Reduced mannosidase MAN1A1 expression leads to aberrant N-glycosylation and impaired survival in breast cancer.

BACKGROUND: Alterations in protein glycosylation have been related to malignant transformation and tumour progression. We recently showed that low mRNA levels of Golgi alpha-mannosidase MAN1A1 correlate with poor prognosis in breast cancer patients. METHODS: We analysed the role of MAN1A1 on a protein level using western blot analysis (n=105) and studied the impact of MAN1A1-related glycosylation on the prognostic relevance of adhesion molecules involved in breast cancer using microarray data (n=194). Functional consequences of mannosidase inhibition using the inhibitor kifunensine or MAN1A1 silencing were investigated in breast cancer cells in vitro. RESULTS: Patients with low/moderate MAN1A1 expression in tumours showed significantly shorter disease-free intervals than those with high MAN1A1 levels (P=0.005). Moreover, low MAN1A1 expression correlated significantly with nodal status, grading and brain metastasis. At an mRNA level, membrane proteins ALCAM and CD24 were only significantly prognostic in tumours with high MAN1A1 expression. In vitro, reduced MAN1A1 expression or mannosidase inhibition led to a significantly increased adhesion of breast cancer cells to endothelial cells. CONCLUSIONS: Our study demonstrates the prognostic role of MAN1A1 in breast cancer by affecting the adhesive properties of tumour cells and the strong influence of this glycosylation enzyme on the prognostic impact of some adhesion proteins.

Cloning, Expression and Biochemical Characterization of Endomannanases from Thermobifida Species Isolated from Different Niches.

Thermobifidas are thermotolerant, compost inhabiting actinomycetes which have complex polysaccharide hydrolyzing enzyme systems. The best characterized enzymes of these hydrolases are cellulases from T. fusca, while other important enzymes especially hemicellulases are not deeply explored. To fill this gap we cloned and investigated endomannanases from those reference strains of the Thermobifida genus, which have published data on other hydrolases (T. fusca TM51, T. alba CECT3323, T. cellulosilytica TB100T and T. halotolerans YIM90462T). Our phylogenetic analyses of 16S rDNA and endomannanase sequences revealed that T. alba CECT3323 is miss-classified; it belongs to the T. fusca species. The cloned and investigated endomannanases belong to the family of glycosyl hydrolases 5 (GH5), their size is around 50 kDa and they are modular enzymes. Their catalytic domains are extended by a C-terminal carbohydrate binding module (CBM) of type 2 with a 23-25 residues long interdomain linker region consisting of Pro, Thr and Glu/Asp rich repetitive tetrapeptide motifs. Their polypeptide chains exhibit high homology, interdomain sequence, which don't show homology to each other, but all of them are built up from 3-6 times repeated tetrapeptide motifs) (PTDP-Tc, TEEP-Tf, DPGT-Th). All of the heterologously expressed Man5A enzymes exhibited activity only on mannan. The pH optima of Man5A enzymes from T. halotolerans, T. cellulosilytica and T. fusca are slightly different (7.0, 7.5 and 8.0, respectively) while their temperature optima span within the range of 70-75°C. The three endomannanases exhibited very similar kinetic performances on LBG-mannan substrate: 0.9-1.7mM of KM and 80-120 1/sec of turnover number. We detected great variability in heat stability at 70°C, which was influenced by the presence of Ca2+. The investigated endomannanases might be important subjects for studying the structure/function relation behind the heat stability and for industrial applications to hemicellulose degradation.

Recombinant endo-mannanase (ManB-1601) production using agro-industrial residues: Development of economical medium and application in oil extraction from copra.

