The pupal cuticle of Drosophila: biphasic synthesis of pupal cuticle proteins in vivo and in vitro in response to 20-hydroxyecdysone

We investigated the synthesis and localization of Drosophila pupal cuticle proteins by immunochemical techniques using both a complex antiserum and monoclonal antibodies. A set of low molecular weight (15,000-25,000) pupal cuticle proteins are synthesized by the imaginal disk epithelium before pupation. After pupation, synthesis of the low molecular weight proteins ceases and a set of unrelated high molecular weight proteins (40,000-82,000) are synthesized and incorporated into the pupal cuticle. Ultrastructural changes in the cuticle deposited before and after pupation correlate with the switch in cuticle protein synthesis. A similar biphasic accumulation of low and high molecular weight pupal cuticle proteins is also seen in imaginal discs cultured in vitro. The low molecular weight pupal cuticle proteins accumulate in response to a pulse of the insect steroid hormone 20-hydroxyecdysone and begin to appear 6 h after the withdrawal of the hormone from the culture medium. The high molecular weight pupal cuticle proteins accumulate later in culture; a second pulse of hormone appears to be necessary for the accumulation of two of these proteins.     

The cuticular proteins of Tenebrio molitor: I. Electrophoretic banding patterns during postembryonic development

Buffer-soluble cuticular proteins of the abdomen of the yellow mealworm, Tenebrio molitor, were analyzed by SDS-polyacrylamide gel electrophoresis. Since the abdominal epidermis of Tenebrio persists throughout the insect's life, these cuticular proteins reflect the secretory history of a continuous line of cells during its entire metamorphic developmental program. Twenty-two to thirty-eight bands were detected in extracts of larval cuticle, 11 to 35 in pupal cuticular extracts, and 30 to 41 in extracts from adults. No population polymorphism was apparent, nor was there any sexual dimorphism, in these cuticular proteins. At each metamorphic stage, the cuticular proteins formed a unique banding pattern. Bands unique to the larval and to the adult exocuticular extracts were observed. Extracts from cuticles of freshly ecdysed animals (exocuticle) differed from extracts from animals in which sclerotization and postecdysial (endocuticle) deposition had occurred, both in number of hands and in their molecular weight distributions. Some proteins became less soluble during sclerotization. The majority of the exocuticular bands from all three stages had molecular weights below 25,000; higher-molecular-weight proteins were extracted from postecdysial animals of each stage.     

Drosophila salivary gland proteins and pupation

Pulse-labeling experiments of salivary glands from the prepupal stages of development showed selectively high rates of synthesis of a set of low molecular weight proteins (6K–12K). These proteins are stably maintained in the salivary glands during prepupal development and are subsequently transported to the pupation fluid (found between the pupal case and the prepupal cuticle) when pupation occurs. These small polypeptides are very basic with the major components having isoelectric points of 8.6–8.7 and the minor components having isoelectric points of 9.1–9.5. This study shows the continuing function of the salivary glands—specifically, the synthesis and secretion of a set of proteins with a putative role in pupation.     

CPF and CPFL, two related gene families encoding cuticular proteins of Anopheles gambiae and other insects

Cuticular proteins (CPs) are structural proteins of insects as well as other arthropods. Several CP families have been described, among them a small family defined by a 51 amino acid motif [Andersen, S.O., Rafn, K., Roepstorff, P., 1997. Sequence studies of proteins from larval and pupal cuticle of the yellow meal worm, Tenebrio molitor. Insect Biochem. Mol. Biol. 27, 121-131]. We identified four proteins of this family in Anopheles gambiae that we have named CPF. We have also identified CPFs from other insects by searching databases. Alignment of these CPF proteins showed that the conserved region is only 44 aa long and revealed another conserved motif at the C-terminus. A dendrogram divided the CPF proteins into four groups, one basal and three specialized. We also identified several proteins of another CP family, CPFL, which has similarities to CPFs. CPFs and CPFLs share some protein motifs. Expression studies with real-time qRT-PCR of the A. gambiae CPFs and CPFLs showed that the four CPFs and one CPFL gene are expressed just before pupal or adult ecdysis, suggesting that they are components of the outer layer of pupal and adult cuticles. The other CPFLs appear to contribute to larval cuticle. Recombinant CPF proteins did not bind to chitin in the assay we used.     

