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We describe isolation and characterization of the gene encoding the glucosidase II alpha subunit (GIIα) of the industrially important fungus Trichoderma reesei. This subunit is the catalytic part of the glucosidase II heterodimeric enzyme involved in the structural modification within the endoplasmic reticulum (ER) of N-linked oligosaccharides present on glycoproteins. The gene encoding GIIα (gls2α) in the hypercellulolytic strain Rut-C30 contains a frameshift mutation resulting in a truncated gene product. Based on the peculiar monoglucosylated N-glycan pattern on proteins produced by the strain, we concluded that the truncated protein can still hydrolyze the first α-1,3-linked glucose residue but not the innermost α-1,3-linked glucose residue from the Glc2Man9GlcNAc2 N-glycan ER structure. Transformation of the Rut-C30 strain with a repaired T. reesei gls2α gene changed the glycosylation profile significantly, decreasing the amount of monoglucosylated structures and increasing the amount of high-mannose N-glycans. Full conversion to highmannose carbohydrates was not obtained, and this was probably due to competition between the endogenous mutant subunit and the introduced wild-type GIIα protein. Since glucosidase II is also involved in the ER quality control of nascent polypeptide chains, its transcriptional regulation was studied in a strain producing recombinant tissue plasminogen activator (tPA) and in cultures treated with the stress agents dithiothreitol (DTT) and brefeldin A (BFA), which are known to block protein transport and to induce the unfolded protein response. While the mRNA levels were clearly upregulated upon tPA production or BFA treatment, no such enhancement was observed after DTT addition. [ABSTRACT FROM AUTHOR]

Abstract: The yeast transcriptional response to murine Bax expression was compared with the changes induced by H2O2 treatment via microarray technology. Although most of the Bax-responsive genes were also triggered by H2O2 treatment, OYE3, ICY2, MLS1 and BTN2 were validated to have a Bax-specific transcriptional response not shared with the oxidative stress trigger. In knockout experiments, only deletion of OYE3, coding for yeast Old yellow enzyme, attenuated the rate of Bax-induced growth arrest, cell death and NADPH decrease. Lipid peroxidation was completely absent in ¿OYE3 expressing Bax. However, the absence of OYE3 sensitized yeast cells to H2O2-induced cell death, and increased the rate of NADPH decrease and lipid peroxidation. Our results clearly indicate that OYE3 interferes with Bax- and H2O2-induced lipid peroxidation and cell death in Saccharomyces cerevisiae. [Copyright &y& Elsevier]

Ferritin is one of the major eukaryotic proteins involved in regulating iron metabolism and maintaining iron homeostasis. However, Saccaromyces cerevisiae is an exception, possessing no ferritin and using other means to store excess iron. The only potential iron storage protein identified in yeast so far is the homologue of human frataxin (YFH1p). In this study, we found that dysfunction of yeast frataxin shortens mean lifespan by 49% compared to the WT control. Interestingly, the human ferritin L gene can, at least partially, complement the function of yeast frataxin, extending lifespan and protecting cells from death induced by oxidative stress or excess iron. Our findings indicate that ferritin L can perform functions in yeast that are similar to its functions in mammals, and suggest that common mechanisms may exist for preventing iron and oxidative damage in single- and multi-cellular eukaryotic organisms. Clearly, elucidation of the function of human ferritin in yeast would help in gaining a better understanding the molecular basis of iron storage diseases. [Copyright &y& Elsevier]

The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an α-l,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and β-l,4- galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides. [ABSTRACT FROM AUTHOR]

We applied our 'clinical glycomics' technology, based on DNA sequencer/fragment analyzers, to generate profiles of serum protein N-glycans of liver disease patients. This technology yielded a biomarker that distinguished compensated cirrhotic from noncirrhotic chronic liver disease patients, with 79% sensitivity and 86% specificity (100% sensitivity and specificity for decompensated cirrhosis). In combination with the clinical chemistry-based Fibrotest biomarker, compensated cirrhosis was detected with 100% specificity and 75% sensitivity. The current 'gold standard' for liver cirrhosis detection is an invasive, costly, often painful liver biopsy. Consequently, the highly specific set of biomarkers presented could obviate biopsy in many cirrhosis patients. This biomarker combination could eventually be used in follow-up examinations of chronic liver disease patients, to yield a warning that cirrhosis has developed and that the risk of complications (such as hepatocellular carcinoma) has increased considerably. Our clinical glycomics technique can easily be implemented in existing molecular diagnostics laboratories. [ABSTRACT FROM AUTHOR]