Expression of pRSETA manb-1601 construct in Hi-Control Escherichia coli BL21 (DE3) cells improved recombinant endo-mannanase (ManB-1601) production by 2.73-fold (1821±100U/ml). A low-cost, agro-industrial residue supplemented industrial medium for enhanced and economical production of ManB-1601 was developed in two mutual phases. Phase-I revealed the potential of various pre- (induction time: 5h, induction mode: lactose 0.5mM) and post-induction [peptone supplementation: 0.94%(w/v), glycerol 0.123%(v/v)] parameters for enhanced production of ManB-1601 and resulted in 4.61-fold (8406±400U/ml) and 2.53-fold (3.30g/l) higher ManB-1601 and biomass production, respectively. Under phase-II, economization of phase-I medium was carried out by reducing/replacing costly ingredients with solubilized-defatted flax seed meal (S-DFSM), which resulted in 3.25-fold (5926U/ml) higher ManB-1601 production. Industrial potential of ManB-1601 was shown in oil extraction from copra as enzyme treatment led to cracks, peeling, fracturing and smoothening of copra, which facilitated higher (18.75%) oil yield.

α-Mannosidase I Protein Expression in Peripheral Blood Mononuclear Cells Is Upregulated During Hepatitis B Virus Infection.

Hepatitis B virus (HBV) has been reported to be recognized by dendritic cell-specific ICAM-3-grabbing nonintegrin in the presence of the α-mannosidase I inhibitor kifunensine, whereas native HBV is not. The aim of our study was to determine whether changes in α-mannosidase I expression in peripheral blood mononuclear cells (PBMCs) occur in patients with HBV infection. Peripheral blood was collected from 90 HBV-infected patients (grouped into immune tolerance, chronic hepatitis B, or inactive carrier group based on their clinical states) and 30 healthy donors. Expression of the three α-mannosidase I subtypes, MAN1A1, MAN1A2, and MAN1C1, was measured using western blot analyses. Compared with the healthy controls, significant increases in the MAN1A1, MAN1A2, and MAN1C1 expression levels were observed in the three HBV-infected groups, among whom the immune tolerance group showed the largest increase. For the patients in the immune tolerance phase, the expression levels of both MAN1A1 and MAN1A2 were linearly and positively correlated with the hepatitis B e antigen (HBeAg) titer and HBV DNA level, although a positive correlation was only found between MAN1C1 expression and the HBeAg titer. These results indicate that increased α-mannosidase I expression in PBMCs may play an important role in HBV immune escape and that its expression level is closely related to viral replication activity.

Cloning, Expression, and Characterization of Capra hircus Golgi α-Mannosidase II.

Golgi α-mannosidase II (GMII), a key glycosyl hydrolase in the N-linked glycosylation pathway, has been demonstrated to be closely associated with the genesis and development of cancer. In this study, we cloned cDNA-encoding Capra hircus GMII (chGMII) and expressed it in Pichia pastoris expression system. The chGMII cDNA contains an open reading frame of 3432 bp encoding a polypeptide of 1144 amino acids. The deduced molecular mass and pI of chGMII was 130.5 kDa and 8.04, respectively. The gene expression profile analysis showed GMII was the highest expressed gene in the spleen. The recombinant chGMII showed maximum activity at pH 5.4 and 42 °C and was activated by Fe(2+), Zn(2+), Ca(2+), and Mn(2+) and strongly inhibited by Co(2+), Cu(2+), and EDTA. By homology modeling and molecular docking, we obtained the predicted 3D structure of chGMII and the probable binding modes of chGMII-GnMan5Gn, chGMII-SW. A small cavity containing Tyr355 and zinc ion fixed by residues Asp290, His176, Asp178, and His570 was identified as the active center of chGMII. These results not only provide a clue for clarifying the catalytic mechanism of chGMII but also lay a theoretical foundation for subsequent investigations in the field of anticancer therapy for mammals.

Dual functions for cytosolic α-mannosidase (Man2C1): its down-regulation causes mitochondria-dependent apoptosis independently of its α-mannosidase activity.

