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The genetically encoded calcium sensor protein Cameleon YC3.6 has previously been applied for functional G protein-coupled receptor screening using receptor cell arrays. However, different types of sensors are available, with a wide range in [Ca2+] sensitivity, Hill coefficients, calcium binding domains, and fluorophores, which could potentially improve the performance of the assay. Here, we compared the responses of 3 structurally different calcium sensor proteins (Cameleon YC3.6, Nano140, and Twitch2B) simultaneously, on a single chip, at different cytosolic expression levels and in combination with 2 different bitter receptors, TAS2R8 and TAS2R14. Sensor concentrations were modified by varying the amount of calcium sensor DNA that was printed on the DNA arrays prior to reverse transfection. We found that ~2-fold lower concentrations of calcium sensor protein, by transfecting 4 times less sensor-coding DNA, resulted in more sensitive bitter responses. The best results were obtained with Twitch2B, where, relative to YC3.6 at the default DNA concentration, a 4-fold lower DNA concentration increased sensitivity 60-fold and signal strength 5- to 10-fold. Next, we compared the performance of YC3.6 and Twitch2B against an array with 11 different bitter taste receptors. We observed a 2- to 8-fold increase in sensitivity using Twitch2B compared with YC3.6. The bitter receptor arrays contained 300 spots and could be exposed to a series of 18 injections within 1 h resulting in 5400 measurements. These optimized sensor conditions provide a basis for enhancing receptomics calcium assays for receptors with poor Ca2+ signaling and will benefit future high-throughput receptomics experiments., (© The Author(s) 2019. Published by Oxford University Press.)

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, β1,2-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., (© 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

β1,4-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 β1,4-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.

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., (© 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)