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PubMed1996

Soluble adenylyl cyclase from Spodoptera frugiperda (Sf9) cells. Purification and biochemical characterization.

An insect ovarian cell, Spodoptera frugiperda (Sf9), has been widely used to express recombinant proteins, including adenylyl cyclase, as a host cell in the baculovirus expression system. We report the presence and characterization of a soluble adenylyl cyclase (sAC) distinct from a membrane-bound form of adenylyl cyclase (mAC) that is also present in Sf9 cells. sAC was purified 3,500-fold to near homogeneity; a single band at 25 kDa on SDS-polyacrylamide gel electrophoresis correlated well with adenylyl cyclase catalytic activity. The purified enzyme had a catalytic activity of 0.1 micromol/min.mg and the Km of 0.55 mM for the substrate ATP. In contrast to mAC, sAC was heat-stable. Enzymatic activity of sAC was not stimulated by forskolin and was inhibited by salts at high concentrations. sAC utilized both manganese- and magnesium-ATP as substrate. Di- or triphosphate-containing nucleotides, such as GTP and GDP, as well as pyrophosphate, noncompetitively inhibited sAC. Our data suggest that the physical and biochemical characteristics of sAC are different from those of mAC in Sf9 cells as well as from those of other known forms of adenylyl cyclase in animal cells; sAC in Sf9 cells may constitute a new member of adenylyl cyclase found in animals.

Adenylyl Cyclase Inhibitors↗
PubMed1996

Human cathepsin O2, a matrix protein-degrading cysteine protease expressed in osteoclasts. Functional expression of human cathepsin O2 in Spodoptera frugiperda and characterization of the enzyme.

Cathepsin O2, a human cysteine protease predominantly present in osteoclasts, has been functionally expressed in Spodoptera frugiperda Sf9 cells using the Autographa californica nuclear polyhedrosis virus. Following in vitro activation at pH 4.0 with pepsin, active enzyme with an apparent molecular weight of 29,000 was obtained. N-terminal sequencing revealed the typical processing site for cysteine proteases of the papain family with a proline in the position adjacent to the N-terminal alanine residue. The S2P2 subsite specificity of human cathepsin O2 is similar to cathepsin S but distinguished from cathepsins L and B. Similar to cathepsin S, cathepsin O2 is characterized by a bellshaped pH activity profile and is stable at pH 6.5 for 30 min at 37 degrees C. Cathepsin O2 is further distinguished by its potent collagenolytic activity against Type I collagen between pH 5 and 6, and elastinolytic activity against insoluble elastin at pH 7.9. Its capacity to efficiently degrade Type I collagen and its high expression in osteoclasts suggest that cathepsin O2 may play a major role in human osteoclastic bone resorption.

Amino Acid Sequence↗
PubMed1997

Functional Co-expression of the canine cardiac Ca2+ pump and phospholamban in Spodoptera frugiperda (Sf21) cells reveals new insights on ATPase regulation.

The utility of the baculovirus cell expression system for investigating Ca2+-ATPase and phospholamban regulatory interactions was examined. cDNA encoding the canine cardiac sarco(endo)plasmic Ca2+-ATPase pump (SERCA2a) was cloned for the first time and expressed in the presence and absence of phospholamban in Spodoptera frugiperda (Sf21) insect cells. The recombinant Ca2+ pump was produced in high yield, contributing 20% of the total membrane protein in Sf21 microsomes. At least 70% of the expressed pumps were active. Co-expression of wild-type, pentameric phospholamban with the Ca2+-ATPase decreased the apparent affinity of the ATPase for Ca2+, but had no effect on the maximum velocity of the enzyme, similar to phospholamban's action in cardiac sarcoplasmic reticulum vesicles. To investigate the importance of the oligomeric structure of phospholamban in ATPase regulation, SERCA2a was co-expressed with a monomeric mutant of phospholamban, in which leucine residue 37 was changed to alanine. Surprisingly, monomeric phospholamban suppressed SERCA2a Ca2+ affinity more strongly than did wild-type phospholamban, demonstrating that the pentamer is not essential for Ca2+ pump inhibition and that the monomer is the more active species. To test if phospholamban functions as a Ca2+ channel, Sf21 microsomes expressing either SERCA2a or SERCA2a plus phospholamban were actively loaded with Ca2+ and then assayed for unidirectional 45Ca2+ efflux. No evidence for a Ca2+ channel activity of phospholamban was obtained. We conclude that the phospholamban monomer is an important regulatory component inhibiting SERCA2a in cardiac sarcoplasmic reticulum membranes, and that the channel activity of phospholamban previously observed in planar bilayers is not involved in the mechanism of ATPase regulation.