Cytosolic α-mannosidase (Man2C1) trims free oligosaccharides in mammalian cells, and its down-regulation reportedly delays cancer growth by inducing mitotic arrest or apoptosis. However, the mechanism by which Man2C1 down-regulation induces apoptosis is unknown. Here, we demonstrated that silencing of Man2C1 via small hairpin RNAs induced mitochondria-dependent apoptosis in HeLa cells. Expression of CHOP (C/EBP homologous protein), a transcription factor critical to endoplasmic reticulum stress-induced apoptosis, was significantly up-regulated in Man2C1 knockdown cells. However, this enhanced CHOP expression was not caused by endoplasmic reticulum stress. Interestingly, Man2C1 catalytic activity was not required for this regulation of apoptosis; introduction of mutant, enzymatically inactive Man2C1 rescued apoptotic phenotypes of Man2C1 knockdown cells. These results show that Man2C1 has dual functions: one in glycan catabolism and another in apoptotic signaling.

Gene expression of coffee seed oxidation and germination processes during drying.

Coffee (Coffea arabica L.) seeds are sensitive to desiccation and oxidative stress during drying processes. We investigated the effect of drying and moisture levels on germination-related gene expressions associated with enzymatic systems that prevent oxidative stress in coffee seeds. Coffee seeds collected at physiological maturity were subjected to slow and quick drying to 40, 30, 20, and 12% moisture levels (wet basis), and as the control, seeds without drying were used. The seeds' physiological quality was calculated as percentage of normal seedlings at 15 and 30 days, normal vigorous seedlings at 30 days, and cotyledonary leaves at 45 days. The isoenzymes esterase, catalase (CAT), peroxidase (POX), and endo-ß-mannanase expressions were electrophoretically analyzed. CAT and POX expressions were analyzed using RT-qPCR with specific primers constructed from the target gene sequences from the Brazilian Coffee Genome Database. Slow drying showed better physiological quality for seeds at 40 and 12% moisture levels, while quick drying was the most effective for seeds with 20% moisture. Sensitivity to water loss was confirmed by quick drying and activation of enzymes. CAT and POX transcriptions reduced during drying. RT-qPCR revealed a complex gene-expression pattern during the oxidative process, with high gene expression in wet seeds.

Studies on extraction of mannanase enzyme by Aspergillus terreus SUK-1 from fermented palm kernel cake.

Microbial mannanases have become biotechnologically important in industry but their application is limited due to high production cost. In presents study, the extraction of mannanase from fermented Palm Kernel Cake (PKC) in the Solid State Fermentation (SSF) was optimized. Local isolate of Aspergillus terreus SUK-1 was grown on PKC in (SSF) using column bioreactor. The optimum condition were achieved after two washes of fermented PKC by adding of 10% glycerol (v/v) soaked for 10 h at the room temperature with solvent to ratio, 1:5 (w/v).

A highly active endo-ß-1,4-mannanase produced by Cellulosimicrobium sp. strain HY-13, a hemicellulolytic bacterium in the gut of Eisenia fetida.

A xylanolytic gut bacterium isolated from Eisenia fetida, Cellulosimicrobium sp. strain HY-13, produced an extracellular glycoside hydrolase capable of efficiently degrading mannose-based substrates such as locust bean gum, guar gum, mannotetraose, and mannopentaose. The purified mannan-degrading enzyme (ManK, 34,926 Da) from strain HY-13 was found to have an N-terminal amino acid sequence of DEATTDGLHVVDD, which has not yet been identified. Under the optimized reaction conditions of 50°C and pH 7.0, ManK exhibited extraordinary high specific activities of 7109 IU/mg and 5158 IU/mg toward locust bean gum and guar gum, respectively, while the enzyme showed no effect on sugars substituted with p-nitrophenol and various non-mannose carbohydrates. Thin layer chromatography revealed that the enzyme degraded locust bean gum to mannobiose and mannotetraose. No detectable amount of mannose was produced from hydrolytic reactions with the substrates. ManK strongly attached to Avicel, ß-cyclodextrin, lignin, and poly(3-hydroxybutyrate) granules, but not bound to chitin, chitosan, curdlan, or insoluble oat spelt xylan. The aforementioned characteristics of ManK suggest that it is a unique endo-ß-1,4-mannanase without additional carbohydrolase activities, which differentiates it from other well-known carbohydrolases.

