Nico Callewaert

Genome sequence of the recombinant protein production host Pichia pastoris.

The methylotrophic yeast Pichia pastoris is widely used for the production of proteins and as a model organism for studying peroxisomal biogenesis and methanol assimilation. P. pastoris strains capable of human-type N-glycosylation are now available, which increases the utility of this organism for biopharmaceutical production. Despite its biotechnological importance, relatively few genetic tools or engineered strains have been generated for P. pastoris. To facilitate progress in these areas, we present the 9.43 Mbp genomic sequence of the GS115 strain of P. pastoris. We also provide manually curated annotation for its 5,313 protein-coding genes.

Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models

Necrostatin-1 (Nec-1) is widely used in disease models to examine the contribution of receptor-interacting protein kinase (RIPK) 1 in cell death and inflammation. We studied three Nec-1 analogs: Nec-1, the active inhibitor of RIPK1, Nec-1 inactive (Nec-1i), its inactive variant, and Nec-1 stable (Nec-1s), its more stable variant. We report that Nec-1 is identical to methyl-thiohydantoin-tryptophan, an inhibitor of the potent immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Both Nec-1 and Nec-1i inhibited human IDO, but Nec-1s did not, as predicted by molecular modeling. Therefore, Nec-1s is a more specific RIPK1 inhibitor lacking the IDO-targeting effect. Next, although Nec-1i was ∼100 × less effective than Nec-1 in inhibiting human RIPK1 kinase activity in vitro, it was only 10 times less potent than Nec-1 and Nec-1s in a mouse necroptosis assay and became even equipotent at high concentrations. Along the same line, in vivo, high doses of Nec-1, Nec-1i and Nec-1s prevented tumor necrosis factor (TNF)-induced mortality equally well, excluding the use of Nec-1i as an inactive control. Paradoxically, low doses of Nec-1 or Nec-1i, but not Nec -1s, even sensitized mice to TNF-induced mortality. Importantly, Nec-1s did not exhibit this low dose toxicity, stressing again the preferred use of Nec-1s in vivo. Our findings have important implications for the interpretation of Nec-1-based data in experimental disease models.

In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans in Pichia pastoris

ABSTRACT 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-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and β-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.

Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology

Here we provide a protocol for engineering the N-glycosylation pathway of the yeast Pichia pastoris. The general strategy consists of the disruption of an endogenous glycosyltransferase gene (OCH1) and the stepwise introduction of heterologous glycosylation enzymes. Each engineering step results in the introduction of one glycosidase or glycosyltransferase activity into the Pichia endoplasmic reticulum or Golgi complex and consists of a number of stages: transformation with the appropriate GlycoSwitch vector, small-scale cultivation of a number of transformants, sugar analysis and heterologous protein expression analysis. If desired, the resulting clone can be further engineered by repeating the procedure with the next GlycoSwitch vector. Each engineering step takes ∼3 weeks. The conversion of any wild-type Pichia strain into a strain that modifies its glycoproteins with Gal2GlcNAc2Man3GlcNAc2N-glycans requires the introduction of five GlycoSwitch vectors. Three examples of the full engineering procedure are provided to illustrate the results that can be expected.

Alteration of protein glycosylation in liver diseases

Chronic liver diseases are a serious health problem worldwide. The current gold standard to assess structural liver damage is through a liver biopsy which has several disadvantages. A non-invasive, simple and non-expensive test to diagnose liver pathology would be highly desirable. Protein glycosylation has drawn the attention of many researchers in the search for an objective feature to achieve this goal. Glycosylation is a posttranslational modification of many secreted proteins and it has been known for decades that structural changes in the glycan structures of serum proteins are an indication for liver damage. The aim of this paper is to give an overview of this altered protein glycosylation in different etiologies of liver fibrosis / cirrhosis and hepatocellular carcinoma. Although individual liver diseases have their own specific markers, the same modifications seem to continuously reappear in all liver diseases: hyperfucosylation, increased branching and a bisecting N-acetylglucosamine. Analysis at mRNA and protein level of the corresponding glycosyltransferases confirm their altered status in liver pathology. The last part of this review deals with some recently developed glycomic techniques that could potentially be used in the diagnosis of liver pathology.

