Sf9 Cell Line Research Articles

Aminotransferase from insect cells

The presence of transaminase activity is demonstrated in the cultured insect cell line Sf21 (Spodoptera frugiperda) at a level sufficient to enable replacement of rat liver in an undergraduate student experiment designed to demonstrate enzyme activity responsible for amino acid interconversions.

Characterization of the cDNA encoding the 90 kDa heat-shock protein in the Lepidoptera Bombyx mori and Spodoptera frugiperda

This report presents the first hsp90 complete cDNA sequences from two Lepidoptera. The Bombyx mori full sequence was reconstituted from 15 partial cDNA clones belonging to expressed sequence tag libraries obtained from different tissues or cultured cells, thus showing the ubiquitous expression of the hsp90 gene. The Spodoptera frugiperda cDNA was isolated as a full-length clone from a cDNA library established from the Sf9 cell line. Both cDNAs are highly homologous and display the classical amino acid (aa) stretches representing the HSP90 signature. They potentially encode a 716 aa ( B. mori) and a 717 aa ( S. frugiperda) protein, with a calculated molecular mass of 83 kDa similar to the Drosophila homologous protein. We show that, unlike the vertebrates, hsp90 is a unique gene in both S. frupiperda and B. mori genomes. Sequencing of the corresponding genomic region shows that, contrary to the dipteran homologous gene, the lepidopteran hsp90 gene does not display any intron. Phylogenetic analysis based on the two lepidopteran and 23 other HSP90 aa sequences displays a high consistency with known phylogeny at both high and low taxonomic levels. Transcriptional analysis performed in S. frugiperda shows that the induction of the hsp90 gene only occurs 14°C above physiological growth conditions (42°C).

Antioxidant defense systems of two lipidopteran insect cell lines

Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines were found to contain unique assemblages of antioxidant enzymes. Specifically, the Sf-9 insect cell line contained Manganese and Copper-Zinc superoxide dismutase (MnSOD and CuZnSOD) for reducing the superoxide radical (O2•−) to hydrogen peroxide (H2O2) and ascorbate peroxidase (APOX) for reducing the resulting H2O2 to H2O. Approximately one third of the total SOD activity was found to be MnSOD. The Tn-5B1-4 cells were also found to contain MnSOD (∼ two thirds of the total SOD activity), CuZnSOD and APOX activities. However, the Tn-5B1-4 cell line, in contrast to the Sf-9 cell line, contained catalase (CAT) activity for reducing H2O2 to H2O. Both the Sf-9 and Tn-5B1-4 cell lines contained glutathione reductase and dehydroascorbic acid reductase activities for regenerating the reduced forms of glutathione and ascorbic acid, respectively. In addition, both cell lines contained glutathione S-transferase peroxidase activity towards hydroperoxides other than H2O2. Finally, neither cell line contains the glutathione peroxidase activity that is ubiquitous in mammalian cells.

Stable cell lines expressing baculovirus P35: resistance to apoptosis and nutrient stress, and increased glycoprotein secretion.

The baculovirus P35 protein is a caspase inhibitor that prevents the induction of apoptosis during infection of Sf21 cells by Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). P35 inhibits the induction of apoptosis in a broad range of cells and circumstances. In this study, we examined the effects of constitutive cellular P35 expression on the response of cells to stressful culture conditions and on protein production in AcMNPV infected cells. Sf9 cell lines expressing AcMNPV P35 or an epitope-tagged P35 protein were generated using a double selection technique, involving selection in the antibiotic G418, followed by a second round of selection by exposure to actinomycin D, a potent inducer of apoptosis in Sf9 cells. Clonal cell lines were generated and examined for (1) resistance to actinomycin D induced apoptosis, (2) resistance to nutrient deprivation, and (3) baculovirus expression of intracellular and secreted proteins. When compared with Sf9 cells, two P35-expressing cell lines (Sf9P35AcV5-1 and Sf9P35AcV5-3) showed increased resistance to actinomycin D-induced apoptosis and a profound resistance to nutrient deprivation. When these cell lines were infected with a recombinant baculovirus expressing a secreted glycoprotein (secreted alkaline phosphatase), expression of the glycoprotein from these cells exceeded that from the parental Sf9 cells and was comparable to expression levels obtained from Tn5B1-4 cells, the best available cell line for high-level expression. Increased levels of protein secretion in Sf9P35AcV5-1 and Sf9P35AcV5-3 cells appear to result from a prolonged infection cycle and accumulation of the secreted glycoprotein.