Expression and characterization of an extremely thermostable ß-glycosidase (mannosidase) from the hyperthermophilic archaeon Pyrococcus furiosus DSM3638.

Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF0356) similar to the enzymes in glycoside hydrolase family 1. This ß-glycosidase, designated PFTG (P. furiosus thermostable glycosidase), was cloned and expressed in Escherichia coli. The expressed enzyme was purified by heat treatment and Ni-NTA affinity chromatography. The gene was composed of 1,452 bp encoding 483 amino acids for a protein with a predicted molecular mass of 56,326 Da. The temperature and pH optima were 100°C and 5.0 in sodium citrate buffer, respectively. The substrate specificity of PFTG suggests that it possesses characteristics of both ß-galactosidase and ß-mannosidase activities. However, through kinetic studies by ITC (Isothermal Titration Colorimetry) which is very sensitive method for enzyme kinetics, PF0356 enzyme revealed the highest catalytic efficiency toward p-nitrophenyl-ß-d-mannopyranoside (3.02 k(cat)/K(m)) and mannobiose (4.32 k(cat)/K(m)). The enzyme showed transglycosylation and transgalactosylation activities toward cellobiose, lactose and mannooligosaccharides that could produce GOS (galactooligosaccharides) and MOS (maltooligosaccharides). This novel hyperthermostable ß-glycosidase may be useful for food and pharmaceutical applications.

Overexpression of Man2C1 leads to protein underglycosylation and upregulation of endoplasmic reticulum-associated degradation pathway.

Unfolded glycoproteins retained in the endoplasmic reticulum (ER) are degraded via the ER-associated degradation (ERAD) pathway. These proteins are subsequently transported to the cytosol and degraded by the proteasomal complex. Although the sequential events of ERAD are well described, its regulation remains poorly understood. The cytosolic mannosidase, Man2C1, plays an essential role in the catabolism of cytosolic free oligomannosides, which are released from the degraded proteins. We have investigated the impact of Man2C1 overexpression on protein glycosylation and the ERAD process. We demonstrated that overexpression of Man2C1 led to modifications of the cytosolic pool of free oligomannosides and resulted in accumulation of small Man(2-4)GlcNAc(1) glycans in the cytosol. We further correlated this accumulation with incomplete protein glycosylation and truncated lipid-linked glycosylation precursors, which yields an increase in N-glycoprotein en route to the ERAD. We propose a model in which high mannose levels in the cytosol interfere with glucose metabolism and compromise N-glycan synthesis in the ER. Our results show a clear link between the intracellular mannose-6-phosphate level and synthesis of the lipid-linked precursors for protein glycosylation. Disturbance in these pathways interferes with protein glycosylation and upregulated ERAD. Our findings support a new concept that regulation of Man2C1 expression is essential for maintaining efficient protein N-glycosylation.

Efficient recombinant expression and secretion of a thermostable GH26 mannan endo-1,4-beta-mannosidase from Bacillus licheniformis in Escherichia coli.

