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.     

Developmental Studies in Drosophila

The salivary gland chromosomes of 3rd instar Drosophila pseudoobscura larvae were observed for puffing changes after injection of larvae with ecdysterone solution. Chromosomes from the salivary glands of 3rd instar larvae and prepupae were similarly examined after incubation in ecdysterone-containing medium. The larvae, after treatment, showed advancement of the puffing process with the occurrence of a pattern similar to that observed during the pre-spiracle eversion period of normal development. At least 92 puffs showed changes in size. For the prepupae, the puffing changes resembled those occurring normally during the late prepupal period. A group of puffs were selected for detailed study. Among these were four puffs on the XR chromosome which exhibited large increases before spiracle eversion and pupation in normal development. As in normal development, two of these became the most prominent puffs observed within h after hormone treatment. In chromosomes from larval glands, the other two XR chromosome puffs were among the largest puffs to appear later in the sequence. However, in chromosomes from prepupal glands one of these later puffs failed to appear. The significance of this large number of hormone-inducible puffing changes at two different periods in development is discussed.     

The prepupal salivary glands of Drosophila melanogaster

Evidence is presented in support of the concept that the larval salivary gland of Drosophila melanogaster continues to function as an important secretory organ throughout prepupal stages and after pupation. Just after puparium formation, and at other later periods, the glands appear to be in the process of disintegration, but each time they recover until after pupation. Nuclear blebbing occurs through the time of survival of the glands, but is shown not to involve transport of RNA out of the nucleus. Transport in and out of the nucleus is clearly rapid and in a steady state as compared to the massive and intermittent export of cytoplasmic substance into the lumen of the gland.This work was supported by grants from the National Science Foundation (GB-23343, PCH-02044).     

A male accessory gland peptide with protease inhibitory activity in Drosophila funebris

Male accessory glands of Drosophila funebris synthesize and secrete a peptide that shows a protease-inhibiting activity. Amino acid sequencing of the purified peptide revealed that the peptide consists of 63 amino acid residues. It is a serine protease inhibitor belonging to the pancreatic trypsin inhibitor (Kunitz) family. The inhibitory function and the kinetic characteristics of the inhibition have been examined with various substrates. The peptide possibly plays a role as an acrosin inhibitor involved in Drosophila reproduction.     

Sequence and Conformational Specificity in Substrate Recognition: SEVERAL HUMAN KUNITZ PROTEASE INHIBITOR DOMAINS ARE SPECIFIC SUBSTRATES OF MESOTRYPSIN*

Mesotrypsin is an isoform of trypsin that is uniquely resistant to polypeptide trypsin inhibitors and can cleave some inhibitors rapidly. Previous studies have shown that the amyloid precursor protein Kunitz protease inhibitor domain (APPI) is a specific substrate of mesotrypsin and that stabilization of the APPI cleavage site in a canonical conformation contributes to recognition by mesotrypsin. We hypothesized that other proteins possessing potential cleavage sites stabilized in a similar conformation might also be mesotrypsin substrates. Here we evaluated a series of candidate substrates, including human Kunitz protease inhibitor domains from amyloid precursor-like protein 2 (APLP2), bikunin, hepatocyte growth factor activator inhibitor type 2 (HAI2), tissue factor pathway inhibitor-1 (TFPI1), and tissue factor pathway inhibitor-2 (TFPI2), as well as E-selectin, an unrelated protein possessing a potential cleavage site displaying canonical conformation. We find that Kunitz domains within APLP2, bikunin, and HAI2 are cleaved by mesotrypsin with kinetic profiles of specific substrates. TFPI1 and TFPI2 Kunitz domains are cleaved less efficiently by mesotrypsin, and E-selectin is not cleaved at the anticipated site. Cocrystal structures of mesotrypsin with HAI2 and bikunin Kunitz domains reveal the mode of mesotrypsin interaction with its canonical substrates. Our data suggest that major determinants of mesotrypsin substrate specificity include sequence preferences at the P₁ and P′₂ positions along with conformational stabilization of the cleavage site in the canonical conformation. Mesotrypsin up-regulation has been implicated previously in cancer progression, and proteolytic clearance of Kunitz protease inhibitors offers potential mechanisms by which mesotrypsin may mediate pathological effects in cancer.     