Persistent expression of a newly characterized Hyposoter didymator polydnavirus gene in long-term infected lepidopteran cell lines.

An Hyposoter didymator ichnovirus (HdIV) gene was stably maintained and efficiently transcribed in lepidopteran cell lines more than 3 years after HdIV infection. This K-gene had two introns and the fully spliced cDNA, named K19, comprised a short open reading frame and a long 3'-untranslated region with 13 imperfectly repeated sequences (44 to 102 nt). Transcripts related to the K-gene were detected in several long-term infected cell lines (Sf9, Spodoptera littoralis haemocytes, Trichoplusia ni). Conversely, no transcripts related to seven other viral cDNAs were detected, suggesting that the K-related DNA is selectively retained in long-term infected Sf9 cells. The function of the K-gene product and its association with stably transformed insect cell lines remains to be investigated.

A GP64-null baculovirus pseudotyped with vesicular stomatitis virus G protein.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein is an essential virion protein that is involved in both receptor binding and membrane fusion during viral entry. Genetic studies have shown that GP64-null viruses are unable to move from cell to cell and this results from a defect in the assembly and production of budded virions (BV). To further examine requirements for virion budding, we asked whether a GP64-null baculovirus, vAc(64-), could be pseudotyped by introducing a heterologous viral envelope protein (vesicular stomatitis virus G protein [VSV-G]) into its membrane and whether the resulting virus was infectious. To address this question, we generated a stably transfected insect Sf9 cell line (Sf9(VSV-G)) that inducibly expresses the VSV-G protein upon infection with AcMNPV Sf9(VSV-G) and Sf9 cells were infected with vAc(64-), and cells were monitored for infection and for movement of infection from cell to cell. vAc(64-) formed plaques on Sf9(VSV-G) cells but not on Sf9 cells, and plaques formed on Sf9(VSV-G) cells were observed only after prolonged intervals. Passage and amplification of vAc(64-) on Sf9(VSV-G) cells resulted in pseudotyped virus particles that contained the VSV-G protein. Cell-to-cell propagation of vAc(64-) in the G-expressing cells was delayed in comparison to wild-type (wt) AcMNPV, and growth curves showed that pseudotyped vAc(64-) was generated at titers of approximately 10(6) to 10(7) infectious units (IU)/ml, compared with titers of approximately 10(8) IU/ml for wt AcMNPV. Propagation and amplification of pseudotyped vAc(64-) virions in Sf9(VSV-G) cells suggests that the VSV-G protein may either possess the signals necessary for baculovirus BV assembly and budding at the cell surface or may otherwise facilitate production of infectious baculovirus virions. The functional complementation of GP64-null viruses by VSV-G protein was further demonstrated by identification of a vAc(64-)-derived virus that had acquired the G gene through recombination with Sf9(VSV-G) cellular DNA. GP64-null viruses expressing the VSV-G gene were capable of productive infection, replication, and propagation in Sf9 cells.

Biosynthesis and subcellular localization of a lepidopteran insect alpha 1,2-mannosidase

Like lower and higher eucaryotes, insects have α 1,2-mannosidases which function in the processing of N-glycans. We previously cloned and characterized an insect α 1,2-mannosidase cDNA and demonstrated that it encodes a member of a family of N-glycan processing α 1,2-mannosidases ( Kawar, Z., Herscovics, A., Jarvis, D.L., 1997. Isolation and characterisation of an alpha 1,2-mannosidase cDNA from the lepidopteran insect cell line Sf9. Glycobiology 7, 433–443). These enzymes have similar protein sequences, require calcium for their activities, and are sensitive to 1-deoxymannojirimycin, but can have different substrate specificities and intracellular distributions. We recently determined the substrate specificity of the insect α 1,2-mannosidase, SfManI ( Kawar, Z., Romero, P., Herscovics, A., Jarvis, D.L., 2000. N-glycan processing by a lepidopteran insect and 1,2-mannosidase. Glycobiology 10, 347–355). Now, we have examined the biosynthesis and subcellular localization of SfManI. We found that SfManI is partially N-glycosylated and that N-glycosylation is dramatically enhanced if the wild type sequon is changed to one that is highly utilized in a mammalian system. We also found that an SfManI-GFP fusion protein had a punctate cytoplasmic distribution in insect cells. Colocalization studies indicated that this fusion protein is localized in the Golgi apparatus, not in the endoplasmic reticulum or lysosomes. Finally, N-glycosylation had no influence over the substrate specificity or subcellular localization of SfManI.