BACKGROUND: Mannans are one of the key polymers in hemicellulose, a major component of lignocellulose. The Mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannanase (EC 3.2.1.78), commonly named beta-mannanase, is an enzyme that can catalyze random hydrolysis of beta-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans. The enzyme has found a number of applications in different industries, including food, feed, pharmaceutical, pulp/paper industries, as well as gas well stimulation and pretreatment of lignocellulosic biomass for the production of second generation biofuel. Bacillus licheniformis is a Gram-positive endospore-forming microorganism that is generally non-pathogenic and has been used extensively for large-scale industrial production of various enzymes; however, there has been no previous report on the cloning and expression of mannan endo-1,4-beta-mannosidase gene (manB) from B. licheniformis. RESULTS: The mannan endo-1,4-beta-mannosidase gene (manB), commonly known as beta-mannanase, from Bacillus licheniformis strain DSM13 was cloned and overexpressed in Escherichia coli. The enzyme can be harvested from the cell lysate, periplasmic extract, or culture supernatant when using the pFLAG expression system. A total activity of approximately 50,000 units could be obtained from 1-l shake flask cultures. The recombinant enzyme was 6 x His-tagged at its C-terminus, and could be purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The specific activity of the purified enzyme when using locust bean gum as substrate was 1672 +/- 96 units/mg. The optimal pH of the enzyme was between pH 6.0 - 7.0; whereas the optimal temperature was at 50 - 60 degrees C. The recombinant beta-mannanase was stable within pH 5 - 12 after incubation for 30 min at 50 degrees C, and within pH 6 - 9 after incubation at 50 degrees C for 24 h. The enzyme was stable at temperatures up to 50 degrees C with a half-life time of activity (tau1/2) of approximately 80 h at 50 degrees C and pH 6.0. Analysis of hydrolytic products by thin layer chromatography revealed that the main products from the bioconversion of locus bean gum and mannan were various manno-oligosaccharide products (M2 - M6) and mannose. CONCLUSION: Our study demonstrates an efficient expression and secretion system for the production of a relatively thermo- and alkali-stable recombinant beta-mannanase from B. licheniformis strain DSM13, suitable for various biotechnological applications.

Optimization of the production of thermostable endo-beta-1,4 mannanases from a newly isolated Aspergillus niger gr and Aspergillus flavus gr.

The aim of this work was to establish optimal conditions for the maximum production of endo-beta-1,4 mannanases using cheaper sources. Eight thermotolerant fungal strains were isolated from garden soil and compost samples collected in and around the Gulbarga University campus, India. Two strains were selected based on their ability to produce considerable endo-beta-1,4 mannanases activity while growing in liquid medium at 37 degrees C with locust bean gum (LBG) as the only carbon source. They were identified as Aspergillus niger gr and Aspergillus flavus gr. The experiment to evaluate the effect of different carbon sources, nitrogen sources, temperatures and initial pH of the medium on maximal enzyme production was studied. Enzyme productivity was influenced by the type of polysaccharide used as the carbon source. Copra meal defatted with n-hexane showed to be a better substrate than LBG and guar gum for endo-beta-1,4 mannanases production by A. niger gr (40.011 U/ml), but for A. flavus gr (33.532 U/ml), the difference was not significant. Endo-beta-1,4 mannanases produced from A. niger gr and A. flavus gr have high optimum temperature (65 and 60 degrees C) and good thermostability in the absence of any stabilizers (maintaining 50% of residual activity for 8 and 6 h, respectively, at 60 degrees C) and are stable over in a wide pH range. These new strains offer an attractive alternative source of enzymes for the food and feed processing industries.

Screening and identification of endomannanase-producing microfungi from hypersaline environments.

A culture-dependent enrichment technique was used to isolate endo-1,4-beta-mannanase-producing fungi from a hypersaline environment. Galactomannan was used as carbon source and resulted in isolation of strains of Scopulariopsis brevicaulis, S. candida, and Verticillium dahliae. The Scopulariopsis isolates were found to be more dominant and could be isolated from consecutive evaporation ponds, whereas Verticillium was only isolated from one pond. The Scopulariopsis strains exhibited only endomannanase activity, whereas Verticillium displayed broad-activity spectrum by secreting endoxylanases and cellulases in addition to endomannanases. S. candida LMK004 and LMK008 produced 7,420 and 14,750 nkat g(-1) biomass, respectively. Endomannanase production in these strains increased with an increase in NaCl concentration up to 10% (w/v), after which both growth and enzyme production was decreased. V. dahliae LMK006 grew and produced up to 5,000 nkat g(-1) biomass endomannanase in the absence of NaCl. Increased NaCl concentration had a negative effect on this strain. S. brevicaulis LMK002 showed poor endomannanase production but a similar growth trend as the other Scopulariopsis strains. In general, the Scopulariopsis strains exhibited better halotolerance than V. dahliae and could grow in the presence of 20% NaCl on solid medium.