Amino Acid Sequence↗
PubMed1997

Spodoptera frugiperda caspase-1, a novel insect death protease that cleaves the nuclear immunophilin FKBP46, is the target of the baculovirus antiapoptotic protein p35.

Employing the degenerate primer-dependent polymerase chain reaction approach used recently to clone human Mch2, we have identified and cloned the insect Spodoptera frugiperda target of the baculovirus antiapoptotic protein p35. This protein named Sf caspase-1 belongs to the family of caspases and is highly related to human Mch3 and CPP32 in sequence and specific activity. The proenzyme of Sf caspase-1 is 299 amino acids in length and can undergo autocatalytic processing in Escherichia coli to an active enzyme heterocomplex. Autoprocessing occurs at Asp-28, Asp-184, and Asp-195 to generate the large p19/p18 and small p12 subunits. Sf caspase-1 is able to induce apoptosis in Sf9 cells and is capable of cleaving p35 to similar sized fragments as observed with extracts from p35 null mutant baculovirus-infected Sf9 cells. Sf caspase-1 activity is potently inhibited by p35, suggesting that it is an important target of this antiapoptotic protein. Finally, the Sf9 nuclear immunophilin FKBP46 was identified as a death-associated substrate for Sf caspase-1.

Amino Acid Sequence↗
PubMed1997

Agonist-independent activation of Gz by the 5-hydroxytryptamine1A receptor co-expressed in Spodoptera frugiperda cells. Distinguishing inverse agonists from neutral antagonists.

The human 5-hydroxytryptamine1A receptor, when expressed in Spodoptera frugiperda (Sf9) cells, facilitates the binding of [35S]GTPgammaS to a co-expressed GTP-binding regulatory protein, Gz, consistent with constitutive activity. The antagonists 4-(2'-methoxyphenyl)-1-[2'(n-2"-pyridinyl)-p-iodobenzamido]ethyl-p ipe razine (p-MPPI) and the related fluorobenzamido analogue p-MPPF had little (p-MPPI) or no (p-MPPF) effect on this activity. In contrast, a third antagonist, the neuroleptic spiperone, produced an almost complete suppression. Thus, using G protein activation as an index of receptor activity, p-MPPF was classified as a neutral antagonist, p-MPPI as a partial inverse agonist, and spiperone as essentially a full inverse agonist. As predicted, spiperone displayed properties consistent with a special form of noncompetitive antagonism when used to displace the agonist [125I]R-(+)-trans-8-hydroxy-2-[N-n-propyl-N-(3'-iodo-2'-propenyl)amin o]tetralin. Our data profile Sf9 cells as a unique vehicle for the characterization of inverse agonists, as these cells support a systematic pairing of mammalian receptors and G proteins, quantitative assays of G protein activation, and unambiguously labeled populations of coupled and uncoupled receptors.

Aminopyridines↗
PubMed1999

Molecular cloning and characterization of hemolymph 3-dehydroecdysone 3beta-reductase from the cotton leafworm, Spodoptera littoralis. A new member of the third superfamily of oxidoreductases.

The primary product of the prothoracic glands of last instar larvae of Spodoptera littoralis is 3-dehydroecdysone (3DE). After secretion, 3DE is reduced to ecdysone by 3DE 3beta-reductase in the hemolymph. We have previously purified and characterized 3DE 3beta-reductase from the hemolymph of S. littoralis. In this study, cDNA clones encoding the enzyme were obtained by reverse transcription-polymerase chain reaction, employing primers based on the amino acid sequences, in conjunction with 5'- and 3'-rapid amplification of cDNA ends. Multiple polyadenylation signals and AT-rich elements were found in the 3'-untranslated region, suggesting that this region may have a role in regulation of expression of the gene. Conceptual translation and amino acid sequence analysis suggest that 3DE 3beta-reductase from S. littoralis is a new member of the third superfamily of oxidoreductases. Northern analysis shows that 3DE 3beta-reductase mRNA transcripts are widely distributed, but are differentially expressed, in some tissues. The developmental profile of the mRNA revealed that the gene encoding 3DE 3beta-reductase is only transcribed in the second half of the last larval instar and that this fluctuation in expression accounts for the change in the enzyme activity during the instar. Southern analysis indicates that the 3DE 3beta-reductase is encoded by a single gene, which probably contains at least one intron.