Biological Characterization of a Nucleopolyhedrovirus of Spodoptera litura (Lepidoptera: Noctuidae) Isolated from the Philippines

Abstract A Philippine Spodoptera litura nucleopolyhedrovirus (SltMNPV) was isolated by plaque purification from an uncloned NPV population in a formulated product that exhibits high insecticidal activity against the onion-attacking common cutworm, S. litura. Its estimated genome size was approximately 142 kb. Comparison with Autographa californica MNPV and Spodoptera exigua NPV by restriction fragmentation analysis showed that it was a distinct isolate. Light microscopy examination showed that SltMNPV-P7 infected insect cell lines derived from Spodoptera littoralis (CLS-79), Spodoptera frugiperda (Sf9), and S. litura (TUAT-SpLi221). It produced typical cytopathic effects in cell lines established from Bombyx mori and S. exigua and induced apoptotic-like cytopathology in Lymantria dispar -derived cell line; it exerted no observable cytopathic effect on Spilosoma imparilis cell line. Significant increase in budded virus (BV) production was observed in CLS-79, Sf9, and TUAT-SpLi221 cell lines, in which TUAT-SpLi221 cell line supported the highest BV titer. No significant BV production was observed in the remaining four cell lines. Similarly, viral DNA replication and polyhedrin production proceeded only in these three spodopteran cell lines. Results showed that SltMNPV-P7 is a unique isolate which can be propagated in vitro for further studies and thus has a potential for development into a more effective microbial insecticide.

Characterization of an iridescent virus isolated from Gryllus bimaculatus (Orthoptera: Gryllidae).

We have isolated an iridescent virus from commercially produced colonies of Gryllus bimaculatus in Germany, which showed apparent mortality. Transmission electron microscopy studies on adult cricket specimens revealed the paracrystalline assembly of icosahedral virus particles in the cytoplasm of hypertrophied abdominal fat body cells. The infecting agent could be cultivated in the lepidopteran cell line sf-9, where it caused cytopathogenic effects such as cell hypertrophy, cytoplasmic vacuolization, and cell death within 8 days postinfection. Infection titers of the first virus passage reached 10(7.5) TCID(50)/ml. Negatively stained virus particles (n = 100) had dimensions of 172 +/- 6 nm (apex to apex) and 148 +/- 5 nm (side to side). SDS-polyacrylamide gel electrophoresis of virus proteins showed more than 20 distinct polypeptides with a major species of approximately 50 kDa. Analysis of the restriction fragment length profiles from digestion of purified viral DNA with the endonucleases EcoRI, BamHI, and HindIII showed marked differences from the profiles of iridoviruses of lower vertebrates (genus Ranavirus), e.g., Rana esculenta Iridovirus and Frog virus 3. Restriction enzyme digests with the endonucleases MspI and HpaII indicated the lack of methylation of viral DNA. Polymerase chain reaction led to the amplification of a 420-bp gene fragment with 97% sequence homology to the major capsid protein gene of Chilo iridescent virus. These data indicate that this new isolate, which we propose to be termed Gryllus bimaculatus iridescent virus, belongs to the genus Iridovirus of the family Iridoviridae.

A baculovirus superinfection system: efficient vehicle for gene transfer into Drosophila S2 cells.

The baculovirus expression vector system is considered to be a safe, powerful, but cell-lytic heterologous protein expression system in insect cells. We show here that there is a new baculovirus system for efficient gene transfer and expression using the popular and genetically well-understood Drosophila S2 cells. The recombinant baculovirus was constructed to carry an enhanced green fluorescent protein under the control of polyhedrin promoter as a fluorescent selection marker in the Sf21 cell line. Recombinant baculoviruses were then used to transduce S2 cells with target gene expression cassettes containing a Drosophila heat shock protein 70, an actin 5C, or a metallothionein promoter. Nearly 100% of the S2 cells showed evidence of gene expression after infection. The time course for the optimal protein expression peaked at 24 to 36 h postinfection, which is significantly earlier than a polyhedrin-driven protein expression in Sf21 cells. Importantly, S2 cells did not appear to be lysed after infection, and the protein expression levels are comparable to those of proteins under the control of polyhedrin promoter in several lepidopteran cell lines. Most surprisingly, S2 cells permit repetitive infections of multiple baculoviruses over time. These findings clearly suggest that this baculovirus-S2 system may effect the efficient gene transfer and expression system of the well-characterized Drosophila S2 cells.

