Expression of natural human b1,4-GalT1 variants and of non-mammalian homologues in plants leads to differences in galactosylation of N-glycans
Hesselink, T. ; Rouwendal, G.J.A. ; Henquet, M.G.L. ; Florack, D.E.A. ; Helsper, J.P.F.G. ; Bosch, H.J. - \ 2014
Transgenic Research 23 (2014)5. - ISSN 0962-8819 - p. 717 - 728.
golgi-apparatus - murine beta-1,4-galactosyltransferase - beta 1,4-galactosyltransferase - transgenic plants - gene - cells - localization - antibodies - oligosaccharides - glycoproteins
b1,4-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 b1,4-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 biantennary 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 a2,6-sialyltransferase yielded a gene whose expression enhanced the level of bi-antennary galactosylation even further.
Phosphorescence Imaging of Living Cells with Amino Acid-Functionalized Tris(2-phenylpyridine)iridium(III) Complexes
Steunenberg, P. ; Ruggi, A. ; Berg, N.S. van den; Buckle, T. ; Kuil, J. ; Leeuwen, F.W.B. van; Velders, A.H. - \ 2012
Inorganic Chemistry 51 (2012)4. - ISSN 0020-1669 - p. 2105 - 2114.
cyclometalated iridium complexes - light-emitting-diodes - golgi-apparatus - energy-transfer - emission - ligand - microscopy - ir(iii) - accumulation - derivatives
A series of nine luminescent cyclometalated octahedral iridium(III) tris(2-phenylpyridine) complexes has been synthesized, functionalized with three different amino acids (glycine, alanine, and lysine), on one, two, or all three of the phenylpyridine ligands. All starting complexes and final compounds have been fully analyzed by one-dimensional (ID) and two-dimensional (2D) NMR spectroscopy, and photophysical data have been obtained for all the mono-, bis-, and tri- substituted iridium(III) complexes. Cellular uptake and localization have been studied with flow cytometry and confocal microscopy, respectively. Confocal experiments demonstrate that all nine substituted iridium(III) complexes show variable uptake in the tumor cells. The monosubstituted iridium(III) complexes give the highest cellular uptake, and the series substituted with lysines shows the highest toxicity. This systematic study of amino acid-functionalized Ir(ppy)(3) complexes provides guidelines for further functionalization and possible implementation of luminescent iridium complexes, for example, in (automated) peptide synthesis or biomarker specific targeting.
Comprehensive overview of the vertebrate p24 family: identification of a novel tissue-specifically expressed member
Strating, J. ; Bakel, N.H.M. van; Leunissen, J.A.M. ; Martens, G.J.M. - \ 2009
Molecular Biology and Evolution 26 (2009)8. - ISSN 0737-4038 - p. 1707 - 1714.
multiple sequence alignment - putative cargo receptors - coated transport vesicles - endoplasmic-reticulum - secretory pathway - transmembrane protein - golgi-apparatus - coatomer - emp24p - complex
The members of the p24 protein family have an important but unclear role in transport processes in the early secretory pathway. The p24 family consists of four subfamilies (alpha, beta, gamma, and delta), whereby the exact composition of the family varies among species. Despite more than 15 years of p24 research, the vertebrate p24 family is still surprisingly ill characterized. Here, we describe the human, mouse, Xenopus, and zebrafish orthologues of 10 p24 family members and a new member that we term p24 gamma(5). Of these eleven p24 family members, nine are conserved throughout the vertebrate lineage, whereas two (p24 gamma(4) and p24 delta(2)) occur in some but not all vertebrates. We further show that all p24 proteins are widely expressed in mouse, except for p24 alpha(1) and p24 gamma(5) that display restricted expression patterns. Thus, we present for the first time a comprehensive overview of the phylogeny and expression of the vertebrate p24 protein family.
Molecular cloning of two Arabidopsis UDP-galactose transporters by complementation of a deficient Chinese hamster ovary cell line
Bakker, H. ; Routier, F. ; Oelmann, S. ; Jordi, W.J.R.M. ; Lommen, A. ; Gerardy-Schahn, R. ; Bosch, H.J. - \ 2005
Glycobiology 15 (2005)2. - ISSN 0959-6658 - p. 193 - 201.
cmp-sialic acid - nucleotide-sugar transporters - gdp-mannose transporter - expression cloning - n-acetylgalactosamine - golgi-apparatus - gene family - 5'-phosphosulfate transporter - functional expression - carbohydrate epitope
Nucleotide-sugar transporters (NSTs) form a family of structurally related transmembrane proteins that transport nucleotide-sugars from the cytoplasm to the endoplasmic reticulum and Golgi lumen. In these organelles, activated sugars are substrates for various glycosyltransferases involved in oligo- and polysaccharide biosynthesis. The Arabidopsis thaliana genome contains more than 40 members of this transporter gene family, of which only a few are functionally characterized. In this study, two Arabidopsis UDP-galactose transporter cDNAs (UDP-GalT1 and UDP-GalT2) are isolated by expression cloning using a Chinese hamster ovary cell line (CHO-Lec8) deficient in UDP-galactose transport. The isolated genes show only 21% identity to each other and very limited sequence identity with human and yeast UDP-galactose transporters and other NSTs. Despite this low overall identity, the two proteins clearly belong to the same gene family. Besides complementing Lec8 cells, the two NSTs are shown to transport exclusively UDP-galactose by an in vitro NST assay. The most homologous proteins with known function are plant transporters that locate in the inner chloroplast membrane and transport triose-phosphate, phosphoenolpyruvate, glucose-6-phosphate, and xylulose 5-phosphate. Also, the latter proteins are members of the same family, which therefore has been named the NST/triose-phosphate transporter family