André Dinter

Golgi-disturbing agents

Abstract Pharmacological agents have proven useful for gaining fundamental insights into the biology of the Golgi apparatus. This review summarizes pertinent and recent work on the effects on this organelle of monensin, brefeldin A, bafilomycin, ilimaquinone, okadaic acid, retinoic acid, and nocodazole. The molecular targets of monensin, brefeldin A, ilimaquinone, and retinoic acid remain to be elucidated whereas those for bafilomycin (vacuolar H+-ATPase), okadaic acid (serine/threonine phosphatases types 1, 2a, and 2b), and nocodazole (microtubules) are reasonably well understood. The molecular target of brefeldin has not been defined, but has been suggested to involve guanine nucleotide exchange proteins acting on ADP-ribosylation factor 1. Whether a defined molecular target can be found for monensin must be questioned since its main action consists in exchanging protons for Na+ which leads to osmotic swelling of post-Golgi endosomal structures and Golgi subcompartments by virtue of its membrane-associated effect as a cationophore. Brefeldin A was one of the most thoroughly investigated Golgi-disturbing agents and proved instrumental in unraveling retrograde flow mechanisms in the secretory pathways. Okadaic acid attracted interest for its properties mimicking mitotic fragmentation of the Golgi apparatus. Nocodazole was instrumental in establishing the cytoskeletal anchoring of the Golgi apparatus close to the microtubular organizing center.

Genomic cloning and expression of three murine UDP-galactose: beta-N-acetylglucosamine beta1,3-galactosyltransferase genes.

Based on the detection of expressed sequence tags that are similar to known galactosyltransferase sequences, we have isolated three novel UDP-galactose:β-N-acetylglucosamine β1,3-galactosyltransferase (β3GalT) genes from a mouse genomic library. The three genes, named β3GalT-I, -II, and -III, encode type II transmembrane proteins of 326, 422, and 331 amino acids, respectively. The three proteins constitute a distinct subfamily as they do not share any sequence identity with other eucaryotic galactosyltransferases. Also, the entire protein-coding region of the three β3GalT genes was contained in a single exon, which contrasts with the genomic organization of the β1,4- and α1,3-galactosyltransferase genes. The three β3GalT genes were mainly expressed in brain tissue. The expression of the full-length murine genes as recombinant baculoviruses in insect cells revealed that the β3GalT enzymes share the same acceptor specificity for β-linked GlcNAc, although they differ in their K m for this acceptor and the donor UDP-Gal. The identification of β3GalT genes emphasizes the structural diversity present in the galactosyltransferase gene family.

The Regulation of Cell-and Tissue-Specific Expression of Glycans by Glycosyltransferases

Recent developments in the rapidly evolving field of glycobiology led to the notion of spatially and temporally regulated expression of glycan structures on the cell surface. By virtue of the concise informational package of complex glycans and their relative distribution and densities, biospecific interactions between cells are rendered possible. Thus, glycan expression is likely to have a major impact on morphogenesis during ontogenesis. This hypothesis has gained major support by recent work showing that mice in which the GnT-I gene (Mgat-I) was rendered nonfunctional displayed frequent inversions of the left-right asymmetry before exerting its effect as a lethal factor [94]. Moreover, a newly discovered inborn error of metabolism affecting GnT-II seems to be responsible for pronounced dysmorphic features [46].

Completely enzymic synthesis of the mucin-type sialyl Lewis x epitope, involved in the interaction between PSGL-1 and P-selectin