Retinoic acid stimulates erythropoietin gene transcription in embryonal carcinoma cells through the direct repeat of a steroid/thyroid hormone receptor response element half-site in the hypoxia-response enhancer

We have previously reported that expression of the erythropoietin (Epo) gene in mouse embryonal cells was not induced by hypoxia, although hypoxia induced other hypoxia-inducible genes. This study identifies retinoic acid (RA) as an inducer for Epo production in the embryonal carcinoma cell lines P19 and F9. RA induced Epo production through the transcriptional activation of the Epo gene in an oxygen-independent manner. With the use of reporter assays in P19 cells, it is shown that a direct repeat of the nuclear hormone receptor-binding motif separated by a 2-bp spacer (DR-2) in the hypoxia-response enhancer was responsible for the transcriptional activation by RA. Electrophoretic mobility shift assays show that nuclear extracts from P19 cells contained RA receptor complexes that bound to DR-2. In human hepatoma Hep3B cells, an orphan receptor, hepatocyte nuclear factor-4, strongly augmented hypoxic induction of the Epo gene in cooperation with hypoxia-inducible factor-1 (HIF-1) by binding to DR-2, whereas in P19 cells, the interaction of RA receptors with DR-2 was sufficient for RA-induced transcriptional activation of the Epo gene without the requirement of the HIF-1 site. These results suggest that DR-2 regulates expression of the Epo gene by acting as the binding site for different transcription factors in different types of cells.

Retinoic acid stimulates erythropoietin gene transcription in embryonal carcinoma cells through the direct repeat of a steroid/thyroid hormone receptor response element half-site in the hypoxia-response enhancer

AbstractWe have previously reported that expression of the erythropoietin (Epo) gene in mouse embryonal cells was not induced by hypoxia, although hypoxia induced other hypoxia-inducible genes. This study identifies retinoic acid (RA) as an inducer for Epo production in the embryonal carcinoma cell lines P19 and F9. RA induced Epo production through the transcriptional activation of the Epo gene in an oxygen-independent manner. With the use of reporter assays in P19 cells, it is shown that a direct repeat of the nuclear hormone receptor-binding motif separated by a 2-bp spacer (DR-2) in the hypoxia-response enhancer was responsible for the transcriptional activation by RA. Electrophoretic mobility shift assays show that nuclear extracts from P19 cells contained RA receptor complexes that bound to DR-2. In human hepatoma Hep3B cells, an orphan receptor, hepatocyte nuclear factor-4, strongly augmented hypoxic induction of the Epo gene in cooperation with hypoxia-inducible factor-1 (HIF-1) by binding to DR-2, whereas in P19 cells, the interaction of RA receptors with DR-2 was sufficient for RA-induced transcriptional activation of the Epo gene without the requirement of the HIF-1 site. These results suggest that DR-2 regulates expression of the Epo gene by acting as the binding site for different transcription factors in different types of cells.

Inhibition of [ 3H]ponasterone A binding by ecdysone agonists in the intact Sf-9 cell line

Ecdysone agonists, including dibenzoylhydrazines, significantly inhibited the binding of [ 3H]ponasterone A ([ 3H]PoA) in intact Sf-9 cells ( Spodoptera frugiperda). The amount of [ 3H]PoA binding varied in a concentration-dependent manner. According to the IC 50, concentration at which there is 50% inhibition, the order of potency of typical ecdysone agonists is tebufenozide (RH-5992) > methoxyfenozide (RH-2485) > PoA > 20-hydroxyecdysone > cyasterone > RH-5849, makisterone A ≥ inokosterone > ecdysone. The ranking is consistent with that obtained from a cultured integument system of the rice stem borer Chilo suppressalis except for methoxyfenozide. Other compounds whose modes of action are different from that of ecdysteroids, for example respiration inhibitors, plant steroid hormones, and chitin synthesis inhibitors, did not inhibit the binding of [ 3H]PoA significantly. The mammalian hormones estradiol and diethylstilbestrol, and a secondary bile acid, lithocholic acid, significantly inhibited the binding of [ 3H]PoA at 25 μM. However, their binding activity in terms of pIC 50 was either very low or not evaluated.