We developed a high-throughput functional genomic screening system that allows identification of genes prolonging life-span in the baker''s yeast Saccharomyces cerevisiae. The method is based on isolating yeast mother cells with extended number of cell divisions as indicated by the increased number of bud scars on their surface. Fluorescently labelled Wheat Germ Agglutinin was used for specific staining of chitin, a major component of bud scars. Screening of a human HepG2 cDNA expression library in yeast resulted in the isolation of 12 yeast transformants with a potentially prolonged life-span. The transgene in one of the lines was identified as ferritin light chain (FTL) and studied in more detail. Yeast cells containing FTL showed an enhanced iron and H2O2 resistance, a reduced cell death rate and an increased number of cell divisions. Overexpression of FTL in the nematode Caenorhabditis elegans resulted in a life-span increase of 8% confirming our yeast observations in a multicellular organism. Our data demonstrate that this method permits a fast screening of libraries for hunting genes involved in ageing processes. [Copyright &y& Elsevier]

All currently existing eukaryotic protein expression systems are based on autonomous life forms. To exploit the potential practical benefits associated with parasitic organisms we have developed a new protein expression system based on Leishmania tarentolae (Trypanosomatidae), a protozoan parasite of lizards. To achieve strong transcription, the genes of interest were integrated into the small subunit ribosomal RNA gene. Expression levels obtained were up to 30 mg of recombinant protein per liter of suspension culture and increased linearly with the number of integrated gene copies. To assess the system''s potential for production of post-translationally modified proteins, we have expressed human erythropoietin in L. tarentolae. The recombinant protein isolated from the culture supernatants was biologically active, natively processed at the N-terminus, and N-glycosylated. The N-glycosylation was exceptionally homogenous, with a mammalian-type biantennary oligosaccharide and the Man3GlcNAc2 core structure accounting for >90% of the glycans present. L. tarentolae is thus the first described biotechnologically useful unicellular eukaryotic organism producing biantennary fully galactosylated, core-α-1,6-fucosylated N-glycans. [Copyright &y& Elsevier]

A20 is a Cys2/Cys2 zinc finger protein which is induced by a variety of inflammatory stimuli and which has been characterized as an inhibitor of cell death by a yet unknown mechanism. In order to clarify its molecular mechanism of action, we used the yeast two-hybrid system to screen for proteins that interact with A20. A cDNA fragment was isolated which encoded a portion of a novel protein (TXBP151), which was recently found to be a human T-cell leukemia virus type-I (HTLV-I) Tax-binding protein. The full-length 2386 bp TXBP151 mRNA encodes a protein of 86 kDa. Like A20, overexpression of TXBP151 could inhibit apoptosis induced by tumour necrosis factor (TNF) in NIH3T3 cells. Moreover, transfection of antisense TXBP151 partially abolished the anti-apoptotic effect of A20. Furthermore, apoptosis induced by TNF or CD95 (Fas/APO-1) was associated with proteolysis of TXBP151. This degradation could be inhibited by the broad-spectrum caspase inhibitor zVAD-fmk or by expression of the cowpox virus-derived inhibitor CrmA, suggesting that TXBP151 is a novel substrate for caspase family members. TXBP151 was indeed found to be specifically cleaved in vitro by members of the caspase-3-like subfamily, viz. caspase-3, caspase-6 and caspase-7. Thus TXBP151 appears to be a novel A20-binding protein which might mediate the anti-apoptotic activity of A20, and which can be processed by specific caspases. [ABSTRACT FROM AUTHOR]

In addition to eugenetic changes, cancerous cells exhibit extensive modifications in the expression levels of a variety of genes. The phenotypic switch observed after inoculation of T lymphoma cells into syngenic mice illustrates the active participation of tumoral environment in the induction of an aberrant gene expression pattern. To further substantiate this contribution, we performed polymerase chain reaction (PCR)-based subtraction suppression hybridization (SSH) to identify genes that are differentially expressed in tumor-derived EL4/13.3 cells compared to the same cells isolated from cultures. Besides a number of unknown genes, the subtracted library contained several known genes that have been reported to be expressed at increased levels in tumors and/or to contribute to carcinogenesis. Apart from clones representing translated transcripts, the subtracted library also contained a high number of clones representing B2 repeat elements, viz. short interspersed repetitive elements that are transcribed by RNA polymerase III. Northern blotting confirmed the induction of B2 transcripts in tumor tissue and also revealed induction of chimeric, B2 repeat-containing mRNA. The appearance of chimeric transcripts was accompanied by aberrant, shorter-than-full-length transcripts, specifically from upregulated genes. Accordingly, in addition to altered gene expression, tumoral environmental triggers constitute a potent mechanism to create an epigenetic diversity in cancers by inducing extensive transcript anomalies. [ABSTRACT FROM AUTHOR]

The glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized application-customized glycan structures. In several expression hosts, this has been quite successful, but one caveat is that the new N-glycan structures inadvertently might be substrates for one or more of the multitude of endogenous glycosyltransferases in such heterologous background. This then results in the formation of novel, undesired glycan structures, which often remain insufficiently characterized. When expressing mouse interleukin-22 in a Pichia pastoris (syn. Komagataella phaffii) GlycoSwitchM5 strain, which had been optimized to produce Man 5 GlcNAc 2 N-glycans, glycan profiling revealed two major species: Man 5 GlcNAc 2 and an unexpected, partially α-mannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis, and nuclear magnetic resonance revealed that this novel glycan was Man 5 GlcNAc 2 modified with a Glcα-1,2-Manβ-1,2-Manβ-1,3-Glcα-1,3-R tetrasaccharide. Expression of a Golgi-targeted GlcNAc transferase-I strongly inhibited the formation of this novel modification, resulting in more homogeneous modification with the targeted GlcNAcMan 5 GlcNAc 2 structure. Our findings reinforce accumulating evidence that robustly customizing the N-glycosylation pathway in P. pastoris to produce particular human-type structures is still an incompletely solved synthetic biology challenge, which will require further innovation to enable safe glycoprotein pharmaceutical production., (© 2020 Wiley Periodicals LLC.)

Background and Aims: There is a demand for serum markers that can routinely assess the progression of liver cancer. DENA (diethylnitrosamine), a hepatocarcinogen, is commonly used in an experimental mouse model to induce liver cancer that closely mimics a subclass of human hepatocellular carcinoma (HCC). However, blood monitoring of the progression of HCC in mouse model has not yet been achieved. In this report, we studied glycomics during the development of mouse HCC induced by DENA., Methods: Mouse HCC was induced by DENA. Serum N-glycans were profiled using the sequencer assisted-Fluorophore-assisted carbohydrate electrophoresis technique developed in our laboratory. Possible alteration in the transcription of genes relevant to the synthesis of the changed glycans was analysed by real-time polymerase chain reaction., Results: In comparison with the control mice that received the same volume of saline, a tri-antennary glycan (peak 8) and a biantennary glycan (peak 4) in serum total glycans of DENA mice increased gradually but significantly during progression of liver cancer, whereas a core-fucosylated biantennary glycan (peak 6) decreased. Expression of alpha-1,6-fucosyltransferase 8 (Fut8), which is responsible for core fucosylation, decreased in the liver of DENA mice compared with that of age-matched control mice. Likewise, the expression level of Mgat4a, which is responsible for tri-antennary, significantly increased in the liver of DENA mice (P<0.001)., Conclusions: The changes of N-glycan levels in the serum could be used as a biomarker to monitor the progress of HCC and to follow up the treatment of liver tumours in this DENA mouse model.

Background: We previously reported on serum N-glycans as markers for the diagnosis of cirrhosis in patients with chronic hepatitis C infection. Our present study aimed to evaluate the use of serum glycan markers for the diagnosis of liver fibrosis in patients with chronic hepatitis B infection., Methods: Patients with hepatitis B virus (HBV) infection (n=173) were diagnosed by clinical laboratory analysis and histological examination. Liver fibrosis was staged using Ishak score. N-glycan profiles of serum proteins were determined by DNA sequencer-based carbohydrate analytical profiling., Results: We found that in HBV patients, like in hepatitis C virus patients, several serum N-glycans were altered during the development of liver fibrosis. We found higher levels of total agalactosylated biantennary glycans in fibrosis patients with HBV infection than in healthy controls. The biantennary (NA2) and the triantennary (NA3) N-glycans decreased significantly (P<0.001) with increased severity of fibrosis. The diagnostic power of serum glycan marker (GlycoFibroTest) [area under the curve (AUC)=0.735) was similar to that of FibroTest (AUC=0.740) for discriminating between moderate and advanced fibrosis (F3-F6) from non- or mild fibrosis (F0-F2). However, GlycoFibroTest (AUC=0.740) was slightly better than FibroTest (AUC=0.696) for distinguishing fibrotic patients (F1 or more) from non-fibrotic patients (F0)., Conclusions: The assay for serum glycan profiling showed satisfactory reproducibility and is a non-invasive blood test for the diagnosis of liver fibrosis. The changes of N-glycan level in serum can be used to monitor or follow-up the progress of fibrosis using specific N-glycan markers.