The timing of drosophila salivary gland apoptosis displays an l(2)gl-dose response

During Drosophila metamorphosis, larval tissues, such as the salivary glands, are histolysed whereas imaginal tissues differentiate into adult structures forming at eclosion a fly-shaped adult. Inactivation of the lethal(2)giant larvae (l(2)gl) gene encoding the cytoskeletal associated p127 protein, causes malignant transformation of brain neuroblasts and imaginal disc cells with developmental arrest at the larval-pupal transition phase. At this stage, p127 is expressed in wild-type salivary glands which become fully histolysed 12 - 13 h after pupariation. By contrast to wild-type, administration of 20-hydroxyecdsone to l(2)gl-deficient salivary glands is unable to induce histolysis, although it releases stored glue granules and gives rise to a nearly normal pupariation chromosome puffing, indicating that p127 is required for salivary gland apoptosis. To unravel the l(2)gl function in this tissue we used transgenic lines expressing reduced ( approximately 0.1) or increased levels of p127 (3.0). Here we show that the timing of salivary gland histolysis displays an l(2)gl-dose response. Reduced p127 expression delays histolysis whereas overexpression accelerates this process without affecting the duration of third larval instar, prepupal and pupal development. Similar l(2)gl-dependence is noticed in the timing of expression of the cell death genes reaper, head involution defective and grim, supporting the idea that p127 plays a critical role in the implementation of ecdysone-triggered apoptosis. These experiments show also that the timing of salivary gland apoptosis can be manipulated without affecting normal development and provide ways to investigate the nature of the components specifically involved in the apoptotic pathway of the salivary glands.     

The Amyloid Precursor Protein/Protease Nexin 2 Kunitz Inhibitor Domain Is a Highly Specific Substrate of Mesotrypsin

The amyloid precursor protein (APP) is a ubiquitously expressed transmembrane adhesion protein and the progenitor of amyloid-β peptides. The major splice isoforms of APP expressed by most tissues contain a Kunitz protease inhibitor domain; secreted APP containing this domain is also known as protease nexin 2 and potently inhibits serine proteases, including trypsin and coagulation factors. The atypical human trypsin isoform mesotrypsin is resistant to inhibition by most protein protease inhibitors and cleaves some inhibitors at a substantially accelerated rate. Here, in a proteomic screen to identify potential physiological substrates of mesotrypsin, we find that APP/protease nexin 2 is selectively cleaved by mesotrypsin within the Kunitz protease inhibitor domain. In studies employing the recombinant Kunitz domain of APP (APPI), we show that mesotrypsin cleaves selectively at the Arg¹⁵-Ala¹⁶ reactive site bond, with kinetic constants approaching those of other proteases toward highly specific protein substrates. Finally, we show that cleavage of APPI compromises its inhibition of other serine proteases, including cationic trypsin and factor XIa, by 2 orders of magnitude. Because APP/protease nexin 2 and mesotrypsin are coexpressed in a number of tissues, we suggest that processing by mesotrypsin may ablate the protease inhibitory function of APP/protease nexin 2 in vivo and may also modulate other activities of APP/protease nexin 2 that involve the Kunitz domain.     

Biochemical and ontogenetic aspects of glycoprotein synthesis in Drosophila virilis salivary glands

After SDS-polyacrylamide gel electrophoresis two glycosylated glue proteins are found in the salivary glands of Drosophila virilis late third instar larvae. Synthesis of larval glue protein 1 occurs in three successive steps: at first a precursor protein with a molecular weight of about 138,000 daltons is formed. This is modified by two subsequent steps of glycosylation, the first one involving hexosamine, the second one hexoses. Studies with tunicamycin and β-hydroxynorvaline suggest that glycosylation occurs at threonine residues. Larval glue protein 2 has a molecular weight of approximately 15,000 daltons and is weakly glycosylated. The synthesis of glue proteins is stage specific. It starts at about 120 hr after oviposition and attains its maximal rate about 20 hr later. At this time the larvae leave the food. Between ecdysone release and puparium formation (146–151 hr) larval glue protein synthesis is terminated. Throughout the prepupal stage a different set of glycoproteins is synthesized. Thus, the larval-prepupal transition is accompanied by the reprogramming of glycoprotein synthesis in salivary glands. The secretion products formed during the two developmental stages seem to possess different biological functions.     

Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster,: IV. The mid prepupal period

In the mid prepupal period of development of Drosophila melanogaster two major changes in gene activity occur in the salivary glands: (a) The mid prepupal puffs (e.g., 63E, 75CD) are induced and (b) the late prepupal puffs (e.g., 62E, 74EF, 75B, and 93F) acquire the competence to respond to ecdysone. These events can be studied in vitro. Both require that the ecdysone titre be very low (<5 × 10^?9, M) and both events depend upon protein synthesis.     

家蚕蛹-成虫变态期中肠和涎腺超微结构变化与功能

为进一步明确家蚕Bombyx mori变态期间消化系统的生理功能以及溶茧酶的来源器官,通过透射电镜观察和酶活性检测,对家蚕蛹 成虫变态期中肠和涎腺的超微结构、水解酶活力以及中肠内容物的变化进行了观察和分析。结果表明: 蛹第7日到羽化前1日家蚕中肠细胞和刚羽化成虫的涎腺细胞中,均可观察到大量的分泌泡、分泌颗粒、微绒毛等分泌细胞的结构特征以及活跃的分泌现象。潜成虫的中肠和涎腺中都存在活性较高的水解酶活力,其中每毫克中肠组织中蛋白酶活力、酯酶活力和纤溶酶活力分别为726 U、751 U和263 U,每毫克涎腺组织中上述3种酶的活力分别为603 U、523 U和147 U,说明中肠和涎腺可能都具有分泌溶茧酶的功能。家蚕蛹期中肠内容物的主要成分是蛋白质、脂质和糖,三者占内含物总量的95%以上,其中蛋白质含量占78.8%～80.2%。中肠内容物的重量在刚化蛹时为20.1～21.9 mg/头,化蛹后7日内无明显变化,化蛹第9日内容物重量减少63.01%～66.17%,到成虫羽化时已所剩无几,可能是因内容物被消化吸收所致。据此推测,在家蚕变态期中肠还具有贮存和释放营养物质的功能,而溶茧酶的另一个功能可能是分解消化中肠内容物。     

Relationship of developmental stage to initiation of replication in polytene nuclei

The various types of autoradiographic patterns occurring in salivary gland nuclei ofD. melanogaster following a short pulse of tritiated thymidine have been described and the probable order of their sequence within a polytenic replication cycle assigned. On the basis of that assignment, the distribution of those patterns in the nuclei of salivary glands of 160 staged larvae and prepupae has been assessed and the following interpretations made: Throughout the third larval instar and prepupal period there is a continuing decrease in frequency of labeled nuclei. Within the larval period that decrease is reflected in a proportional decrease in label patterns characteristic of both initiation and propagation phases of the replication cycle. At the beginning of the prepupal period there is a rise in the proportion of labeled cells displaying initiation type patterns followed by an abrupt cessation of such patterns. It is proposed that termination of initiation of polytenic replication is amongst the physiologic manifestations of the induction of pupation.     

Drosophila signal peptide peptidase is an essential protease for larval development

We identified the Drosophila melanogaster Signal peptide peptidase gene (Spp) that encodes a multipass transmembrane aspartyl protease. Drosophila SPP is homologous to the human signal peptide peptidase (SPP) and is distantly related to the presenilins. We show that, like human SPP, Drosophila SPP can proteolyze a model signal peptide and is sensitive to an SPP protease inhibitor and that it localizes to the endoplasmic reticulum. Expression of Drosophila SPP was first apparent at germ band extension, and in late embryos it was robust in the salivary glands, proventriculus, and tracheae. Flies bearing mutations in conserved residues or carrying deficiencies for the Spp gene had defective tracheae and died as larvae.