Endogenous glutathione-binding proteins of insect cell lines: characterization and removal from glutathione S-transferase (GST) fusion proteins.

After affinity purification on immobilized glutathione, insect-cell-derived glutathione S-transferase (GST) fusion proteins contain variable amounts of protein contaminants of about 23-24 kDa. We have isolated these glutathione-binding proteins from the widely used Sf9 and Hi5 insect cell lines and characterized them by LC-MS and N-terminal sequencing. Based on the observation that these proteins have higher affinity for glutathione than GST fusions, we have found that by using differential elution conditions the amount of such contaminants in GST fusion preparations can be strongly reduced directly during the affinity purification step. The main interest of these results is that they are not restricted to a specific construct, but rather they seem to apply to various insect-cell-derived GST fusions.

Adaptation of an insect cell line of Spodoptera frugiperda to grow at 37 degrees C: characterization of an endodiploid clone.

Sf21 and Sf9 cell lines established from the lepidoptera Spodoptera frugiperda do not display major induction of heat shock proteins when exposed to a temperature of 37 degrees C. After some months of adaptation at 37 degrees C we obtained two new cell lines, Sf21-HT and Sf9-HT, which have now been established for several years in our laboratory. The Sf9-HT line displays a slightly shorter doubling time at 37 degrees C than the wild type at 28 degrees C, but cell lethality gives rise to an earlier growth arrest. The composition of total lipid extract from heat-adapted cells reveals a higher sphingomyelin to phosphatidylcholine ratio and a higher percentage of saturated fatty acids, which are expected for the lower membrane fluidity, required for thermotolerance. The cell volume of Sf9-HT is doubled, and by flow cytometry we showed that the DNA content is twice that in the parental cell line. Karyotypic examination of metaphasic cells achieved under epifluorescence microscopy revealed a doubled chromosome number in Sf9-HT.

Constitutive Secretion of an Endogenous Insulin-like Peptide Binding Protein with High Affinity for Insulin in Spodoptera frugiperda (Sf9) Cell Cultures

Serum-free medium from batch cultures of Sf9 insect cells was examined for the occurrence of proteins related to the insulin-like growth factor family. We found that the Sf9 cell line constitutively produced and secreted a soluble protein with a MW of 27 kDa that exerted specific binding to human insulin-like growth factor-I (IGF-I) and -II. Moreover, the secreted protein bound human insulin and human proinsulin with higher affinity than IGF-I and -II. The order of affinity to the insulin peptides, determined by competitive inhibition of ligand binding, was: insulin > proinsulin > IGF-I ⪢ IGF-II. The dissociation constant (kd) for IGF-II was 28.5 ± 1.7 nM and for insulin 7.2 ± 1.3 nM, as determined by Scatchard plot analysis. The results suggest that the Sf9 cells produce an insulin binding protein similar to the human insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1).

High throughput detection of retrovirus-associated reverse transcriptase using an improved fluorescent product enhanced reverse transcriptase assay and its comparison to conventional detection methods

The development and application of a novel, sensitive TaqMan fluorescent probe-based product enhanced RT test (F-PERT) for the detection of retrovirus are described. The assay allows discrimination between the amplification signals generated by genuine positive signals that result from retroviral RT activity and the RT-like activity from DNA polymerases. The RT-like activity from DNA polymerases was suppressed by the addition of activated calf-thymus DNA with no reduction in the RT activity. A linear relationship between threshold cycle ( C T) and the number of virus particles was demonstrated, allowing quantification of retroviruses in unknown samples. The F-PERT assay was able to detect a wide range of retroviral RT activities, including that from porcine endogenous retrovirus (PoERV), murine leukaemia virus (MLV), simian foamy virus (SFV), simian immunodeficiency virus (SIV mac) and squirrel monkey retrovirus (SMRV). The detection limit of SMRV, MLV and PoERV was approximately 100 virion particles and the test was able to detect at least 10 2 molecules of purified RT enzyme. RT activity was not detected in cellular lysates and supernatants from MRC-5, BT, VERO, or Raji cells, whereas RT activity was detected in C127I, Mus dunni, K-Balb, BHK-21, CHO-K1, SP2/0-Ag14 and NSO cell supernatants. RT activity was also detected in the Spodoptera cell line Sf9.