Temporal programming of epidermal cell protein synthesis during the larval-pupal transformation ofManduca sexta

Summary During the final larval instar the epidermis of the tobacco hornworm,Manduca sexta, synthesizes the larval cuticular proteins and the pigment insecticyanin. Then at the onset of metamorphosis the cells first become pupally-committed, then later produce the pupal cuticle. The changes in the pattern of epidermal protein synthesis during this period were followed by incubating the integument in vitro with either³H-leucine or³⁵S-methionine, then analyzing the proteins by 2-dimensional gel electrophoresis. Precipitation by larval and pupal cuticular antisera and by insecticyanin antibody identified these proteins. Three distinct changes in epidermal protein synthesis were noted: 1) Stage-specific proteins, some of which are larval cuticular proteins, appear just before and during the change of commitment on day 3. (2) By late the following day (wandering stage), synthesis of these and many other proteins including all the identified larval cuticular proteins and insecticyanin was undetectable. Several noncuticular proteins were transiently synthesized by this pupally committed cell during wandering and sometimes the following day. (3) During the production of pupal cuticle a new set of pupal-specific cuticular proteins as well as some common cuticular proteins (precipitated by both antisera) were synthesized. Some of the latter were also synthesized during the period between pupal commitment and pupal cuticle deposition.In spite of an apparent absence of methionine in both larval and pupal cuticle, many cuticular proteins incorporated³⁵S-methionine. Thus they may be synthesized as proproteins.Insecticyanin was shown to have two forms differing in isoelectric point, the cellular form being more acidic than the hemolymph form. Synthesis of the cellular form ceased before that of the hemolymph form.     

Spatial and temporal variations in cuticle proteins as revealed by monoclonal antibodies. Immunoblotting analysis and ultrastructural immunolocalization in a beetle, Tenebrio molitor

A library of monoclonal antibodies (Mabs) against adult cuticle of Tenebrio was used to visualize the secretion of cuticular antigens during metamorphosis. Immunoblots of water- and urea-soluble proteins, and high resolution immunogold labelling has shown that, except in one clone, the Mabs recognize antigens in the three developmental stages. However, the MW of larval and pupal antigens are different from the adult ones, though sharing common epitopes. Blots of cuticle proteins (CPs) bound to different lectins shown few water-soluble glycosylated proteins weakly or not recognized by the Mabs, suggesting that the majority of the Mabs do not recognize glycosylated epitopes. The immunolocalization of the different antigens suggests a molecular basis for both developmental and regional variations in cuticular architecture and to the modifications due to sclerotization, which differ between pre- and postecdysial cuticles of the three developmental stages.     

Shotgun proteomic analysis of wing discs from the domesticated silkworm (Bombyx mori) during metamorphosis

Proteomic profiles from the wing discs of silkworms at the larval, pupal, and adult moth stages were determined using shotgun proteomics and MS sequencing. We identified 241, 218, and 223 proteins from the larval, pupal, and adult moth stages, respectively, of which 139 were shared by all three stages. In addition, there were 55, 37, and 43 specific proteins identified at the larval, pupal, and adult moth stages, respectively. More metabolic enzymes were identified among the specific proteins expressed in the wing disc of larvae compared with pupae and moths. The identification of FKBP45 and the chitinase-like protein EN03 as two proteins solely expressed at the larval stage indicate these two proteins may be involved in the immunological functions of larvae. The myosin heavy chain was identified in the pupal wing disc, suggesting its involvement in the formation of wing muscle. Some proteins, such as proteasome alpha 3 subunits and ribosomal proteins, specifically identified from the moth stage may be involved in the degradation of old cuticle proteins and new cuticle protein synthesis. Gene ontology analysis of proteins specific to each of these three stages enabled their association with cellular component, molecular function, and biological process categories. The analysis of similarities and differences in these identified proteins will greatly further our understanding of wing disc development in silkworm and other insects.     

Synthesis of the major adult cuticle proteins of Drosophila melanogaster during hypoderm differentiation