3-Hydroxysteroid Dehydrogenases↗
PubMed2002

Silencing of midgut aminopeptidase N of Spodoptera litura by double-stranded RNA establishes its role as Bacillus thuringiensis toxin receptor.

Insecticidal crystal proteins of Bacillus thuringiensis bind to receptors in the midgut of susceptible insects leading to pore formation and death of the insect. The identity of the receptor is not clearly established. Recently a direct interaction between a cloned and heterologously expressed aminopeptidase (slapn) from Spodoptera litura and the Cry1C protein was demonstrated by immunofluorescence and in vitro ligand blot interaction. Here we show that administration of slapn double-stranded RNA to S. litura larvae reduces its expression. As a consequence of the reduced expression, a corresponding decrease in the sensitivity of these larvae to Cry1C toxin was observed. The gene silencing was retained during the insect's moulting and development and transmitted to the subsequent generation albeit with a reduced effect. These results directly implicate larval midgut aminopeptidase N as receptor for Bacillus thuringiensis insecticidal proteins.

Animals↗
PubMed2001

Regulation of ecdysteroid signaling: cloning and characterization of ecdysone oxidase: a novel steroid oxidase from the cotton leafworm, Spodoptera littoralis.

One route of inactivation of ecdysteroids in insects involves ecdysone oxidase-catalyzed conversion into 3-dehydroecdysteroid followed by irreversible reduction by 3-dehydroecdysone 3alpha-reductase to 3-epiecdysone. We have purified from Spodoptera littoralis the first ecdysone oxidase and subjected it to limited amino acid sequencing. A reverse-transcriptase polymerase chain reaction-based approach has been used to clone the cDNA (2.8 kilobases) encoding this 65-kDa protein. Northern blotting showed that the mRNA transcript was expressed in midgut during the prepupal stage of the last larval instar at a time corresponding to an ecdysteroid titer peak. Conceptual translation of the ecdysone oxidase cDNA and data base searching revealed that the enzyme is an FAD flavoprotein that belongs to the glucose-methanol-choline oxidoreductase superfamily. Ecdysone oxidase represents the only oxidase in eukaryotic animals known to catalyze oxygen-dependent oxidation of steroids; by contrast, oxidation of steroids in vertebrates occurs via NAD(P)(+)-linked dehydrogenases. The injection of RH-5992, an ecdysteroid agonist, induced the transcription of ecdysone oxidase, suggesting that ecdysone oxidase is an ecdysteroid-responsive gene. The gene encoding this enzyme, consisting of five exons, has also been isolated. Sequences similar to the binding motifs for Broad-Complex and FTZ-F1 have been found in the 5'-flanking region. Southern blotting indicated that ecdysone oxidase is encoded by a single-copy gene. We have determined the kinetic characteristics of this novel recombinant ecdysone oxidase produced using a baculovirus expression system.

3-Hydroxysteroid Dehydrogenases↗
PubMed2002

Characterization of the apoptosis suppressor protein P49 from the Spodoptera littoralis nucleopolyhedrovirus.