Spodoptera littoralis Type B Nucleopolyhedrovirus Infection of a Grasshopper Cell Line

We determined that the type B nucleopolyhedrovirus of the Egyptian cottonworm, Spodoptera littoralis (SpliNPV), can infect a cell line derived from a grasshopper. We compared the infectivity of SpliNPV in two lepidopteran cell lines (Sf9 and Md210) and in a cell line (MSE4) derived from the western migratory grasshopper, Melanoplus sanguinipes (Orthoptera: Acrididae). Both Sf9 and MSE4 cells were permissive for SpliNPV replication and supported production of viable progeny. Md210 cells were nonpermissive for SpliNPV, and although the virus entered into these cells, they supported neither viral replication nor production of viable progeny. Infection of MSE4 cells with SpliNPV resulted in cytopathic effects within 48 h post infection and complete destruction of the cells within 5 days. Both virions and polyhedra were detected within virus-infected MSE4 cells by transmisstion electron microscopy. Extracellular virions were detected in the culture medium and were infectious to Sf9 cells, indicating that the MSE4 cells supported production of viable virus progeny.

Preliminary assessment of extrastriatal dopamine d-2 receptor binding in the rodent and nonhuman primate brains using the high affinity radioligand, 18F-fallypride

We have identified the value of 18F-fallypride {( S)- N-[(1-allyl-2-pyrrolidinyl)methyl]-5-(3-[ 18F]fluoropropyl)-2,3-dimethoxybenzamide}, as a dopamine D-2 receptor radiotracer for the study of striatal and extrastriatal receptors. Fallypride exhibits high affinities for D-2 and D-3 subtypes and low affinity for D-4 ( 3H-spiperone IC 50s: D-2 = 0.05 nM [rat striata], D-3 = 0.30 nM [SF9 cell lines, rat recombinant], and D-4 = 240 nM [CHO cell lines, human recombinant]). Biodistribution in the rat brain showed localization of 18F-fallypride in striata and extrastriatal regions such as the frontal cortex, parietal cortex, amygdala, hippocampus, thalamus, and hypothalamus. In vitro autoradiographic studies in sagittal slices of the rat brain showed localization of 18F-fallypride in striatal and several extrastriatal regions, including the medulla. Positron emission tomography (PET) experiments with 18F-fallypride in male rhesus monkeys were carried out in a PET VI scanner. In several PET experiments, apart from the specific binding seen in the striatum, specific binding of 18F-fallypride was also identified in extracellular regions (in a lower brain slice, possibly the thalamus). Specific binding in the extrastriata was, however, significantly lower compared with that observed in the striata of the monkeys (extrastriata/cerebellum = 2, striata/cerebellum = 10). Postmortem analysis of the monkey brain revealed significant 18F-fallypride binding in the striata, whereas binding was also observed in extrastriatal regions such as the thalamus, cortical areas, and brain stem.

Use of inhibitors to characterize intermediates in the processing of N-glycans synthesized by insect cells: a metabolic study with Sf9 cell line.

The most frequent type of N-glycan synthesized by lepidopteran Sf9 cells appears to be fucosylated Man3GlcNAc2,and this has been a limitation for a large scale production and utilization of therapeutic glycoproteins in cultured insect cells. The current knowledge of the protein glycosylation pathway derived from structural studies on recombinant glyco-proteins expressed by using baculovirus vectors. In this work we provide more direct evidence for the sequential events occurring in the processing of endogenous N-glycoproteins of noninfected Sf9 cells. By metabolic labeling with radioactive mannose, we characterized the glycan structures which accumulated in the presence of processing inhibitors (castanospermine and swainsonine) and in the presence of an intracellular trafficking inhibitor (monensin). We thus demonstrated that from the glycan precursor Glc3Man9GlcNAc2 to GlcNAcMan5(Fuc)GlcNAc2 intermediate, the processing pathway in Sf9 cells paralleled the one demonstrated in mammalian cells. By using monensin, we demonstrated the formation of Man3(Fuc)GlcNAc2 from GlcNAcMan3(Fuc)GlcNAc2, a reaction which has not been described in mammalian cells. Our results support the idea that the hexosaminidase activity is of physiological relevance to the glycosylation pathway and is Golgi located.