Protein glycosylation, the most common form of co-translational modification of proteins, is the enzymatic addition of sugars or oligosaccharides (glycans) to proteins. Protein glycosylation increases the diversity of the functions of proteins encoded in the genome. The result is that different glycomes of the same protein may have different functional, kinetic or physical properties. The glycosylation pathway is largely regulated by the condition of the cells, which means that the sugar chains can be altered by the physiological or pathophysiological condition of the cell. Thus, the type of glycans produced by cells, tissues, or organism could reflect their current physiological state. We determined the N-glycan profiles of serum proteins by using DNA sequencer-based carbohydrate analytical profiling technology. We show that two N-glycan structures (NGA2F and NA2F) present in human blood glycoproteins change with ageing, and that one triantennary glycan (NA3Fb) is correlated with tumor stage in HCC patients. Therefore, examining alterations in serum glycan fingerprint by using our platform could be a suitable tool for monitoring the healthiness of ageing and for the follow-up of pathophysiological conditions such as liver cancer.

Expression of proteins on the surface of yeasts has a wide range of applications in biotechnology, such as directed evolution of proteins for increased affinity and thermal stability, screening of antibody libraries, epitope mapping, and use as whole-cell biocatalysts. However, hyperglycosylation can interfere with overall protein accessibility on the surface. Therefore, the less elaborate hyperglycosylation in wild type Pichia pastoris and the availability of glycoengineered strains make this yeast an excellent alternative for surface display of glycoproteins. Here, we report the implementation of the well-established a-agglutinin-based yeast surface display technology in P. pastoris. Four heterologous proteins were expressed on the surface of a wild type and a glycoengineered strain. Surface display levels were monitored by Western blot, immunofluorescence microscopy, and FACS analysis. The availability of glycoengineered strains makes P. pastoris an excellent alternative for surface display of glycoproteins and paves the way for new applications.

Chagas' disease is a major tropical disease for which a cure for chronic phase does not exist yet. Trypanosoma cruzi trans-sialidase (TS) seems to be involved in relevant processes such as infectivity, host survival and, very importantly, disease pathogenesis. In this study, we show that mice vaccinated with an engineered enzymatically deficient mutant TS containing the catalytic domain without the immunodominant SAPA (Shed Acute Phase Antigen) repeats, were highly protected against T. cruzi infection. Adult male BALB/c mice were immunized with mutant protein, purified from Pichia pastoris yeast, using three inoculations in Freund's adjuvant. All immunized mice were protected against challenge with a lethal dose of T. cruzi trypomastigotes. The protected immunized mice developed no clinical or tissue evidence of infection throughout the study. In contrast, 60-90% mortality and 100% occurrence of myocardial lesions were observed in the non-immunized counterparts. Titers of circulating antibody against TS did not correlate with protection, while anti-SAPA antibodies were coincident with disease severity. Further studies indicated that a single inoculation of mutant recombinant protein in Freund's complete adjuvant was not associated with blood or organic alterations, per se. Mutant TS vaccination seems to be a promising tool for immune intervention strategies in Chagas' disease, aimed at preventing T. cruzi-related heart tissue damage.

The establishment of a robust technology platform for the expression cloning of carbohydrate-binding proteins remains a key challenge in glycomics. Here we explore the utility of using yeast surface display (YSD) technology in the interaction-based lectin cloning from complete cDNA libraries. This should pave the way for more detailed studies of protein-carbohydrate interactions. To evaluate the performance of this system, lectins representing three different subfamilies (galectins, siglecs, and C-type lectins) were successfully displayed on the surface of Saccharomyces cerevisiae and Pichia pastoris as a-agglutinin and/or alpha-agglutinin fusions. The predicted carbohydrate-binding activity could be detected for three out of five lectins tested (galectin-1, galectin-3, and siaoadhesin). For galectin-4 and E-selectin, no specific carbohydrate-binding activity could be detected. We also demonstrate that proteins with carbohydrate affinity can be specifically isolated from complex metazoan cDNA libraries through multiple rounds of FACS sorting, employing multivalent, fluorescent-labeled polyacrylamide-based glycoconjugates.