Two antiapoptotic types of genes, iap and p35, were found in baculoviruses. P35 is a 35-kDa protein that can suppress apoptosis induced by virus infection or by diverse stimuli in vertebrates or invertebrates. iap homologues were identified in insects and mammals. Recently, we have identified sl-p49, a novel apoptosis suppressor gene and the first homologue of p35, in the genome of the Spodoptera littoralis nucleopolyhedrovirus. Here we show that sl-p49 encodes a 49-kDa protein, confirmed its primary structure that displays 48.8% identity to P35, and performed computer-assisted modeling of P49 based on the structure of P35. We demonstrated that P49 is able to inhibit insect and human effector caspases, which requires P49 cleavage at Asp(94). Finally we identified domains important for P49's antiapoptotic function that include a reactive site loop (RSL) protruding from a beta-barrel domain. RSL begins at an amphipathic alpha1 helix, traverses the beta-sheet central region, exposing Asp(94) at the apex, and rejoins the beta-barrel. Our model predicted seven alpha-helical motifs, three of them unique to P49. alpha-Helical motifs alpha(1), alpha(2), and alpha(4') were required for P49 function. The high structural homology between P49 and P35 suggests that these molecules bear a scaffold common to baculovirus "apoptotic suppressor" proteins. P49 may serve as a novel tool to analyze the contribution of different components of the caspase chain in the apoptotic response in organisms not related phylogenetically.

Amino Acid Sequence↗
PubMed2004

The role of Bacillus thuringiensis Cry1C and Cry1E separate structural domains in the interaction with Spodoptera littoralis gut epithelial cells.

The Bacillus thuringiensis delta-endotoxins Cry1C and Cry1E share toxicity against several important lepidopteran species. Their combined use to delay development of resistance in target insects depends on their differential interaction with the gut epithelial cells. The three structural domains and combinations of two consecutive domains of Cry1C and Cry1E were separately expressed in Escherichia coli, and their interactions with the brush border membrane vesicles (BBMV) of Cry1E-tolerant and -susceptible Spodoptera littoralis larvae were studied. About 80% reduction in binding of Cry1E and each of its separate domains to BBMV of Cry1E-tolerant larvae was observed, whereas Cry1C was toxic to all larvae and bound equally to BBMV derived from both Cry1E-tolerant and -susceptible larvae. These results suggest differential interactions of the two toxins with BBMV encompassing all three domains. Comparable binding assays performed with fluorescent Cry1C and Cry1C domain II showed that Cry1C has higher Bmax and lower Kd than Cry1C domain II and further supported the existence of toxin multisite interactions. Competitive binding assays were used to estimate the sequence of interaction events. Cry1C domain II could compete with domain III binding, whereas domain III did not interfere with domain II binding, indicating sequential interactions of domain III and then domain II with the same membrane site. No competition between domain II of Cry1C and Cry1E was observed, confirming the existence of different domain II binding sites for the two toxins. Taken together, all three domains specifically interact with the epithelial cell membrane. The folding of the three-domain toxin probably dictates the sequence of interaction events.

Amino Acid Sequence↗
PubMed2006

Purification, characterization, and cloning of a Spodoptera frugiperda Sf9 beta-N-acetylhexosaminidase that hydrolyzes terminal N-acetylglucosamine on the N-glycan core.

Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a beta-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a beta-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the beta(1,2)-linked N-acetylglucosamine from the Man alpha(1,3) branch compared with the Man alpha(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal beta-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing beta-N-acetylhexosaminidase acting on N-glycans from Sf9 cells.

Acetylglucosamine↗
PubMed2003

Radar observations of the autumn migration of the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae) and other moths in northern China.

The autumn return migration of the beet armyworm Spodoptera exigua Hübner and other insects in northern China was observed with radar and with a simultaneously-operated searchlight trap and ground light-trap at a site in Langfang, near Beijing. The searchlight trap was found to be an efficient tool for trapping migrants and, operated alongside a ground light-trap, could distinguish migrant from locally-flying species. It was confirmed that S. exigua and some other species were high-altitude nocturnal windborne migrants during September and October in northern China. Maximum density of moths typically occurred below 500 m, and strong layering was often observed at about 200 m above ground level in airflows that would carry the moths towards the south. Descent of S. exigua in the vicinity of the radar site in late September was often associated with rain.

Animal Migration↗
PubMed2000

Efficacy of the botanical extract (myrrh), chemical insecticides and their combinations on the cotton leafworm, Spodoptera littoralis boisd (Lepidoptera : Noctuidae).