Non-pathogenic trypanosomatid protozoa as a platform for protein research and production

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 systems 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 homogeneous, with a mammalian-type biantennary oligosaccharide and the Man(3)GlcNAc(2) 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-alpha-1,6-fucosylated N-glycans.

N-glycomic changes in hepatocellular carcinoma patients with liver cirrhosis induced by hepatitis B virus†

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 α‐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. (HEPATOLOGY 2007.)

Multiple phenotypes in phosphoglucomutase 1 deficiency

BACKGROUND Congenital disorders of glycosylation are genetic syndromes that result in impaired glycoprotein production. We evaluated patients who had a novel recessive disorder of glycosylation, with a range of clinical manifestations that included hepatopathy, bifid uvula, malignant hyperthermia, hypogonadotropic hypogonadism, growth retardation, hypoglycemia, myopathy, dilated cardiomyopathy, and cardiac arrest. METHODS Homozygosity mapping followed by whole-exome sequencing was used to identify a mutation in the gene for phosphoglucomutase 1 (PGM1) in two siblings. Sequencing identified additional mutations in 15 other families. Phosphoglucomutase 1 enzyme activity was assayed on cell extracts. Analyses of glycosylation efficiency and quantitative studies of sugar metabolites were performed. Galactose supplementation in fibroblast cultures and dietary supplementation in the patients were studied to determine the effect on glycosylation. RESULTS Phosphoglucomutase 1 enzyme activity was markedly diminished in all patients. Mass spectrometry of transferrin showed a loss of complete N-glycans and the presence of truncated glycans lacking galactose. Fibroblasts supplemented with galactose showed restoration of protein glycosylation and no evidence of glycogen accumulation. Dietary supplementation with galactose in six patients resulted in changes suggestive of clinical improvement. A new screening test showed good discrimination between patients and controls. CONCLUSIONS Phosphoglucomutase 1 deficiency, previously identified as a glycogenosis, is also a congenital disorder of glycosylation. Supplementation with galactose leads to biochemical improvement in indexes of glycosylation in cells and patients, and supplementation with complex carbohydrates stabilizes blood glucose. A new screening test has been developed but has not yet been validated. (Funded by the Netherlands Organization for Scientific Research and others.).

Engineering of glycosylation in yeast and other fungi: current state and perspectives

With the increasing demand for recombinant proteins and glycoproteins, research on hosts for producing these proteins is focusing increasingly on more cost-effective expression systems. Yeasts and other fungi are promising alternatives because they provide easy and cheap systems that can perform eukaryotic post-translational modifications. Unfortunately, yeasts and other fungi modify their glycoproteins with heterogeneous high-mannose glycan structures, which is often detrimental to a therapeutic protein’s pharmacokinetic behavior and can reduce the efficiency of downstream processing. This problem can be solved by engineering the glycosylation pathways to produce homogeneous and, if so desired, human-like glycan structures. In this review, we provide an overview of the most significant recently reported approaches for engineering the glycosylation pathways in yeasts and fungi.

Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations

The HEK293 human cell lineage is widely used in cell biology and biotechnology. Here we use whole-genome resequencing of six 293 cell lines to study the dynamics of this aneuploid genome in response to the manipulations used to generate common 293 cell derivatives, such as transformation and stable clone generation (293T); suspension growth adaptation (293S); and cytotoxic lectin selection (293SG). Remarkably, we observe that copy number alteration detection could identify the genomic region that enabled cell survival under selective conditions (i.c. ricin selection). Furthermore, we present methods to detect human/vector genome breakpoints and a user-friendly visualization tool for the 293 genome data. We also establish that the genome structure composition is in steady state for most of these cell lines when standard cell culturing conditions are used. This resource enables novel and more informed studies with 293 cells, and we will distribute the sequenced cell lines to this effect.