René Cacan

Protein ubiquitination is modulated by O-GlcNAc glycosylation

During the past two decades, O‐GlcNAc modification of cytosolic and nuclear proteins has been intensively studied. Nevertheless, the function of this post‐translational modification remains unclear. It has been recently speculated that O‐GlcNAc could act as a protective signal against proteasomal degradation, both by modifying target substrates and/or by inhibiting the proteasome itself. In this work, we have investigated the putative relation between O‐GlcNAc and the ubiquitin pathway. First, we showed that the level of both modifications increased rapidly after thermal stress but, unlike ubiquitinated proteins, O‐GlcNAc‐modified proteins failed to be stabilized by inhibiting proteasome function. Increasing O‐GlcNAc levels, using glucosamine or PUGNAc, enhanced ubiquitination. Inversely, when O‐GlcNAc levels were reduced, using forskolin or glucose deprivation, ubiquitination decreased. Targeted‐RNA interference of O‐GlcNAc transferase also reduced ubiquitination and moreover halved cell thermotolerance. Finally, we demonstrated that the ubiquitin‐activating enzyme E1 was O‐GlcNAc modified and that its glycosylation and its interaction with Hsp70 varied according to the conditions of cell culture. Altogether, these results show that O‐GlcNAc and ubiquitin are not strictly antagonistic post‐translational modifications, but rather that the former might regulate the latter, and also suggest that E1 could be one of the common links between the two pathways. —Guinez, C., Mir, A.‐M., Dehennaut, V., Cacan, R., Harduin‐Lepers, A., Michalski, J.‐C., Lefebvre, T. Protein ubiquitination is modulated by O‐GlcNAc glycosylation. FASEB J. 22, 2901–2911 (2008)

The unfolded protein response in a dolichyl phosphate mannose-deficient Chinese hamster ovary cell line points out the key role of a demannosylation step in the quality-control mechanism of N-glycoproteins.

The CHO (Chinese hamster ovary) glycosylation mutant cell line, B3F7, transfers the truncated glycan Glc(3)Man(5)GlcNAc(2) on to nascent proteins. After deglucosylation, the resulting Man(5)GlcNAc(2) glycan is subjected to two reciprocal enzymic processes: the action of an endoplasmic-reticulum (ER) kifunensine-sensitive alpha1,2-mannosidase activity to yield a Man(4)GlcNAc(2) glycan, and the reglucosylation involved in the quality-control system which ensures that only correctly folded glycoproteins leave the ER. We show that the recombinant secreted alkaline phosphatase (SeAP) produced in stably transfected B3F7 cells, is co-immunoprecipitated with the GRP78 (glucose-regulated protein 78), a protein marker of the unfolded protein response (UPR). The level of GRP78 transcription has been evaluated by reverse transcription-PCR (RT-PCR) and we demonstrate that B3F7 cells present a constitutively higher level of UPR in the absence of inductors, compared with Pro(-5) cells. Interestingly, a decrease was observed in the UPR and an increase in SeAP secretion in the kifunensine-treated B3F7 cells. Altogether, these data highlight the relationships between the glycan structure, the quality control system and the UPR. Moreover, they support the idea that a specific demannosylation step is a key event of the glycoprotein quality control in B3F7 cells.

Transport of CMP‐N‐glycoloylneuraminic acid into mouse liver Golgi vesicles

CMP‐Neu5Gc has been shown to be transported into mouse liver Golgi vesicles by a specific carrier the characteristics of which were investigated in detail. In the system employed, CMP‐Neu5Gc enters the Golgi vesicles within 2 min; transport was saturable with high concentrations of the sugar‐nucleotide and was dependent on temperature. A kinetic analysis gave an apparent K m of 1.3 μM and a maximal transport velocity of 335 pmol/mg protein per min. Almost identical values were obtained with CMP‐Neu5Ac, under the same incubation conditions. Furthermore, the uptake of CMP‐Neu5Gc was inhibited by CMP‐Neu5Ac, a substrate analogue. Conversely, the uptake of CMP‐Neu5Ac was inhibited by CMP‐Neu5Gc to the same extent, leading to the conclusion that the transport of CMP‐Neu5Ac and CMP‐Neu5Gc is mediated by the same carrier molecule. This transport system for CMP‐Neu5Gc involves both CMP and CMP‐Neu5Gc since intravesicular CMP induced the entry of external CMP‐Neu5Gc.

Permeabilized cells as a way of gaining access to intracellular organelles: an approach to glycosylation reactions.