Differentiating imaginal hypodermal cells of Drosophila melanogaster form adult cuticle during the second half of the pupal stage (about 40 to 93 hr postpupariation). A group of proteins with molecular weights of 23,000, 20,000, and 14,000 is identified as putative major wing cuticle proteins with the following biological properties: These proteins are abundant components of cuticle and are major synthetic products of cuticle-secreting hypodermal cells. They are leucine-rich and methionine-free and are the most prominent proteins of this type synthesized by wing hypoderm at 65 hr, during the period of procuticle formation. Electron microscopic autoradiography shows that leucine-rich, methionine-free proteins specifically localize to the apical cell surface and newly secreted cuticle of 65-hr wing cells. This strongly suggests the export of these proteins to the cuticle. Lastly, these proteins undergo a reduction in extractability just after eclosion, during the period of cuticle protein crosslinking (sclerotization). The synthesis of these major hypoderm proteins is temporally regulated in development. In wing cells, the 14-kDa proteins are synthesized first, from 53 to 78 hr, and the 20- and 23-kDa proteins are synthesized from 63 to 93 hr. The pattern of synthesis for these proteins is similar in abdominal cells but delayed by 6 to 10 hr. Two-dimensional gel electrophoresis shows that each of the 23-, 20-, and 14-kDa size classes contains at least two component polypeptides. Patterns of protein synthesis in cells of the imaginal hypodermis are regulated in a precise temporal sequence during the production of adult cuticle. Their study yields a useful system for the analysis of molecular events in gene control and cell differentiation.     

Degradation of cuticle during larval-pupal and pupal-adult development of the housefly, Musca domestica

Abstract. The patterns of changes in cuticle weight, its chitin content and chitinase activity have been studied during postembryonic development of the housefly, Musca domestica L. During pupariation the larval cuticle loses weight. During the early part of this weight-loss the decline in chitin content parallels the overall change in cuticle weight. A simultaneous elevation in chitinase activity suggests that at this time the larval cuticle is being enzymatically degraded. Later weight loss may be due to sclerotization. No significant changes in cuticle weight or its chitin content occur in pharate cuticle until one day before eclosion. However, a peak of chitinase activity found at mid-late pupal stage suggests the timing of pupal cuticle breakdown.     

Heterogeneity of immunoreactive prolactin in the rat brain

Three immunoreactive prolactin proteins (24 Kd, 16 Kd, and 12 Kd) were identified in the rat brain using sodium dodecyl sulphate polyacrylamide gel electrophoresis, and western blot analyses. In male and female brains, the primary prolactin protein has a molecular weight of 24 Kd which is similar to that of pituitary prolactin. Two additional proteins with apparent molecular weights of 16 Kd and 12 Kd were also identified and were found in greater concentrations in the brain than in the pituitary, and were more predominant in the female brain. In addition, brain extracts proteolytically modify the 24K dalton PRL resulting in the formation of two fragments with apparent molecular weights of 16 and 8 Kd. These data indicate that the prolactin identified in the rat brain is similar to pituitary prolactin, and suggests, that like other PRL target tissues the brain may have the capacity to proteolytically modify prolactin.     

Purification,properties, and titer of a hemolymph trophic factor in larvae and pupae of Manduca sexta

Purification of a hemolymph protein (hemolymph trophic factor, or HTF) from last instar larvae of Manduca sexta was achieved using Sephadex G15-120 gel filtration and DEAE anion exchange chromatography. Homogeneity was visualized using SDS gel electrophoresis and ampholytic chromatofocusing. HTF was estimated to be a tetrameric protein with a molecular weight of 286 K and a Stokes' radius of 55.3 × 10⁻⁸ cm by agarose bead gel filtration; chromatofocusing suggests an isoionic point > 10. Polyclonal antibodies to HTF were prepared in rabbits and an ELISA was developed. The ELISA was used to titer HTF during the last larval instar and day 1 and 14 of the pupal stage and estimates a maximum of 1.5 mg/ml larval hemolymph on day 6 with a smaller larval peak of 0.75 mg/ml at day 3 and titers of 0.70 and 0.35 mg/ml on the 2 pupal days, respectively. ELISA of aqueous extracts of larval fat body, epidermis, and cuticle demonstrate that HTF comprises nearly a third of the soluble fat body protein and is a lesser component of epidermis and cuticle. The physiological role of HTF has not yet been determined.     

The cuticle proteins of Drosophila melanogaster: stage specificity

Five stage-specific cuticles are produced during the development of Drosophila. Urea-soluble proteins were extracted from each developmental stage and compared by gel electrophoresis. Proteins from first and second instar cuticle are identical except for minor differences in two proteins. Each subsequent stage, third instar, pupa, and adult, has a unique set of cuticle proteins. Qualitative changes within stages are seen in proteins from third instar and adult cuticle. Third instar cuticle proteins can be divided into “early” [proteins 2a, 3, 4, 5, 7, and 8] and “late” [proteins 2 and 1] groups. Adult cuticle proteins change in relative amounts during pharate adult development and change mobility at eclosion. The lower abdominal pupal cuticle lacks a protein found in the pupal cuticle covering the head and thorax. Cuticle proteins from each stage are immunologically related. Nonetheless, electrophoretic variants of three larval proteins do not affect any major changes in the electrophoretic mobility of proteins from other stages. We propose that each stage (except first and second instar) has proteins encoded by discrete genes.     