Heterologous production, assembly, and secretion of a minicellulosome by Clostridium acetobutylicum ATCC 824.

The gene man5K encoding the mannanase Man5K from Clostridium cellulolyticum was cloned alone or as an operon with the gene cipC1 encoding a truncated scaffoldin (miniCipC1) of the same origin in the solventogenic Clostridium acetobutylicum. The expression of the heterologous gene(s) was under the control of a weakened thiolase promoter Pthl. The recombinant strains of the solventogenic bacterium were both found to secrete active Man5K in the range of milligrams per liter. In the case of the strain expressing only man5K, a large fraction of the recombinant enzyme was truncated and lost the N-terminal dockerin domain, but it remained active towards galactomannan. When man5K was coexpressed with cipC1 in C. acetobutylicum, the recombinant strain secreted almost exclusively full-length mannanase, which bound to the scaffoldin miniCipC1, thus showing that complexation to the scaffoldin stabilized the enzyme. The secreted heterologous complex was found to be functional: it binds to crystalline cellulose via the carbohydrate binding module of the miniscaffoldin, and the complexed mannanase is active towards galactomannan. Taken together, these data show that C. acetobutylicum is a suitable host for the production, assembly, and secretion of heterologous minicellulosomes.

Intact {alpha}-1,2-endomannosidase is a typical type II membrane protein.

Rat endomannosidase is a glycosidic enzyme that catalyzes the cleavage of di-, tri-, or tetrasaccharides (Glc(1-3)Man), from N-glycosylation intermediates with terminal glucose residues. To date it is the only characterized member of this class of endomannosidic enzymes. Although this protein has been demonstrated to localize to the Golgi lumenal membrane, the mechanism by which this occurs has not yet been determined. Using the rat endomannosidase sequence, we identified three homologs, one each in the human, mouse, and rat genomes. Alignment of the four encoded protein sequences demonstrated that the newly identified sequences are highly conserved but differed significantly at the N-terminus from the previously reported protein. In this study we have cloned two novel endomannosidase sequences from rat and human cDNA libraries, but were unable to amplify the open reading frame of the previously reported rat sequence. Analysis of the rat genome confirmed that the 59- and 39-termini of the previously reported sequence were in fact located on different chromosomes. This, in combination with our inability to amplify the previously reported sequence, indicated that the N-terminus of the rat endomannosidase sequence previously published was likely in error (a cloning artifact), and that the sequences reported in the current study encode the intact proteins. Furthermore, unlike the previous sequence, the three ORFs identified in this study encode proteins containing a single N-terminal transmembrane domain. Here we demonstrate that this region is responsible for Golgi localization and in doing so confirm that endomannosidase is a type II membrane protein, like the majority of other secretory pathway glycosylation enzymes.

Monitoring of the tissue distribution of fibroblast growth factor containing a high mannose-type sugar chain produced in mutant yeast.