The selective plasma membrane permeabilization of animal cells is a way of introducing non permeable substrates into the cytoplasmic space. This technique facilitates the introduction of a wide range of labelled precursors and avoids the drawbacks of subcellular fractionation. We review here various physical and chemical methods successfully used in different metabolic studies, and as an example, note the advantages of permeabilized cells in glycosylation studies.

The Dual Origin of Toxoplasma gondii N-Glycans

N-Linked glycosylation is the most frequent modification of secreted proteins in eukaryotic cells that plays a crucial role in protein folding and trafficking. Mature N-glycans are sequentially processed in the endoplasmic reticulum and Golgi apparatus through a pathway highly conserved in most eukaryotic organisms. Here, we demonstrate that the obligate intracellular protozoan parasite Toxoplasma gondii independently transfers endogenous truncated as well as host-derived N-glycans onto its own proteins.Therefore, we propose that the apicomplexan parasite scavenges N-glycosylation intermediates from the host cells to compensate for the rapid evolution of its biosynthetic pathway, which is primarily devoted to modification of proteins with glycosylphosphatidylinositols rather than N-glycans.

Overexpression of Man2C1 leads to protein underglycosylation and upregulation of endoplasmic reticulum-associated degradation pathway.

Unfolded glycoproteins retained in the endoplasmic reticulum (ER) are degraded via the ER-associated degradation (ERAD) pathway. These proteins are subsequently transported to the cytosol and degraded by the proteasomal complex. Although the sequential events of ERAD are well described, its regulation remains poorly understood. The cytosolic mannosidase, Man2C1, plays an essential role in the catabolism of cytosolic free oligomannosides, which are released from the degraded proteins. We have investigated the impact of Man2C1 overexpression on protein glycosylation and the ERAD process. We demonstrated that overexpression of Man2C1 led to modifications of the cytosolic pool of free oligomannosides and resulted in accumulation of small Man(2-4)GlcNAc(1) glycans in the cytosol. We further correlated this accumulation with incomplete protein glycosylation and truncated lipid-linked glycosylation precursors, which yields an increase in N-glycoprotein en route to the ERAD. We propose a model in which high mannose levels in the cytosol interfere with glucose metabolism and compromise N-glycan synthesis in the ER. Our results show a clear link between the intracellular mannose-6-phosphate level and synthesis of the lipid-linked precursors for protein glycosylation. Disturbance in these pathways interferes with protein glycosylation and upregulated ERAD. Our findings support a new concept that regulation of Man2C1 expression is essential for maintaining efficient protein N-glycosylation.

Membrane transport of sugar donors to the glycosylation sites

The assembly of N-linked glycoproteins in eukaryotic cells begins with the segregation of these molecules within the lumen of intracellular vesicles. Since the sugar nucleotides are cytoplasmic molecules, translocation of the sugar moiety across the membrane appears as a crucial event in the glycoprotein synthesis. This N-glycosylation process occurs in two different cytological sites: in the rough endoplasmic reticulum, the stepwise synthesis of a large lipid-linked oligosaccharide takes place, as well as its transfer to protein; then after trimming the immature glycoprotein is further elongated in the Golgi apparatus. In this paper, a brief review will be given of the present knowledge on the sugar donor transport across the membrane barrier to the glycosylation site. Based upon the transmembrane orientation of oligosaccharide lipid intermediates and on the localization of the glycosyltransferase active sites, the different processes required to translocate the sugar moieties during the preassembly of the dolichyl-pyrophosphate-oligosaccharides will be examined. Combining the different results, obtained in several laboratories, it is suggested that the Man5-GlcNAc2-lipid is synthesized on the cytoplasmic side directly from the sugar-nucleotides and then translocated to the lumenal face where the Glc3-Man9-GlcNAc2-lipid is completed using Man-P-Dol and Glc-P-Dol as transmembrane carriers of these sugars. Concerning the elongation process leading to assembly of the antennae of N-acetyllactosamine type oligosaccharides, specific carriers for sugar nucleotides have been described as Golgi markers. Several authors have characterized such carriers for UDP-Gal, GDP-Fuc, CMP-NeuAc, UDP-GlcNAc and UDP-Glc using microsomal vesicles and similar results have been obtained in our laboratory using plasma membrane permeabilized cells. This carrier-mediated process leads to the formation of an intralumenal pool whose biological significance will be discussed. The translocation process of sugar donors occurring in the rough endoplasmic reticulum via lipid intermediates as well as in the Golgi apparatus via specific carriers would represent a regulation step based on the availability of the substrates for the glycosylation.