Synthesis and processing of sphingolipid activator protein-2 (SAP-2) in cultured human fibroblasts

Sphingolipid activator proteins (SAP) are relatively small molecular weight proteins that stimulate the enzymatic hydrolysis of sphingolipids in the presence of specific lysosomal hydrolases. SAP-2 has previously been demonstrated to activate the hydrolysis of glucosylceramide, galactosylceramide, and, possibly, sphingomyelin. Using monospecific rabbit antibodies against human spleen SAP-2, the synthesis and processing of SAP-2 were studied in cultured human fibroblasts. When [35S]methionine was presented in the medium to control human cells for 4 h, five major areas of radiolabeling were found. These had apparent molecular weights of 73,000, 68,000, 50,000, 12,000, and 9,000. Further studies indicated that the major extracellular product in normal cells given NH4Cl along with the [35S]methionine and in medium from cultures from patients with I cell disease had an apparent molecular weight of 73,000. The Mr = 68,000 and 73,000 species can be converted to a species with an apparent molecular weight of 50,000 by the action of endoglycosidase F. After labeling cells for 1 h followed by a 1-h chase, the Mr = 12,000 and 9,000 species appear. Treatment of the immunoprecipitated mixture with endoglycosidase F resulted in conversion of these species to one band with an apparent molecular weight of 7,600. These studies indicate that this relatively low molecular weight protein is rapidly synthesized from a relatively large molecular weight highly glycosylated precursor.     

Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry

Identification of authenticated cuticular proteins has been based on isolation and sequencing of individual proteins extracted from cleaned cuticles. These data facilitated classification of sequences from conceptual translation of cDNA or genomic sequences. The question arises whether such putative cuticular proteins actually are incorporated into the cuticle. This paper describes the profiling of cuticular proteins from Anopheles gambiae starting with cuticle cleaned by the insect itself in the course of molting. Proteins extracted from cast larval head capsules and cast pupal cuticles were fractionated by 1D SDS gel electrophoresis. Large gel slices were reduced, carbamidomethylated and digested with trypsin. The pellet remaining after SDS extraction was also treated with trypsin. The resulting peptides were separated on a C18 column and then analyzed by tandem mass spectrometry. Two-hundred-ninety-five peptides from putative cuticular proteins were identified; these corresponded to a minimum of 69 and a maximum of 119 different proteins. Each is reported as an authentic Anopheles cuticular protein for the first time. In addition to members of two known cuticular protein families, members of additional families likely to be structural components of the cuticle were identified. Furthermore, other peptides were identified that can be attributed to molting fluid, muscle and sclerotizing agents.     

Composition of major outer membrane proteins of Vibrio vulnificus isolates: effect of different growth media and iron deficiency

The outer membrane proteins of Vibrio vulnificus including isolates from humans, seawater and an asari clam were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A major outer membrane protein with an apparent molecular weight of 48,000 (48K protein) was common to all the strains grown in 3% NaCl-nutrient broth; however this 48K protein was not produced in any of the strains grown in chemically defined medium. Other major outer membrane proteins with molecular weights ranging from 33,000 to 40,000 varied in number, relative amount and molecular weight depending on the strain. One to three new outer membrane proteins with molecular weights ranging from 74,000 to 85,000 were produced in the cells grown in iron-deficient medium. The 48K protein and one or two major proteins with molecular weights ranging from 35,000 to 37,000 in the cells grown in 3% NaCl-nutrient broth were not solubilized by 2% SDS at 60 C for 30 min and were resistant to trypsin, indicating that they are porins. On the other hand, in cells grown in chemically defined medium, one or two major outer membrane proteins with molecular weights ranging from 33,000 to 40,000 might be porins.     

Changes of protein tyrosine phosphorylation in third instar larval integument of the Mediterranean fruit fly, Ceratitis capitata

Developmental analysis of the tyrosine protein phosphorylation levels in larval integument and partial characterization of the endogenous protein tyrosine kinase activity (PTK) in Ceratitis capitata are described in this study. Larval integument contains high levels of PTK activity at the early stages of the third instar, which progressively declines to low levels in the white pupal stage. An integumental 90-kDa polypeptide was identified to have prominent endogenous PTK activity and follow a similar developmental pattern. The major integumental phosphotyrosine-containing polypeptides have apparent molecular weights of 30, 41, 44, 46, and 54 kDa, respectively. Polypeptides with molecular weights of 62 and 73 kDa were identified as Ser/Thr-containing phosphoproteins and were shown to exhibit high levels of phosphorylation at the middle stage of larval development. These differences are likely to be due to the higher activation state of the protein tyrosine kinase(s) at the early stages of larval development.     