Sialyl Lewis x (sLex) is an established selectin ligand occurring on N- and O-linked glycans. Using a completely enzymic approach starting from p-nitrophenyl N-acetyl-α-D-galactosaminide (GalNAc(α1-pNp as core substrate, the sLex-oligosaccharide Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)[Gal(β1-3)]GalNAc(α1-pNp, representing the O-linked form, was synthesized in an overall yield of 32%. In a first step, Gal(β1-3)GalNAc(α1-pNp was prepared in a yield of 52% using UDP-Gal and an enriched preparation of β3-galactosyltransferase (EC 2.4.1.122) from rat liver. UDP-GlcNAc and a recombinant affinity-purified preparation of core 2 β6-N-acetylglucosaminyltransferase (EC 2.4.1.102) fused to Protein A were used to branch the core 1 structure, affording GlcNAc(β1-6)[Gal(β1-3)]GalNAc(α1-pNp in a yield of >85%. The core 2 structure was galactosylated using UDP-Gal and purified human milk β4-galactosyltransferase 1 (EC 2.4.1.38) (yield of >85%), then sialylated using CMP-Neu5Ac and purified recombinant α3-sialyltransferase 3 (EC 2.4.99.X) (yield of 87%), and finally fucosylated using GDP-Fuc and recombinant human α3-fucosyltransferase 6 (EC 2.4.1.152) produced in Pichia pastoris (yield of 100%). Overall 1.5 µmol of product was prepared. MALDI TOF mass spectra, and 1D and 2D TOCSY and ROESY 1H NMR analysis confirmed the obtained structure.

Repressed β-1,3-galactosyltransferase in the Tn syndrome

Abstract The human hematopoietic disorder named Tn syndrome has been ascribed to an acquired stem cell mutation resulting in loss of β -1,3-galactosyltransferase activity in affected Tn+ cells of the hematopoietic lineages. Recently, we could demonstrate that this deficiency is due to a repression of a functional allele of the β -1,3-Gal-T gene since treatment of Tn+ T-lymphocytes from a patient (R.R.) afflicted with the Tn-syndrome with 5-azacytidine or Na n -butyrate resulted in re-expression of the Thomsen–Friedenreich (TF) antigen, the product of β -1,3-Gal-T activity [M. Thurnher, S. Rusconi, E.G. Berger, Persistent repression of functional allele can be responsible for galactosyltransferase deficiency in Tn syndrome, J. Clin. Invest. 91 (1993) 2103–2110]. To reduce these observations to a common pathogenetic mechanism responsible for the Tn-syndrome, more Tn patients need to be investigated. Here, we describe similar Tn+ T-lymphocytes cultured ex vivo from patient M.Z. whose Tn+ syndrome was newly recognized. Tn+ and TF+ T-lymphocyte cultures were characterized by flow cytometry and measurement of β -1,3-Gal-T and shown to be deficient in Tn+ cells. Furthermore, Tn+ cells were treated with 5-azacytidine and Na n -butyrate as described before. Reoccurrence of β -1,3-Gal-T activity dependent epitopes on the cell surface of Tn+ cells was shown by flow cytometry. These support the notion of β -1,3-Gal-T gene repression as a common pathogenetic mechanism underlying the Tn-syndrome.

Immunodetection of Glycosyltransferases: Prospects and Pitfalls

The key element to understand biosynthesis and function of the countless oligosaccharide structures expressed on cell surfaces and soluble glycoconjugates are the enzymes involved in their construction: the glycosyltransferases. Their investigation relies on three basic pillars: i) the biochemical which comprises methods for their purification and assays for enzymic activity; ii) the cell biological which is usually based on antibodies providing means to study their subcellular localization, trafficking and tissue distribution and iii) the approach based on recombinant DNA technology which provides insights in regulation of gene expression, structural data and approaches to test structure/function relationships of heterologously expressed enzymes.

Improved product formation in high density Chinese hamster ovary cell cultures transfected at confluency

Cell-specific productivity was compared in Chinese hamster ovary cell clones transfected at confluency versus subconfluency. Following lipofection of a pCDNA1-derived vector encoding a proteinA-fucosyltransferaseIV fusion protein, selection and single cell sorting by flow cytometry, clones were expanded to tissue flasks and assessed for cell specific productivity. The probability of obtaining high producers at confluency was significantly higher from cells transfected at confluency than at subconfluency (P=0.05).