Effects of the botanical extract (myrrh), Commiphora molmol, and the sublethal treatments of profenofos/chlorofluazuron, fenvalerate and pyriproxyfen on the larvae of S. littoralis were investigated. The results showed that myrrh induced the highest activity after 7 days of treatment with concentration of 10000 ppm and the percentage of mortality reached 44.4%. The chemical insecticide profenofos/chlorofluazuron appeared to be the most effective treatment, giving a mortality of 54% after one day of treatment with a concentration of 100 ppm. Different treatments showed adverse effects on pupation, emerging adults and larval duration. The highest concentration of myrrh (10000 ppm) induced 35% pupation. No pupa was obtained with profenofos/chlorofluazuron while fenvalerate and pyriproxyfen gave 13.3% at 50 ppm. At 10000 ppm of myrrh, 25% adult was emerged, also fenvalerate and pyriproxyfen decreased the emerging moth to 13.3% at 50 ppm. For control there was no larval mortality at 7 days and the percentages of pupation and adult emergence were 90%. Bioassays were also conducted to test the joint action of myrrh applied in binary mixtures with each chemical insecticide on Spodoptera larvae. Results of the bioassay of mixtures indicated antagonistic effects on larval mortality. However, combination of myrrh with profenofos/chlorofluazuron showed additive effect. Also, the degree of pupation and emerging moth varied considerably when myrrh combined with the chemical insecticides. The present work shows the strong efficacy of the botanical extract (myrrh) which could be used alone or in combination with sublethal doses of certain insecticides to control the cotton leafworm.

Animals↗
PubMed2005

Biological studies of azadirachtin and its derivatives against polyphagous pest, Spodoptera litura.

Azadirachtin-A (Aza-A, figure 1) was subjected to a variety of synthetic transformations and the antifeedant activity and toxicity of the azadirachtin derivatives were assessed against second instar larvae Spodoptera litura on castor leaves in comparison with the crude material containing 9.14% Aza-A, as well as relatively pure 91% Aza-A. A probable mechanism for the base initiated fragmentation of the diketocarbonate into a decalin fragment and a spiroketal moiety has been proposed. The present study indicates that the combination of insect toxicity and antifeedancy of azadirachtin provides good crop protection.

Animals↗
PubMed2003

Effects of light and temperature on the circadian system controlling sperm release in moth Spodoptera littoralis.

Reproductive physiology of male moths is regulated by a peripheral circadian system, which controls the timing of sperm release from the testis into the upper vas deferens (UVD) and timing of sperm transfer from the UVD to the seminal vesicles. We investigated various effects of light and temperature on sperm release and transfer rhythms in the moth Spodoptera littoralis. We report that both rhythms persist for up to 1 week in constant darkness without significant dampening and are also temperature compensated in the range from 20 degrees C to 30 degrees C. However, the duration of sperm retention in the UVD is temperature-dependent; consequently, temperature exerts a masking effect on the rhythm of sperm transfer. Experimental manipulations of light and temperature regime demonstrated that light dominates over temperature in entraining the timing of sperm release and transfer. Nevertheless, temperature plays a critical role in the absence of light Zeitgeber. Sperm release and transfer are arrhythmic in constant light (LL); however, both rhythms are restored by temperature cycles.

Animals↗
PubMed2001

Spodoptera exigua: mating disruption, measurement of airborne concentration of pheromone, and use of specialist receptor cell responses for comparison to female pheromone emission.

