitellin was digested with β N- acetyl glycosaminidase H and the oligosaccharides submitted to gel filtration. Coincident elution of radioactivity and carbohydrate suggests that no change occurs in the size of the oligosaccharide between the time the glycoprotein is secreted from the fat body and one day after its uptake by the oocyte. Experiments with Concanavalin A, anti Blattella antibodies and a number of ligands indicate (a) multiple lectin binding sites occur on vitellin, (b) a portion (but possibly not all) of the oligosaccharide chains of vitellin are located on the exterior of the vitellin molecule and (c) the oligosaccharide is not a determinant group of vitellin.

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.

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.