Biomedical subjects

Roland Contreras

Publications and source records attributed to Roland Contreras.

At least 19 recordsLinked to original sources

PubMed2006

Clearance mechanism of a mannosylated antibody-enzyme fusion protein used in experimental cancer therapy.

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.

Animals↗

PubMed2006

Dependence of neurotrophic factor activation of Trk tyrosine kinase receptors on cellular sialidase.

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.

Animals↗

PubMed2006

Secretion of functional human enzymes by Tetrahymena thermophila.

BACKGROUND: The non-pathogenic ciliate Tetrahymena thermophila is one of the best-characterized unicellular eucaryotes used in various research fields. Previous work has shown that this unicellular organism provides many biological features to become a high-quality expression system, like multiplying to high cell densities with short generation times in bioreactors. In addition, the expression of surface antigens from the malaria parasite Plasmodium falciparum and the ciliate Ichthyophthirius multifiliis suggests that T. thermophila might play an important role in vaccine development. However, the expression of functional mammalian or human enzymes remains so far to be seen. RESULTS: We have been able to express a human enzyme in T. thermophila using expression modules that encode a fusion protein consisting of the endogenous phospholipase A1 precursor and mature human DNaseI. The recombinant human enzyme is active, indicating that also disulfide bridges are correctly formed. Furthermore, a detailed N-glycan structure of the recombinant enzyme is presented, illustrating a very consistent glycosylation pattern. CONCLUSION: The ciliate expression system has the potential to become an excellent expression system. However, additional optimisation steps including host strain improvement as wells as measures to increase the yield of expression are necessary to be able to provide an alternative to the common E. coli and yeast-based systems as well as to transformed mammalian cell lines.

Animals↗

PubMed2005

Development of a S. cerevisiae whole cell biocatalyst for in vitro sialylation of oligosaccharides.

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.

Catalysis↗

PubMed2005

Human antimicrobial peptides: defensins, cathelicidins and histatins.

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.

Amino Acid Sequence↗

PubMed2005

Old yellow enzyme interferes with Bax-induced NADPH loss and lipid peroxidation in yeast.

The yeast transcriptional response to murine Bax expression was compared with the changes induced by H(2)O(2) treatment via microarray technology. Although most of the Bax-responsive genes were also triggered by H(2)O(2) 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 DeltaOYE3 expressing Bax. However, the absence of OYE3 sensitized yeast cells to H(2)O(2)-induced cell death, and increased the rate of NADPH decrease and lipid peroxidation. Our results clearly indicate that OYE3 interferes with Bax- and H(2)O(2)-induced lipid peroxidation and cell death in Saccharomyces cerevisiae.

Apoptosis↗

PubMed2005

Cloning and characterization of the glucosidase II alpha subunit gene of Trichoderma reesei: a frameshift mutation results in the aberrant glycosylation profile of the hypercellulolytic strain Rut-C30.

We describe isolation and characterization of the gene encoding the glucosidase II alpha subunit (GIIalpha) 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 GIIalpha (gls2alpha) 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 alpha-1,3-linked glucose residue but not the innermost alpha-1,3-linked glucose residue from the Glc2Man9GlcNAc2 N-glycan ER structure. Transformation of the Rut-C30 strain with a repaired T. reesei gls2alpha gene changed the glycosylation profile significantly, decreasing the amount of monoglucosylated structures and increasing the amount of high-mannose N-glycans. Full conversion to high-mannose carbohydrates was not obtained, and this was probably due to competition between the endogenous mutant subunit and the introduced wild-type GIIalpha 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.

Amino Acid Sequence↗

PubMed2004

Trypanosome trans-sialidase targets TrkA tyrosine kinase receptor and induces receptor internalization and activation.

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.

Animals↗

PubMed2004

Factors influencing glycosylation of Trichoderma reesei cellulases. I: Postsecretorial changes of the O- and N-glycosylation pattern of Cel7A.

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.

Catalytic Domain↗

PubMed2004

Total serum protein N-glycome profiling on a capillary electrophoresis-microfluidics platform.

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.

Electrophoresis, Capillary↗

PubMed2004

Bax-induced cell death in Candida albicans.

Bax is a pro-apoptotic member of the Bcl-2 family of proteins involved in the regulation of genetically programmed cell death in mammalian cells. It has been shown that heterologous expression of Bax in several yeast species, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Pichia pastoris, also induces cell death. In this study we investigated the effects of Bax expression in the pathogenic yeast Candida albicans. Cell death inducing expression of Bax required a synthetic BAX gene that was codon-optimized for expression in Candida albicans. Expression of this BAX gene resulted in growth inhibition and cell death. By fusing Bax with the yeast enhanced green fluorescent protein of Aequoria victoria, the cell death-inducing effect of Bax was increased due to reduced proteolytic degradation of Bax. Using this fusion protein we showed that, upon expression in C. albicans, Bax co-localizes with the mitochondria. Furthermore, we showed for the first time that expression of Bax in yeast causes the mitochondria, which are normally distributed throughout the cell, to cluster in the perinuclear region.

Amino Acid Sequence↗

PubMed2004

Role of oxidative phosphorylation in histatin 5-induced cell death in Saccharomyces cerevisiae.

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.

Apoptosis↗

PubMed2004

Expression of the human ferritin light chain in a frataxin mutant yeast affects ageing and cell death.

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.

Aging↗

PubMed2004

Noninvasive diagnosis of liver cirrhosis using DNA sequencer-based total serum protein glycomics.

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.

Blood Proteins↗

PubMed2004

In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris.

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 alpha-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and beta-1,4-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.

Biotechnology↗

PubMed2004

The bud scar-based screening system for hunting human genes extending life span.

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.

Aging↗

PubMed2003

Increased fucosylation and reduced branching of serum glycoprotein N-glycans in all known subtypes of congenital disorder of glycosylation I.

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.

Adult↗

PubMed2003

A high-throughput screening system for genes extending life-span.

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 H(2)O(2) 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.

Ferritins↗