Calcium Imaging of GPCR Activation Using Arrays of Reverse Transfected HEK293 Cells in a Microfluidic System.

Reverse-transfected cell arrays in microfluidic systems have great potential to perform large-scale parallel screening of G protein-coupled receptor (GPCR) activation. Here, we report the preparation of a novel platform using reverse transfection of HEK293 cells, imaging by stereo-fluorescence microscopy in a flowcell format, real-time monitoring of cytosolic calcium ion fluctuations using the fluorescent protein Cameleon and analysis of GPCR responses to sequential sample exposures. To determine the relationship between DNA concentration and gene expression, we analyzed cell arrays made with variable concentrations of plasmid DNA encoding fluorescent proteins and the Neurokinin 1 (NK1) receptor. We observed pronounced effects on gene expression of both the specific and total DNA concentration. Reverse transfected spots with NK1 plasmid DNA at 1% of total DNA still resulted in detectable NK1 activation when exposed to its ligand. By varying the GPCR DNA concentration in reverse transfection, the sensitivity and robustness of the receptor response for sequential sample exposures was optimized. An injection series is shown for an array containing the NK1 receptor, bitter receptor TAS2R8 and controls. Both receptors were exposed 14 times to alternating samples of two ligands. Specific responses remained reproducible. This platform introduces new opportunities for high throughput screening of GPCR libraries.

Identification of a drimenol synthase and drimenol oxidase from Persicaria hydropiper, involved in the biosynthesis of insect deterrent drimanes.

The sesquiterpenoid polygodial, which belongs to the drimane family, has been shown to be an antifeedant for a number of herbivorous insects. It is presumed to be synthesized from farnesyl diphosphate via drimenol, subsequent C-12 hydroxylation and further oxidations at both C-11 and C-12 to form a dialdehyde. Here, we have identified a drimenol synthase (PhDS) and a cytochrome P450 drimenol oxidase (PhDOX1) from Persicaria hydropiper. Expression of PhDS in yeast and plants resulted in production of drimenol alone. Co-expression of PhDS with PhDOX1 in yeast yielded drimendiol, the 12-hydroxylation product of drimenol, as a major product, and cinnamolide. When PhDS and PhDOX1 were transiently expressed by agro-infiltration in Nicotiana benthamiana leaves, drimenol was almost completely converted into cinnamolide and several additional drimenol derivatives were observed. In vitro assays showed that PhDOX1 only catalyses the conversion from drimenol to drimendiol, and not the further oxidation into an aldehyde. In yeast and heterologous plant hosts, the C-12 position of drimendiol is therefore likely to be further oxidized by endogenous enzymes into an aldehyde and subsequently converted to cinnamolide, presumably by spontaneous hemiacetal formation with the C-11 hydroxyl group followed by oxidation. Purified cinnamolide was confirmed by NMR and shown to be deterrent with an effective deterrent dose (ED50 ) of about 200-400 µg g-1 fresh weight against both whiteflies and aphids. The putative additional physiological and biochemical requirements for polygodial biosynthesis and stable storage in plant tissues are discussed.

Monomeric IgA can be produced in planta as efficient as IgG, yet receives different N-glycans.

The unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. However, in contrast to IgG, reports on plant production of IgA are scarce. We produced IgA1κ and IgG1κ versions of three therapeutic antibodies directed against pro-inflammatory cytokines in Nicotiana benthamiana: Infliximab and Adalimumab, directed against TNF-α, and Ustekinumab, directed against the interleukin-12p40 subunit. We evaluated antibody yield, quality and N-glycosylation. All six antibodies had comparable levels of expression between 3.5 and 9% of total soluble protein content and were shown to have neutralizing activity in a cell-based assay. However, IgA1κ-based Adalimumab and Ustekinumab were poorly secreted compared to their IgG counterparts. Infliximab was poorly secreted regardless of isotype backbone. This corresponded with the observation that both IgA1κ- and IgG1κ-based Infliximab were enriched in oligomannose-type N-glycan structures. For IgG1κ-based Ustekinumab and Adalimumab, the major N-glycan type was the typical plant complex N-glycan, biantennary with terminal N-acetylglucosamine, ß1,2-xylose and core α1,3-fucose. In contrast, the major N-glycan on the IgA-based antibodies was xylosylated, but lacked core α1,3-fucose and one terminal N-acetylglucosamine. This type of N-glycan occurs usually in marginal percentages in plants and was never shown to be the main fraction of a plant-produced recombinant protein. Our data demonstrate that the antibody isotype may have a profound influence on the type of N-glycan an antibody receives.

Expression of natural human ß1,4-GalT1 variants and of non-mammalian homologues in plants leads to differences in galactosylation of N-glycans.