Unlabelled: We evaluated the use of blood serum N-glycan fingerprinting as a tool for the diagnosis of hepatocellular carcinoma (HCC) in patients with cirrhosis induced by hepatitis B virus (HBV). A group of 450 HBV-infected patients with liver fibrosis or cirrhosis with or without HCC were studied. HCC was diagnosed by alpha-fetoprotein (AFP) analysis, ultrasonography, and/or computed tomography and was studied histologically. N-glycan profiles of serum proteins were determined with DNA sequencer-based carbohydrate analytical profiling technology. In this study, we found that a branch alpha(1,3)-fucosylated triantennary glycan was more abundant in patients with HCC than in patients with cirrhosis, patients with fibrosis, and healthy blood donors, whereas a bisecting core alpha(1,6)-fucosylated biantennary glycan was elevated in patients with cirrhosis. The concentration of these 2 glycans and the log ratio of peak 9 to peak 7 (renamed the GlycoHCCTest) were associated with the tumor stage. Moreover, for screening patients with HCC from patients with cirrhosis, the overall sensitivity and specificity of the GlycoHCCTest were very similar to those of AFP., Conclusion: This study indicates that a branch alpha(1,3)-fucosylated glycan is associated with the development of HCC. The serum N-glycan profile is a promising noninvasive method for detecting HCC in patients with cirrhosis and could be a valuable supplement to AFP in the diagnosis of HCC in HBV-infected patients with liver cirrhosis. Its use for the screening, follow-up, and management of patients with cirrhosis and HCC should be evaluated further.

In this study, we used our recently developed screening method, Bud-Scar-based Screening (BSS), to screen a yeast cDNA expression library in an SGS1 deletion BY4742 yeast strain. One gene involved in a nonclassical export pathway, NCE102, was found to extend the life span of Deltasgs1 yeast. Deletion of NCE102 in a wild type yeast strain increased its sensitivity to oxidative stress upon diethylmaleate (DEM) treatment but did not shorten its lifespan, indicating that this gene is not essential in determining yeast lifespan. Transformation of NCE102 into either Deltance102 or Deltasgs1 strains could rescue its tolerance to DEM stress, indicating that NCE102 is protective during oxidative stress. Moreover, overexpression of NCE102 in Deltasgs1 strain leads to reduced protein damage. However, overexpression of NCE102 in wild type yeast strain BY4742 neither protected against oxidative stress due to DEM nor extended yeast lifespan compared to its parental wild type strain, indicating that nonclassical export is redundant and DNA repair is fully sufficient in the wild type strain. We therefore demonstrate that a nonclassical export pathway functions as an alternative clearance/detoxification pathway to eliminate damaged material, when the basic repair pathway is not sufficient.

Trypanosome trans-sialidase (TS) is a sialic acid-transferring enzyme and a novel ligand of tyrosine kinase (TrkA) receptors but not of neurotrophin receptor p75NTR. Here, we show that TS targets TrkB receptors on TrkB-expressing pheochromocytoma PC12 cells and colocalizes with TrkB receptor internalization and phosphorylation (pTrkB). Wild-type TS but not the catalytically inactive mutant TSDeltaAsp98-Glu induces pTrkB and mediates cell survival responses against death caused by oxidative stress in TrkA- and TrkB-expressing cells like those seen with nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). These same effects are not observed in Trk deficient PC12(nnr5) cells, but are re-established in PC12(nnr5) cells stably transfected with TrkA or TrkB, are partially blocked by inhibitors of tyrosine kinase (K-252a), mitogen-activated protein/mitogen-activated kinase (PD98059) and completely blocked by LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K). Both TrkA- and TrkB-expressing cells pretreated with TS or their natural ligands are protected against cell death caused by serum/glucose deprivation or from hypoxia-induced neurite retraction. The cell survival effects of NGF and BDNF against oxidative stress are significantly inhibited by the neuraminidase inhibitor, Tamiflu. Together, these observations suggest that trypanosome TS mimics neurotrophic factors in cell survival responses against oxidative stress, hypoxia-induced neurite retraction and serum/glucose deprivation.

Background: Non-invasive staging of human liver fibrosis is a desirable objective that remains under extensive evaluation. Animal model systems are often used for studying human liver disease and screening antifibrotic compounds. The aim of the present study was to investigate the potential use of serum N-glycan profiles to evaluate liver fibrosis in a rat model., Methods: Liver fibrosis and cirrhosis were induced in rats by oral administration of CCl(4). Liver injury was assessed biochemically (alanine aminotransferase [ALT] activity, aspartate aminotransferase [AST] activity and total bilirubin) and histologically. The N-glycan profile (GlycoTest) was performed using DNA sequencer-assisted-fluorophore-assisted carbohydrate electrophoresis technology. In parallel, the effect of cotreatment with antifibrotic interferon-gamma (IFN-gamma) was studied., Results: The biopsy scoring system showed that CCl(4) induced early fibrosis (F < 1-2) in rats after 3 weeks of treatment, and cirrhosis (F4) after 12 weeks. Significant increases in ALT activity, AST activity and total bilirubin levels were detected only after 12 weeks of CCl(4) treatment. GlycoTest showed three glycans were significantly altered in the CCl(4)-goup. Peak 3 started at week 6, at an early stage in fibrosis development (F < 1-2), whereas peaks 4 and 5 occurred at week 9, at which time mild liver fibrosis (F = 1-2) had developed. The changes in the CCl(4)-IFN-gamma group were intermediate between the CCl(4)- and the control groups., Conclusion: The GlycoTest is much more sensitive than biochemical tests for evaluating liver fibrosis/cirrhosis in the rat model. The test can also be used as a non-invasive marker for screening and monitoring the antifibrotic activity of potential therapeutic compounds.