Synthesis and glycosylation of polyprotein precursors to the internal core proteins of Friend murine leukemia virus

Synthesis and post-translational processing of murine leukemia virus proteins were analyzed in a murine cell line (Eveline) that produces large amounts of Friend lymphatic leukemia virus. Immunoprecipitation of l-[(35)S]methionine-labeled cell extracts demonstrated that several different virus-specific proteins antigenically related to the virion core (gag) proteins p12 and p30 become radioactive within 1 min of labeling and exhibit labeling kinetics characteristic of primary translation products. The most abundant of these were proteins with molecular weights of 75,000 and 65,000. There were, in addition, two large glycosylated polyproteins with apparent molecular weights of 220,000 and 230,000, which were precipitated by antisera to p30 or p12 but not by antiserum to the major envelope glycoproteins gp69/71. Several lines of evidence, including labeling with d-[(3)H]glucosamine and binding to insolubilized lectins, suggested that the 75,000-dalton internal core polyprotein is slowly processed to form a glycoprotein with an apparent molecular weight of 93,000. On the contrary, the 65,000-dalton protein appeared to be an immediate precursor to the virion core proteins. Its processing can involve intermediates containing p30 and p12 antigens with molecular weights of 50,000 and 40,000; however, the latter did not appear to be obligatory intermediates. The detection of the 40,000-dalton protein suggested that the genes for p30 and p12 are adjacent on the viral genome. These results indicated that there are several pathways of synthesis and post-translational processing of polyprotein precursors to the gag proteins and that several of these polyproteins are glycosylated. A comparison of gag precursor processing in rapidly growing, slowly growing, and stationary cells indicated that different pathways are favored under different conditions of cell growth. Our analysis of envelope glycoprotein synthesis has confirmed the existence of two rapidly labeled 90,000-dalton glycoproteins, which appear to be precursors to the envelope glycoproteins gp69/71.     

Structure and properties of human interferon-alpha from Namalwa lymphoblastoid cells.

The chromatographic properties of human interferon-alpha from Namalwa lymphoblastoid cells on Sephadex G-75 are described. The interferons are separated into two groups of four, with apparent molecular weights 19050 and 22000. Some of the latter form dimers at high concentrations. Fractions containing interferon were studied by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Seven of the components had apparent molecular weights in this system, after reduction, of between 18400 and 20900: one component is probably glycosylated and has an apparent molecular weight of 27500. Amino acid sequences of peptides derived from interferon mixtures were determined and are related to published sequences deduced from the nucleotide sequences of cloned complementary DNA coding for interferon-alpha. The results show that the major interferon-alpha species isolated from Namalwa cells do not undergo C-terminal processing. Amino acid analyses of peptides are presented in Supplementary Publication SUP 50117 (28 pages), which has been deposited with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1981) 193, 5.     

Distribution of cuticular protein mRNAs in silk moth integument and imaginal discs

The distributions of mRNAs for two cuticular proteins of Hyalophora cecropia were examined with RT-PCR and in situ hybridization. For major regions of larval and pupal cuticle, there was a strong correspondence between the type of cuticle and the predominant cuticular protein message found. Epidermal cells underlying soft cuticle had mRNA for HCCP12, with a RR-1 consensus attributed to soft cuticle, while the epidermal cells associated with hard cuticle had predominantly mRNA for HCCP66, a protein with the RR-2 consensus attributed to hard cuticle. Both messages were found in all areas of the pupal fore- and hind-wings, with modest area-specific difference in concentration being much less than differences in the relative abundance of these cuticular proteins.

mRNA for HCCP12 was present in imaginal discs of feeding larvae of H cecropia. Data from Bombyx mori available at SilkBase (http://www.ab.a.u-tokyo.ac.jp/silkbase/) revealed that imaginal discs from feeding larvae had abundant mRNA for RR-1 cuticular proteins, representing six distinct gene products. Only discs from spinning larvae had mRNAs that coded for RR-2 proteins arising from 10 distinct genes. Thus, lepidopteran wing imaginal discs can no longer be regarded as inactive in larval cuticle production.