Field experiments were conducted to evaluate Shin-Etsu Yoto-con-S 'rope' pheromone dispensers applied at different site densities in cotton to determine efficacy in disrupting sexual communication and mating of beet armyworm moths, Spodoptera exigua (Hübner); obtain atmospheric measurements of the disruptant pheromone components in and away from treated areas; and relate this information to estimates of the concentration of pheromone in the near vicinity of a calling virgin female moth. The number of pheromone-dispensing sites/ha ranged from 25 with 20 ropes/site up to a total of 247 sites with 2 ropes each. A total of 112 g pheromone of a 70:30 blend of (Z,E)-9,12-tetradecadien-l- ol acetate (ZETA) and (Z)-tetradecen-1-ol (Z9-14:OH) was applied/ha. As few as 25 sites/ha effectively disrupted sexual communication by 91%, as measured by reductions of moth captures in traps baited with pheromone lures, and mating by beet armyworm females by 94% for at least 10 weeks post-treatment. The results compared favorably to an earlier large-scale field experiment in which beet armyworm larval populations were reduced 95% using 500 pheromone-dispensing sites/ha (two ropes/site) and 160 g total pheromone/ha. The airborne concentration of ZETA and Z9-14:OH measured within the pheromone-treated plots that disrupted mating was 1.5 x 10(-13) M and 1.2 x 10(-13) M, respectively. The airborne concentration of ZETA and Z9-14:OH in the vicinity of a female was estimated (by excision and electrophysiological methods) to range between 2.1 x 10(-11) M and 2.4 x 10(-12) M, respectively, in a 1.6 km/h (1 mph) wind speed. Thus, the average ZETA concentration in the treated plots was about 140 times less than that expected from a single female; the average Z9-14:OH in the treated plots was about 20 times less. The data support an argument that disruption of mating by beet armyworm resulted from an imbalance in sensory input because mating was almost completely disrupted in all of the plots for at least 10 weeks with a mixture of ZETA:Z9-14:OH that differed significantly with that from excised female sex pheromone glands. Estimates of these parameters provide a benchmark for the requisite pheromone concentration in the atmosphere to achieve a high level of mating disruption for beet armyworm and possibly other insect pest species.

Air↗
PubMed2002

Spatial representation of odours in the antennal lobe of the moth Spodoptera littoralis (Lepidoptera: Noctuidae).

Glomeruli within the antennal lobe (AL) of moths are convergence sites for a large number of olfactory receptor neurons (ORNs). The ORNs target single glomeruli. In the male-specific cluster of glomeruli, the macroglomerular complex (MGC), the input is chemotypic in that each glomerulus of the MGC receives information about a specific component of the conspecific female sex pheromone. Little is known about how neurons that detect other odorants arborize in and amongst glomeruli. The present study focuses on how sex pheromones and biologically relevant semiochemicals are represented in the ALs of both sexes of the moth Spodoptera littoralis. To assess this, we optically measured odour-evoked changes of calcium concentration in the ALs. Foci of calcium increase corresponded in size and shape with anatomical glomeruli. More than one glomerulus was normally activated by a specific non-pheromonal odorant and the same glomerulus was activated by several odorants. All odorants and pheromone components tested evoked unique patterns of glomerular activity that were highly reproducible at repeated stimulations within an individual. Odour-evoked patterns were similar between individuals for a given odorant, implicating a spatial olfactory code. In addition, we demonstrated that activity patterns evoked by host-plant related volatiles are similar between males and females.

Animals↗
PubMed2002

Biosynthesis and processing of Spodoptera frugiperda alpha-mannosidase III.

We previously cloned a lepidopteran insect cell cDNA that encodes a class II alpha-mannosidase that is localized in the Golgi apparatus but is cobalt-dependent, has a neutral pH optimum, hydrolyzes Man(5)GlcNAc(2) to Man(3)GlcNAc(2), and cannot hydrolyze GlcNAcMan(5)GlcNAc(2). This enzyme was designated SfManIII to distinguish it from Golgi alpha-mannosidase II and indicate its derivation from the fall armyworm Spodoptera frugiperda. In the present study, we prepared a polyclonal antibody and used it to study the biosynthesis and processing of SfManIII. The results showed that Sf9 cells produce at least three different forms of SfManIII. SfManIII is initially synthesized as a precursor glycoprotein, which is slowly converted to two smaller end products with at least some endoglycosidase H-resistant N-glycans. The smallest form of SfManIII is the only one of these two products that accumulates in the extracellular fraction. Tunicamycin blocked the production of SfManIII activity and the secretion of SfManIII protein and activity. Castanospermine blocked production of the larger SfManIII product, retarded production of the smaller, increased intracellular SfManIII activity, and decreased extracellular SfManIII activity. Together, these results indicate that SfManIII is initially synthesized as a high-mannose glycoprotein precursor, its N-glycans are trimmed as it is transported to the Golgi apparatus, and a subpopulation, which appears to be proteolytically cleaved, is secreted in enzymatically active form. N-glycosylation is required for the production of active SfManIII, and N-glycosylation and N-glycan trimming are both required for the efficient secretion of an active form of this protein.

Animals↗