ß1,4-Galactosylation of plant N-glycans is a prerequisite for commercial production of certain biopharmaceuticals in plants. Two different types of galactosylated N-glycans have initially been reported in plants as the result of expression of human ß1,4-galactosyltransferase 1 (GalT). Here we show that these differences are associated with differences at its N-terminus: the natural short variant of human GalT results in hybrid type N-glycans, whereas the long form generates bi-antennary complex type N-glycans. Furthermore, expression of non-mammalian, chicken and zebrafish GalT homologues with N-termini resembling the short human GalT N-terminus also induce hybrid type N-glycans. Providing both non-mammalian GalTs with a 13 amino acid N-terminal extension that distinguishes the two naturally occurring forms of human GalT, acted to increase the levels of bi-antennary galactosylated N-glycans when expressed in tobacco leaves. Replacement of the cytosolic tail and transmembrane domain of chicken and zebrafish GalTs with the corresponding region of rat α2,6-sialyltransferase yielded a gene whose expression enhanced the level of bi-antennary galactosylation even further.

N-glycan occupancy of Arabidopsis N-glycoproteins.

Most secreted proteins in eukaryotes are modified on the amino acid consensus sequence NxS/T by an N-glycan through the process of N-glycosylation. The N-glycans on glycoproteins are processed in the endoplasmic reticulum (ER) to different mannose-type N-glycans or, when the protein passes through the Golgi apparatus, to different complex glycan forms. Here we describe the capturing of N-glycopeptides from a trypsin digest of total protein extracts of Arabidopsis plants and release of these captured peptides following Peptide N-glycosidase (PNGase) treatment for analysis of N-glycan site-occupancy. The mixture of peptides released as a consequence of the PNGase treatment was analyzed by two dimensional nano-LC-MS. As the PNGase treatment of glycopeptides results in the deamidation of the asparagine (N) in the NxS/T site of the released peptide, this asparagine (N) to aspartic acid (D) conversion is used as a glycosylation 'signature'. The efficiency of PNGase F and PNGase A in peptide release is discussed. The identification of proteins with a single glycopeptide was limited by the used search algorithm but could be improved using a reference database including deamidated peptide sequences. Additional stringency settings were used for filtering results to minimize false discovery. This resulted in identification of 330 glycopeptides on 173 glycoproteins from Arabidopsis, of which 28 putative glycoproteins, that were previously not annotated as secreted protein in The Arabidopsis Information Resource database (TAIR). Furthermore, the identified glycosylation site occupancy helped to determine the correct topology for membrane proteins. A quantitative comparison of peptide signal was made between wild type and complex-glycan-less (cgl) mutant Arabidopsis from three replicate leaf samples using a label-free MS peak comparison. As an example, the identified membrane protein SKU5 (AT4G12420) showed differential glycopeptide intensity ratios between WT and cgl indicating heterogeneous glycan modification on single protein. BIOLOGICAL SIGNIFICANCE: Proteins that enter the secretory pathway are mostly modified by N-glycans. The function of N-glycosylation has been well studied in mammals. However, in plants the function of N-glycosylation is still unclear, because glycosylation mutants in plants often do not have a clear phenotype. Here we analyzed which proteins are modified by N-glycans in plants by developing a glycopeptide enrichment method for plant proteins. Subsequently, label free comparative proteomics was employed using protein fractions from wild type and from a mutant which is blocked in modification of the N-glycan into complex glycans. The results provide new information on N-glycosylation sites on numerous secreted proteins. Results allow for specific mapping of multiple glycosylation site occupancy on proteins, which provides information on which glycosylation sites are protected or non-used from downstream processing and thus presumably are buried into the protein structure. Glycoproteomics can therefore contribute to protein structure analysis. Indeed, mapping the glycosylation sites on membrane proteins gives information on the topology of protein folds over the membrane. We thus were able to correct the topology prediction of three membrane proteins. Besides, these studies also identified limitations in the software that is used to identify single modified peptide per protein. This article is part of a Special Issue entitled: Translational Plant Proteomics.

N-glycoproteomics in plants: perspectives and challenges.

In eukaryotes, proteins that are secreted into the ER are mostly modified by N-glycans on consensus NxS/T sites. The N-linked glycan subsequently undergoes varying degrees of processing by enzymes which are spatially distributed over the ER and the Golgi apparatus. The post-ER N-glycan processing to complex glycans differs between animals and plants, with consequences for N-glycan and glycopeptide isolation and characterization of plant glycoproteins. Here we describe some recent developments in plant glycoproteomics and illustrate how general and plant specific technologies may be used to address different important biological questions.