Off-target glycans encountered along the synthetic biology route toward humanized N-glycans in Pichia pastoris.

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 Man5 GlcNAc2 N-glycans, glycan profiling revealed two major species: Man5 GlcNAc2 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 Man5 GlcNAc2 modified with a Glcα-1,2-Manß-1,2-Manß-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 GlcNAcMan5 GlcNAc2 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.

Comparative proteomic profiling in compatible and incompatible interactions between hop roots and Verticillium albo-atrum.

Verticillium wilt, caused by the soil borne fungal pathogen Verticillium albo-atrum, is a serious threat to hop (Humulus lupulus L.) production in several hop-growing regions. A proteomic approach was applied to analyse the response of root tissue in compatible and incompatible interactions between hop and V. albo-atrum at 10, 20 and 30 days after inoculation, using two-dimensional difference gel electrophoresis (2D-DIGE) coupled with de novo sequencing of derivatized peptides. Approximately 1200 reproducible spots were detected on the gels, of which 102 were identified. In the compatible interaction, 252 spots showed infection-specific changes in spot abundance and an accumulation of defence-related proteins, such as chitinase, ß-glucanase, thaumatin-like protein, peroxidase and germin-like protein, was observed. However, no significant infection-specific changes were detected in the incompatible interaction. The results indicate that resistance in this pathosystem may be conferred by constitutive rather than induced defence mechanisms. The identification and high abundance of two mannose/glucose-specific lectin isoforms present only in the roots of the resistant cultivar suggests function of lectins in hop resistance against V. albo-atrum.

Biochemical characterization of Sfh-I, a subclass B2 metallo-beta-lactamase from Serratia fonticola UTAD54.

The subclass B2 metallo-ß-lactamase (MBL) Sfh-I from Serratia fonticola UTAD54 was cloned and overexpressed in Escherichia coli. The recombinant protein binds one equivalent of zinc, as shown by mass spectrometry, and preferentially hydrolyzes carbapenem substrates. However, compared to other B2 MBLs, Sfh-I also shows limited hydrolytic activity against some additional substrates and is not inhibited by a second equivalent of zinc. These data confirm Sfh-I to be a subclass B2 metallo-ß-lactamase with some distinctive properties.

The use of 2D-electrophoresis and de novo sequencing to characterize inter- and intra-cultivar protein polymorphisms in an allopolyploid crop.

Polyploidy and allopolyploidy have played an important role in the evolution of many plants and crops. Several techniques exist to characterize allopolyploid varieties. Analyzing the consequences of genomic reorganization at the gDNA level is a prerequisite but a better insight into the consequences for the phenotype is also primordial. As such, protein polymorphism analysis is important in understanding plant and crop biodiversity and is a driving force behind crop improvement. Our strategy to analyze protein isoforms and to detect possible gene silencing or deletion in bananas was based on protein analysis. Bananas are a good representative of a complex allopolyploid and important crop. We combined two-dimensional electrophoresis (2DE) and 2D DIGE with de novo MS/MS sequence determination to characterize a range of triploid varieties. Via Principal Component Analysis (PCA) and hierarchical clustering we were able to blindly classify the different varieties according to their presumed genome constitution. We report for the first time the application of an automated approach for the derivatization of peptides for facilitated MS/MS de novo sequence determination. We conclude that the proteome does not always correspond to the presumed genome formulae and that proteomics is a powerful tool to characterize varieties. The observations at the protein level provide good indications for a more complex genome structure and genomic rearrangement in some banana varieties.

Protein differential expression induced by endocrine disrupting compounds in a terrestrial isopod.

Endocrine disrupting compounds (EDCs) have been studied due to their impact on human health and increasing awareness of their impact on wildlife species. Studies concerning the organ-specific molecular effects of EDC in invertebrates are important to understand the mechanisms of action of this class of toxicants but are scarce in the literature. We have used a dose/response approach to unravel the protein expression in different organs of isopods exposed to bisphenol A (BPA) and vinclozolin (Vz) and assess their potential use as surrogate species. Male isopods were exposed to a range of Vz or of BPA concentrations. After animal dissection, proteins were extracted from gut, hepatopancreas and testes. Protein profiles were analysed by electrophoresis and differentially expressed proteins were identified by MALDI mass spectrometry. EDCs affected proteins involved in the energy metabolism (arginine kinase), proteins of the heat shock protein family (Hsp70 and GRP78) and most likely microtubule dynamics (tubulin). Different proteins expressed at different concentrations in different organs are indicative of the organ-specific effects of BPA and Vz. Additionally, several proteins were up-regulated at lower but not higher BPA or Vz concentrations, bringing new data to the non-monotonic response curve controversy. Furthermore, our findings suggest that some common responses to EDCs in both vertebrates and invertebrates may exist.