We developed a high-throughput functional genomic screening system that allows identification of genes prolonging lifespan in the baker's yeast Saccharomyces cerevisiae. The method is based on isolating yeast mother cells with a higher than average number of cell divisions as indicated by the number of bud scars on their surface. Fluorescently labeled wheat germ agglutinin (WGA) was used for specific staining of chitin, a major component of bud scars. The critical new steps in our bud-scar-sorting system are the use of small microbeads, which allows successive rounds of purification and regrowth of the mother cells (M-cell), and utilization of flow cytometry to sort and isolate cells with a longer lifespan based on the number of bud scars specifically labeled with WGA.

A direct link between receptor glycosylation and activation following natural ligand interaction has not been observed. Here, we discover a membrane sialidase-controlling mechanism that depends on ligand binding to its receptor to induce enzyme activity which targets and desialylates the receptor and, consequently, causes the induction of receptor dimerization and activation. We also identify a specific sialyl alpha-2,3-linked beta-galactosyl sugar residue of TrkA tyrosine kinase receptor, which is rapidly targeted and hydrolyzed by the sialidase. Trk-expressing cells and primary cortical neurons following stimulation with specific neurotrophic growth factors express a vigorous membrane sialidase activity. Neuraminidase inhibitors, Tamiflu, BCX1812, and BCX1827, block sialidase activity induced by nerve growth factor (NGF) in TrkA-PC12 cells and by brain-derived neurotrophic factor (BDNF) in primary cortical neurons. In contrast, the neuraminidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, specific for plasma membrane ganglioside Neu3 and Neu2 sialidases has no inhibitory effect on NGF-induced pTrkA. The GM1 ganglioside specific cholera toxin subunit B applied to TrkA-PC12 cells has no inhibitory effect on NGF-induced sialidase activity. Neurite outgrowths induced by NGF-treated TrkA-PC12 and BDNF-treated PC12(nnr5) stably transfected with TrkB receptors (TrkB-nnr5) cells are significantly inhibited by Tamiflu. Our results establish a novel mode of regulation of receptor activation by its natural ligand and define a new function for cellular sialidases.

MFECP1 is a mannosylated antibody-enzyme fusion protein used in antibody-directed enzyme prodrug therapy (ADEPT). The antibody selectively targets tumor cells and the targeted enzyme converts a prodrug into a toxic drug. MFECP1 is obtained from expression in the yeast Pichia pastoris and produced to clinical grade. The P. pastoris-derived mannosylation of the fusion protein aids rapid normal tissue clearance required for successful ADEPT. The work presented provides evidence that MFECP1 is cleared by the endocytic and phagocytic mannose receptor (MR), which is known to bind to mannose-terminating glycans. MR-transfected fibroblast cells internalize MFECP1 as revealed by flow cytometry and confocal microscopy. Immunofluorescence microscopy shows that in vivo clearance in mice occurs predominantly by MR on liver sinusoidal endothelial cells, although MR is also expressed on adjacent Kupffer cells. In the spleen, MFECP1 is taken up by MR-expressing macrophages residing in the red pulp and not by dendritic cells which are found in the marginal zone and white pulp. Clearance can be inhibited in vivo by the MR inhibitor mannan as shown by increased enzyme activities in blood. The work improves understanding of interactions of MFECP1 with normal tissue, shows that glycosylation can be exploited in the design of recombinant anticancer therapeutics and opens the ways for optimizing pharmacokinetics of mannosylated recombinant therapeutics.

Glycosylation is an important issue in heterologous protein production for therapeutic applications. Glycoproteins produced in Pichia pastoris contain high mannose glycan structures that can hamper downstream processing, might be immunogenic, and cause rapid clearance from the circulation. This chapter describes a method that helps solving these glycosylation-related problems by inactivation of OCH1, overexpression of an HDEL-tagged mannosidase, and overexpression of a Kre2/GlcNAc-transferase I chimeric enzyme. Different plasmids are described as well as glycan analysis methods.