Most therapeutic glycoproteins have been produced in mammalian cell lines. However, the mammalian cell culture system has various disadvantages, i.e., a high culture cost, difficulty in performing a large scale-up because of complicated handling requirements, and the risk of contamination by prion or other unknown pathogenic components through cultivation in the presence of bovine serum. There is thus a growing need for other host cells in which the recombinant glycoproteins can be produced. Recently, we successfully developed a mutant yeast strain engineered in a glycosylation system. The sugar chain produced in the mutant yeast is not immunogenic to the human immuno-surveillance system. In the present study, we selected fibroblast growth factor (FGF) as a model glycoprotein and assessed the bioactivity of FGF produced in yeast in terms of its proliferating activity and tissue distribution in mammalian cells and in the whole body. Structural changes in the sugar chains of FGFs derived from mutant yeast, as compared with those from mammalian cells, did not affect the proliferating activity remarkably. However, the tissue distribution in the mouse differed significantly; a high-mannose type sugar chain was the major determinant of the specific distribution of FGF to the kidney. The mechanism of this phenomenon is still unclear, but our observations suggest that recombinant glycoproteins derived from mutant yeasts producing high-mannose type sugar chains would be applicable for tissue-targeting therapy.

Engineering the protein N-glycosylation pathway in insect cells for production of biantennary, complex N-glycans.

Insect cells, like other eucaryotic cells, modify many of their proteins by N-glycosylation. However, the endogenous insect cell N-glycan processing machinery generally does not produce complex, terminally sialylated N-glycans such as those found in mammalian systems. This difference in the N-glycan processing pathways of insect cells and higher eucaryotes imposes a significant limitation on their use as hosts for baculovirus-mediated recombinant glycoprotein production. To address this problem, we previously isolated two transgenic insect cell lines that have mammalian beta1,4-galactosyltransferase or beta1,4-galactosyltransferase and alpha2,6-sialyltransferase genes. Unlike the parental insect cell line, both transgenic cell lines expressed the mammalian glycosyltransferases and were able to produce terminally galactosylated or sialylated N-glycans. The purpose of the present study was to investigate the structures of the N-glycans produced by these transgenic insect cell lines in further detail. Direct structural analyses revealed that the most extensively processed N-glycans produced by the transgenic insect cell lines were novel, monoantennary structures with elongation of only the alpha1,3 branch. This led to the hypothesis that the transgenic insect cell lines lacked adequate endogenous N-acetylglucosaminyltransferase II activity for biantennary N-glycan production. To test this hypothesis and further extend the N-glycan processing pathway in Sf9 cells, we produced a new transgenic line designed to constitutively express a more complete array of mammalian glycosyltransferases, including N-acetylglucosaminyltransferase II. This new transgenic insect cell line, designated SfSWT-1, has higher levels of five glycosyltransferase activities than the parental cells and supports baculovirus replication at normal levels. In addition, direct structural analyses showed that SfSWT-1 cells could produce biantennary, terminally sialylated N-glycans. Thus, this study provides new insight on the glycobiology of insect cells and describes a new transgenic insect cell line that will be widely useful for the production of more authentic recombinant glycoproteins by baculovirus expression vectors.

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.

alpha-Mannosidosis in the guinea pig: cloning of the lysosomal alpha-mannosidase cDNA and identification of a missense mutation causing alpha-mannosidosis.

alpha-Mannosidosis is a lysosomal storage disorder caused by deficient activity of the lysosomal alpha-mannosidase. We report here the sequencing and expression of the lysosomal alpha-mannosidase cDNA from normal and alpha-mannosidosis guinea pigs. The amino acid sequence of the guinea pig enzyme displayed 82-85% identity to the lysosomal alpha-mannosidase in other mammals. The cDNA of the alpha-mannosidosis guinea pig contained a missense mutation, 679C>T, leading to substitution of arginine by tryptophan at amino acid position 227 (R227W). The R227W allele segregated with the alpha-mannosidosis genotype in the guinea pig colony and introduction of R227W into the wild-type sequence eliminated the production of recombinant alpha-mannosidase activity in heterologous expression studies. Furthermore, the guinea pig mutation has been found in human patients. Our results strongly indicate that the 679C>T mutation causes alpha-mannosidosis and suggest that the guinea pig will be an excellent model for investigation of pathogenesis and evaluation of therapeutic strategies for human alpha-mannosidosis.