Resistance of the dopamine D4 receptor to agonist-induced internalization and degradation.

Dopamine receptors are G-protein-coupled receptors involved in the control of motivation, learning, and fine-tuning of motor movement, as well as modulation of neuroendocrine signalling. Stimulation of G-protein-coupled receptors normally results in attenuation of signalling through desensitization, followed by internalization and down-regulation of the receptor. These processes allow the cell to regain homeostasis after exposure to extracellular stimuli and offer protection against excessive signalling. Here, we have investigated the agonist-mediated attenuation properties of the dopamine D4 receptor. We found that several hallmarks of signal attenuation such as receptor phosphorylation, internalization and degradation showed a blunted response to agonist treatment. Moreover, we did not observe recruitment of beta-arrestins upon D4 receptor stimulation. We also provide evidence for the constitutive phosphorylation of two serine residues in the third intracellular loop of the D4 receptor. These data demonstrate that, when expressed in CHO, HeLa and HEK293 cells, the human D4 receptor shows resistance to agonist-mediated internalization and down-regulation. Data from neuronal cell lines, which have been reported to show low endogenous D4 receptor expression, such as the hippocampal cell line HT22 and primary rat hippocampal cells, further support these observations.

Identification of a gene coding for a deglycosylating enzyme in Hypocrea jecorina.

An enzyme with mannosyl glycoprotein endo-N-acetyl-beta-D-glucosaminidase (ENGase)-type activity was partially purified from the extracellular medium of the mould Hypocrea jecorina (Trichoderma reesei). Internal peptides were generated and used to identify the gene in the T. reesei genome. The active enzyme is processed both at the N- and at the C-terminus. High-mannose-type glycoproteins are good substrates, whereas complex-type glycans are not hydrolysed. The enzyme represents the first fungal member of glycoside hydrolase family 18 with ENGase-type activity. Bacterial ENGases and the fungal chitinases belonging to the same family show very low homology with Endo T. Database searches identify several highly homologous genes in fungi and the activity is also found within other Trichoderma species. This ENGase activity, not coregulated with cellulase production, could be responsible for the extensive N-deglycosylation observed for several T. reesei cellulases.

Multiplicity of aspartic proteinases from Cynara cardunculus L.

Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine. This is, to our knowledge, one of the highest number of APs purified from a single organism, consistent with a specific and important biological function of these protein within C. cardunculus. These enzymes, cardosins E, F, G and H, are dimeric, glycosylated, pepstatin-sensitive APs, active at acidic pH, with a maximum activity around pH 4.3. Their primary structures were partially determined by N- and C-terminal sequence analysis, peptide mass fingerprint analysis on a MALDI-TOF/TOF instrument and by LC-MS/MS analysis on a Q-TRAP instrument. All four enzymes are present on C. cardunculus L. pistils, along with cyprosins and cardosins A and B. Their micro-heterogeneity was detected by 2D-electrophoresis and mass spectrometry. The enzymes resemble cardosin A more than they resemble cardosin B or cyprosin, with cardosin E and cardosin G being more active than cardosin A, towards the synthetic peptide KPAEFF(NO(2))AL. The specificity of these enzymes was investigated and it is shown that cardosin E, although closely related to cardosin A, exhibits different specificity.

C-terminal sequence analysis of 2DE-separated proteins.

The overall study of post-translational modifications (PTMs) of proteins is gaining strong interest. Beside phosphorylation and glycosylation, truncations of the nascent polypeptide chain at the N- or C-terminus are by far the most common types of PTMs. Nevertheless, little attention has been paid to the development of approaches that allow a systematic analysis of these proteolytic processing events. Here we present a protocol that allows the identification of the C-terminal sequence of proteins separated by two-dimensional polyacrylamide gel electrophoresis (2DE). For each purified protein, a peptide mixture is generated by cleavage of the protein with cyanogen bromide. During incubation with carboxypeptidases only the original C-terminal fragment forms a ladder. Ladder readout is performed using MALDI mass spectrometry. 2DE-separated proteins from Shewanella oneidensis were chosen as a model system to investigate the effectiveness of the approach.

De novo sequence analysis of N-terminal sulfonated peptides after in-gel guanidination.

In this protocol, we describe an approach in which two-dimensional electrophoresis (2DE)-separated proteins are guanidinated in-gel prior to enzymatic cleavage. In contrast to previously described techniques, this procedure allows the extracted tryptic peptides to be N-terminally sulfonated without any further sample purification. The protocol was applied on a proteomic study of 2DE-separated proteins from Halorhodospira halophila, an extremophilic eubacterium with an unsequenced genome at the moment of analysis.