Absence of sialylation on recombinant glycoproteins compromises their efficacy as therapeutic agents, as it results in rapid clearance from the human bloodstream. To circumvent this, several strategies are followed, including the implementation of a post-secretion glycosylation step. In this paper we describe the engineering of yeast cells expressing active surface exposed Trypanosoma cruzi trans-sialidase (TS) fused to the yeast Aga2 protein, and the use of this yeast in the sialylation of synthetic oligosaccharides. In an attempt to improve overall protein accessibility on the yeast surface, we abolished hyperglycosylation on the yeast cell wall proteins. This was achieved by disrupting the OCH1 gene of the TS surface expressing strain, which resulted in increased enzymatic activity. Using a fluorescence-based activity assay and DSA-FACE structural analysis, we obtained almost complete conversion to a fully sialylated acceptor, whereas in the wild type situation this conversion was only partial. Increasing protein accessibility on the yeast surface by modifying the glycosylation content thus proved to be a valuable approach in increasing the cell wall associated activity of an immobilised enzyme, hence resulting in a more effective biocatalyst system.

Antimicrobial peptides, which have been isolated from many bacteria, fungi, plants, invertebrates and vertebrates, are an important component of the natural defenses of most living organisms. The isolated peptides are very heterogeneous in length, sequence and structure, but most of them are small, cationic and amphipathic. These peptides exhibit broad-spectrum activity against Gram-positive and Gram-negative bacteria, yeasts, fungi and enveloped viruses. A wide variety of human proteins and peptides also have antimicrobial activity and play important roles in innate immunity. In this review we discuss three important groups of human antimicrobial peptides. The defensins are cationic non-glycosylated peptides containing six cysteine residues that form three intramolecular disulfide bridges, resulting in a triple-stranded beta-sheet structure. In humans, two classes of defensins can be found: alpha-defensins and beta-defensins. The defensin-related HE2 isoforms will also be discussed. The second group is the family of histatins, which are small, cationic, histidine-rich peptides present in human saliva. Histatins adopt a random coil conformation in aqueous solvents and form alpha-helices in non-aqueous solvents. The third group comprises only one antimicrobial peptide, the cathelicidin LL-37. This peptide is derived proteolytically from the C-terminal end of the human CAP18 protein. Just like the histatins, it adopts a largely random coil conformation in a hydrophilic environment, and forms an alpha-helical structure in a hydrophobic environment.

The susceptibility of Saccharomyces cerevisiae to the anti-microbial peptide, histatin 5, was tested after pre-growth in fermentable and non-fermentable carbon sources and in the absence or presence of the uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP). S. cerevisiae was more resistant to histatin 5 when grown on a fermentable carbon source compared to growth on a non-fermentable carbon source, indicating an important role for oxidative phosphorylation in histatin 5-induced cell death. Oxidative phosphorylation is a pre-requisite for histatin 5-induced cell death in Candida albicans but this is not the case in S. cerevisiae. Incubation of CCCP-treated S. cerevisiae cells with histatin 5 still resulted in cell death. These results suggest that histatin 5-induced cell death in S. cerevisiae differs from that in C. albicans.

Trypanosome trans-sialidase (TS) is a sialic acid-transferring enzyme that hydrolyzes alpha2,3-linked sialic acids and transfers them to acceptor molecules. Here we show that a highly purified recombinant TS derived from T. cruzi parasites targets TrkA receptors on TrkA-expressing PC12 cells and colocalizes with TrkA internalization and phosphorylation (pTrkA). Maackia amurensis lectin II (MAL-II) and Sambucus nigra lectin (SNA) block TS binding to TrkA-PC12 cells in a dose-dependent manner with subsequent inhibition of TS colocalization with pTrkA. Cells treated with lectins alone do not express pTrkA. The catalytically inactive mutant TSDeltaAsp98-Glu also binds to TrkA-expressing cells, but is unable to induce pTrkA. TrkA-PC12 cells treated with a purified recombinant alpha2,3-neuraminidase (Streptococcus pneumoniae) express pTrkA. Wild-type TS but not the mutant TSDeltaAsp98-Glu promotes neurite outgrowth in TrkA-expressing PC12 cells. In contrast, these effects are not observed in TrkA deficient PC12nnr5 cells but are reestablished in PC12nnr5 cells stably transfected with TrkA and are significantly blocked by inhibitors of tyrosine kinase (K-252a) and MAP/MEK protein kinase (PD98059). Together these observations suggest for the first time that hydrolysis of sialyl alpha2,3-linked beta-galactosyl residues of TrkA receptors plays an important role in TrkA receptor activation, sufficient to promote cell differentiation (neurite outgrowth) independent of nerve growth factor.