Peculiar behavior of ectosialyltransferase toward exogenous acceptors

The presence of sialic acid in the cell surface glycoconjugates has a crucial importance in a variety of biological cell properties. It has been involved in the electrokinetic potential of the cell [l] and in the permeability of the membrane [2]. More recently, it has been related to the social life of the cell: cell-cell recognition [3], contact inhibition [4] and crypticity of immunogenic loci [5] . Moreover, changes in membrane-bound sialic acid have been reported to occur upon oncogenic transformation [6]. Further- more, the sialic acid level at the cell surface seems to be correlated to the life-time of the cell [7] and to regulate the activity of some ecto-enzymes [8]. These facts raise the question as to how the surface sialic acid level is maintained or modulated: does it need an intracellular biosynthesis of the complete glycoconjugates and their further integration in the membrane or may it be due to the activity of an ectosialyltransferase as a repair phenomenon? In fact, ectosialyltransferase activity has been widely reported [9-121 and ultrastructural evidence has been obtained [13] . In this paper, we demonstrate that rat spleen lymphocyte possesses an ectosialyltransferase which is able to transfer N-acetylneuraminic acid to its own membrane but is not able to transfer it to a macro- molecular exogenous acceptor. However, when the size of the same exogenous acceptor is reduced by proteolytic cleavage, it can reach the active site of the ecto-enzyme and is efficiently glycosylated. 2.

Membrane glycoprotein IIb is the major endogenous acceptor for human platelet ectosialyltransferase

1. Introduction Platelet surface glycoconjugates have been suggested to play roles in the major biological functions of this cell, namely aggregation and adhesion [ 141. Bosmann [5] first showed that platelet suspension as well as plasma membrane fraction possessed sialyltransferase activity which would play a role in platelet aggregation. In fact, a parallel inhibition of aggregation and sialyl- transferase activity by aspirin [5,6] and an apparent stimulation of sialyltransferase activity with various platelet aggregating agents such as collagen, epinephrin and ADP plus fibrinogen [7] have been noted, while cancer patients with a high incidence of thrombosis have markedly elevated levels of platelet sialyltransfer- ase activity [6]. Despite the possible functional signi- ficance of the above studies, the presence of ectosial- yltransferase activity at the human platelet surface has not been rigorously defined. This is due to possible errors introduced into most assay systems by: (i) Precursor degradation and intracellular utilisation of the free radioactive sialic acid: (ii) The liberation of intracellular enzymes as a result of cell lysis during the assay; (iii) Phagocytosis of added exogenous acceptors [8]. Using exogenous non-phagocytosable acceptors [9] and the methodology developed to prove the presence of ectoglycosyltransferases at the surface of other cell types [ lo,1 11, we demonstrate here that the human blood platelet exhibits ectosialyltransferase activity and that the major endogenous acceptor is the plama membrane glycoprotein GP IIb.

Delineation of the minimal catalytic domain of human Galβ1-3GalNAc α2,3-sialyltransferase (hST3Gal I)

The CMP-Neu5Ac:GalL1-3GalNAc K2,3-sialyltransferase (ST3Gal I, EC 2.4.99.4) is a Golgi membrane-bound type II glycoprotein that catalyses the transfer of sialic acid residues to GalL1-3GalNAc disaccharide structures found on O-glycans and glycolipids. In order to gain further insight into the structure/function of this sialyltransferase, we studied protein expression, N-glycan processing and enzymatic activity upon transient expression in the COS-7 cell line of various constructs deleted in the N-terminal portion of the protein sequence. The expressed soluble polypeptides were detected within the cell and in the cell culture media using a specific hST3Gal I monoclonal antibody. The soluble forms of the protein consisting of amino acids 26^340 (hST3-v25) and 57^340 (hST3-v56) were efficiently secreted and active. In contrast, further deletion of the N-terminal region leading to hST3-v76 and hST3-v105 gave also rise to various polypeptides that were not active within the transfected cells and not secreted in the cell culture media. The kinetic parameters of the active secreted forms were determined and shown to be in close agreement with those of the recombinant enzyme already described (H. Kitagawa, J.C. Paulson, J. Biol. Chem. 269 (1994)). In addition, the present study demonstrates that the recombinant hST3Gal I polypeptides transiently expressed in COS-7 cells are glycosylated with complex and high mannose type glycans on each of the five potential N-glycosylation sites. fl 2001 Elsevier Science B.V. All rights reserved.