Matrix-assisted laser desorption ionisation-time-of of-flight mass spectrometry of intact cells allows rapid identification of Burkholderia cepacia complex.

The present study examined the potential of intact-cell matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid identification of Burkholderia cepacia complex (Bcc) bacteria using an Applied Biosystems 4700 Proteomics Analyser. Two software packages were used to analyse mass profiles based on densitometric curves and peak positions. The 75 strains examined, represented the nine established Bcc species and some commonly misidentified species, closely related or biochemically similar to Bcc and relevant in the context of cystic fibrosis microbiology. All Bcc strains clustered together, separated from non-Bcc strains. Within Bcc, most Bcc strains grouped in species specific clusters, except for Burkholderia anthina and Burkholderia pyrrocinia strains which constituted a single cluster. The present study demonstrates that MALDI-TOF MS is a powerful approach for the rapid identification of Bcc bacteria.

Proteome analysis of non-model plants: a challenging but powerful approach.

Biological research has focused in the past on model organisms and most of the functional genomics studies in the field of plant sciences are still performed on model species or species that are characterized to a great extent. However, numerous non-model plants are essential as food, feed, or energy resource. Some features and processes are unique to these plant species or families and cannot be approached via a model plant. The power of all proteomic and transcriptomic methods, that is, high-throughput identification of candidate gene products, tends to be lost in non-model species due to the lack of genomic information or due to the sequence divergence to a related model organism. Nevertheless, a proteomics approach has a great potential to study non-model species. This work reviews non-model plants from a proteomic angle and provides an outline of the problems encountered when initiating the proteome analysis of a non-model organism. The review tackles problems associated with (i) sample preparation, (ii) the analysis and interpretation of a complex data set, (iii) the protein identification via MS, and (iv) data management and integration. We will illustrate the power of 2DE for non-model plants in combination with multivariate data analysis and MS/MS identification and will evaluate possible alternatives.

Crystal structure of a cold-adapted class C beta-lactamase.

In this study, the crystal structure of a class C beta-lactamase from a psychrophilic organism, Pseudomonas fluorescens, has been refined to 2.2 A resolution. It is one of the few solved crystal structures of psychrophilic proteins. The structure was compared with those of homologous mesophilic enzymes and of another, modeled, psychrophilic protein. The elucidation of the 3D structure of this enzyme provides additional insights into the features involved in cold adaptation. Structure comparison of the psychrophilic and mesophilic beta-lactamases shows that electrostatics seems to play a major role in low-temperature adaptation, with a lower total number of ionic interactions for cold enzymes. The psychrophilic enzymes are also characterized by a decreased number of hydrogen bonds, a lower content of prolines, and a lower percentage of arginines in comparison with lysines. All these features make the structure more flexible so that the enzyme can behave as an efficient catalyst at low temperatures.

Variability of polymorphic families of three types of xylanase inhibitors in the wheat grain proteome.

Cereals contain proteinaceous inhibitors of endo-beta-1,4-xylanases (E.C.3.2.1.8, xylanases). Since these xylanase inhibitors (XIs) are only active against xylanases of microbial origin and do not interact with plant endogenous xylanases, they are believed to act as a defensive barrier against phytopathogenic attack. So far, three types of XIs have been identified, i.e. Triticum aestivum XI (TAXI), xylanase inhibiting protein (XIP), and thaumatin-like XI (TLXI) proteins. In this study the variation in XI forms present in wheat grain was elucidated using high-resolution 2-DE in combination with LC-ESI-MS/MS and biochemical techniques. Reproducible 2-DE fingerprints of TAXI-, XIP-, and TLXI-type XIs, selectively purified from whole meal of three European wheat cultivars using cation exchange chromatography followed by affinity chromatography, were obtained using a pH-gradient of 6 to 11 and a molecular mass range of 10 to 60 kDa. Large polymorphic XI families, not known to date, which exhibit different pI- and/or molecular mass values, were visualised by colloidal CBB staining. Identification of distinct genetic variants by MS/MS-analysis provides a partial explanation for the observed XI heterogeneity. Besides genetic diversity, PTMs, such as glycosylation, account for the additional complexity of the 2-DE patterns.

Biochemical Characterization of SFC-1, a class A carbapenem-hydrolyzing beta-lactamase.