We implemented 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled asparagine-linked glycan (N-glycan) profiling on a microfluidic electrophoresis platform. Using 11.5 cm effective length etched channels and 4% linear polyacrylamide as the separation matrix, the major N-glycans in human serum were profiled in 12 min with a resolution comparable to what is achieved for these analytes on gel-based DNA sequencers. This demonstration suggests a practical clinical application for high-speed compact analyzers which might be uniquely based on microfluidic devices., (Copyright 2004 WILEY-VCH Verlag GmbH & Co.)

The glycosylation of Cel7A (CBH I) from Trichoderma reesei varies considerably when the fungus is grown under different conditions. As shown by ESI-MS and PAG-IEF analyses of both intact protein and the isolated catalytic core module, the microheterogeneity originates mainly from the variable ratio of single N-acetylglucosamine over high-mannose structures on the three N-glycosylation sites and from the presence or absence of phosphate residues. Fully N- and O-glycosylated Cel7A can only be isolated from minimal medium and probably reflects the initial complexity of the protein on leaving the glycosynthetic pathway. Extracellular activities are responsible for postsecretorial modifications in other cultivation conditions: alpha-(1-->2)-mannosidase, alpha-(1-->3)-glucosidase and an Endo H type activity participate in N-deglycosylation (core), whereas a phosphatase and a mannosidase are probably responsible for hydrolysis of O-glycans (linker). The effects are most prominent in corn steep liquor-enriched media, where the pH is closer to the pH optimum (5-6) of these extracellular hydrolases. In minimal medium, the low pH and the presence of proteases could explain for the absence of such activities. On the other hand, phosphodiester linkages in the catalytic module are only observed under specific conditions. The extracellular trigger is still unknown, but mannophosphorylation may be regulated intracellularly by alpha-(1-->2)-mannosidases and phosphomannosyl transferases competing for the same intermediate in the glycosynthetic pathway.

We developed a high-throughput screening system that allows identification of genes prolonging life span in the budding yeast Saccharomyces cerevisiae. The method is based on isolating yeast mother cells with an extended number of cell divisions as indicated by the increased number of bud scars on their surface. Fluorescently labeled wheat germ agglutinin (WGA) was used for specific staining of bud scars. Screening of a human HepG2 cDNA expression library in yeast resulted in the isolation of several yeast transformants with a potentially prolonged life span. The budding yeast S. cerevisiae, one of the favorite models used to study aging, has been studied extensively for the better understanding of the mechanisms of human aging. Because human disease genes often have yeast counterparts, they can be studied efficiently in this organism. One interesting example is the WRN gene, the human DNA helicase, which participates in the DNA repair pathway. The mutation of the WRN gene causes Werner syndrome showing premature-aging phenotype. Budding yeast contains WRN homologue, SGS1, and its mutation results in shortening yeast life span. The knowledge gained from the studies of budding yeast will benefit studies in humans for better understanding of aging and aging-related disease.

The N-glycans present on the total mixture of serum glycoproteins (serum N-glycome) were analyzed in 24 subjects with congenital disorder of glycosylation type I (CDG-I) and 7 healthy, age-matched individuals. No new N-glycan structures were observed in the sera of CDG-I patients as compared with normal sera. However, we observed in all subtypes a significantly increased degree of core alpha-1,6-fucosylation of the biantennary glycans as compared to normal, as well as a significant decrease in the amount of triantennary glycans. These serum N-glycome changes appear to be a milder manifestation of some of the changes observed in adult liver cirrhosis patients, which is compatible with the reported steatosis and fibrosis in CDG-I patients. In the CDG-Ia subgroup, the extent of the serum N-glycome changes correlates with the aberration of the serum transferrin isoelectric focusing pattern, which measures the severity of the lack of entire N-glycan chains (primary consequence of CDG-I) in the liver and is the standard diagnostic test for this category of inherited diseases.

An HEK293S cell line resistant to ricin was prepared by mutagenesis by using ethyl methanesulfonate. It was shown to lack N-acetylglucosaminyltransferase I (GnTI) activity, and consequently unable to synthesize complex N-glycans. The tetracycline-inducible opsin expression system was assembled into this GnTI(-) HEK293S cell line. Stable cell lines were isolated that gave tetracycline/sodium butyrate-inducible expression of the WT opsin gene at levels comparable with those observed in the parent tetracycline-inducible HEK293S cell line. Analysis of the N-glycan in rhodopsin expressed by the HEK293S GnTI(-) stable cell line showed it to be Man(5)GlcNAc(2). In a larger-scale expression experiment (1.1 liter) a WT opsin production level of 6 mg/liter was obtained. Further, the toxic constitutively active rhodopsin mutant, E113Q/E134Q/M257Y, previously shown to require inducible expression, has now been expressed in an HEK293S GNTI(-)-inducible cell line at levels comparable with those obtained with WT rhodopsin.