The carbapenem-hydrolyzing beta-lactamase SFC-1 from Serratia fonticola UTAD54 was overexpressed in Escherichia coli, purified, and characterized. The enzyme exhibited an apparent molecular mass of 30.5 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SFC-1 hydrolyzes penicillins, cephalosporins, aztreonam, and carbapenems and is inhibited by clavulanic acid, sulbactam, and tazobactam.

Functional proteome analysis of the banana plant (Musa spp.) using de novo sequence analysis of derivatized peptides.

We report the use of chemical derivatization with MALDI-MS/MS analysis for de novo sequence analysis. Using three frequently used homology-based search algorithms, we were able to identify more than 40 proteins from banana, a nonmodel plant with unsequenced genome. Furthermore, this approach allowed the identification of different isoforms. We also observed that the identification score obtained varied according to the position of the peptide sequences in the query using the MS-Blast algorithm.

MALDI-TOF/TOF de novo sequence analysis of 2-D PAGE-separated proteins from Halorhodospira halophila, a bacterium with unsequenced genome.

Because protein identifications rely on matches with sequence databases, high-throughput proteomics is currently largely restricted to those species for which comprehensive sequence databases are available. The identification of proteins derived from organisms with unsequenced genomes mainly depends on homology searching. Here, we report the use of a simplified, gel-based, chemical derivatization strategy for de novo sequence analysis using a MALDI-TOF/TOF mass spectrometer. This approach allows the determination of de novo peptide sequences of up to 20 amino acid residues in length. The protocol was applied on a proteomic study of 2-D PAGE-separated proteins from Halorhodospira halophila, an extremophilic eubacterium with yet unsequenced genome. Using three different homology-based search algorithms, we were able to identify more than 30 proteins from this organism using subpicomole quantities of protein.

A method for C-terminal sequence analysis in the proteomic era (proteins cleaved with cyanogen bromide).

There is growing interest in the overall study of post-translational modifications (PTMs) of proteins. Beside phosphorylation and glycosylation, truncations of the nascent polypeptide chain at the N or C termini are by far the most common types of PTMs found in proteins. However, little attention has been paid to the development of approaches that allow a systematic analysis of these proteolytic processing events. Here we present a protocol that allows the identification of the C-terminal sequences of proteins. A peptide mixture is generated by cleavage of the protein with cyanogen bromide and is incubated with carboxypeptidase Y. The enzyme is only able to act on the C-terminal fragment, because this is the only peptide without a homoserine lactone residue at its C terminus. The resulting fragments, forming a peptide ladder, are analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The entire protocol, including the CNBr cleavage, takes 21 h and can be applied to proteins purified either by SDS-PAGE or by 2D PAGE or in solution.

De novo sequence analysis of N-terminal sulfonated peptides after in-gel guanidination.

Here we report a novel approach in which gel-separated proteins are guanidinated in-gel prior to enzymatic cleavage. In contrast to previously described techniques, this procedure allows the extracted tryptic peptides to be N-terminal sulfonated without any further sample purification. The derivatized peptides were subsequently fragmented using a matrix-assisted laser desorption/ionization time of flight/time of flight instrument. The approach facilitates the de novo sequence analysis and allows obtaining longer stretches of amino acid sequence information. We demonstrate that the obtained information can be used to identify proteins using a sequence similarity search algorithm. The technique was compared to the standard peptide mass fingerprint approach, applied either in-gel or in solution, using a number of sodium dodecyl sulfate-polyacrylamide gel electrophoresis separated model proteins. Finally, the new protocol was applied on a proteomic study of two-dimensional PAGE separated proteins from Shewanella oneidensis. More than 50 proteins from this organism were identified using sub-picomol quantities of protein, and peptide sequences of up to 20 amino acid residues in length have been determined.

A new method for C-terminal sequence analysis in the proteomic era.

The overall study of post-translational modifications (PTMs) of proteins is gaining strong interest. Beside phosphorylation and glycosylation, truncations of the nascent polypeptide chain at the amino or carboxy terminus are by far the most common types of PTMs in proteins. In contrast to the analysis of phosphorylation and glycosylation sites, relatively little attention has been paid to the development of approaches for the systematic analysis of proteolytic processing events. Here we present a new mass spectrometry (MS)-based strategy that allows the identification of the C-terminal sequence of proteins. The method can be directly applied to proteins cleaved with cyanogen bromide (CNBr) and purified either by SDS-PAGE, by two-dimensional (2D) PAGE or in solution, and it therefore eliminates the need for specific isolation of the C-terminal peptide. Using Shewanella oneidensis as a model system, we have demonstrated that this approach can be used for C-terminal sequence analysis at a proteomic scale. We also applied the method to study the C-terminal proteolytic processing of procardosin A.