Hugo J. F. Maccioni

Ganglioside Glycosyltransferases Organize in Distinct Multienzyme Complexes in CHO-K1 Cells

The synthesis of gangliosides is compartmentalized in the Golgi complex. In most cells, glycosylation of LacCer, GM3, and GD3 to form higher order species (GA2, GM2, GD2, GM1, GD1b) is displaced toward the most distal aspects of the Golgi and the trans-Golgi network, where the involved transferases (GalNAcT and GalT2) form physical and functional associations. Glycosylation of the simple species LacCer, GM3, and GD3, on the other hand, is displaced toward more proximal Golgi compartments, and we investigate here whether the involved transferases (GalT1, SialT1, and SialT2) share the property of forming physical associations. Co-immunoprecipitation experiments from membranes of CHO-K1 cells expressing epitope-tagged versions of these enzymes indicate that GalT1, SialT1, and SialT2 associate physically in a SialT1-dependent manner and that their N-terminal domains participate in these interactions. Microscopic fluorescence resonance energy transfer and fluorescence recovery after photobleaching in living cells confirmed the interactions, and in addition to showing a Golgi apparatus localization of the complexes, mapped their formation to the endoplasmic reticulum. Neither co-immunoprecipitation nor fluorescence resonance energy transfer detected interactions between either GalT2 or GalNAcT and GalT1 or SialT1 or SialT2. These results, and triple color imaging of Golgi-derived microvesicles in nocodazole-treated cells, suggest that ganglioside synthesis is organized in distinct units each formed by associations of particular glycosyltransferases, which concentrate in different sub-Golgi compartments.

Biosynthesis of Brain Gangliosides

The pathways of synthesis of brain gangliosides have been inferred from results of usual enzymic experiments in which all the substrates are from exogenous origen and from results of experiments in which endogenous acceptors present in subcellular membranes are labelled by using exogenous, water soluble donors. For the synthesis of most gangliosides the results obtained with both methods are in agreement but in the synthesis of GDIb they are discrepant. The pathways obtained with each method are summarized in Fig. 1a and 1b. In the present communication we will deal preferentially with progress made in this field since the ganglioside conference at Strasbourg in 1973 (see ref. 12).

THE SITE OF SYNTHESIS OF GANGLIOSIDES IN THE CHICK OPTIC SYSTEM

Abstract– In the retinas of 1‐day‐old chickens that received an intraocular injection of N‐[3H]acetylmannosamine the labelling of N‐acetylneuraminic acid and CMP‐N‐acetylneuraminic acid increased for at least 8 h and that of gangliosides for at least 24 h after injection. In the optic tectum contralateral to the injected eye at 8 h after the intraocular injection, the labelling of gangliosides exceeded the labelling of gangliosides in the ipsilateral tectum by approx 20‐fold. In the contralateral tectum the highest concentration of labelled gangliosides was in subfractions enriched in synaptosomes and synaptic plasma membranes. No significant contralateral ipsilateral differences were found in the acid soluble substances of the tectum. In the optic tectum, labelled gangliosides appeared earlier in the neuronal perikarya than in synaptosomes when the injection was intracranial. Conversely, when the injection was intraocular the labelling appeared earlier in the synaptosomes than in the neuronal perikarya. The radioactivity pattern of the optic tectum gangliosides resembled the pattern of retina gangliosides when N‐[3H]acetylmannosamine was injected intraocularly, but when N‐[3H]acetylmannosamine was given intracerebrally the radioactivity pattern resembled that of optic tectum gangliosides. Intraocular injection of colchicine or vinblastine did not affect the labelling of retinal gangliosides from N‐[3H]acetylmannosamine injected into the same eye but prevented the appearance of labelled gangliosides in the optic tectum. In vitro the ganglioside glycosylating activity of optic tectum synaptosomes and synaptic plasma membranes was between 6 and 10‐fold lower than that found in the optic tectum neuronal perikarya. These findings support the notion that the main subcellular site of synthesis of neuronal gangliosides is in the neuronal perikarya, from which they are translocated to the nerve endings.

Biosynthesis and expression of gangliosides during differentiation of chick embryo retina cells in vitro

Cells from neural retina from 7‐day chick embryos were cultured on polylysine‐coated dishes up to 7 days. The small, round‐shaped cells at seeding differentiated progressively, and after 4 days in vitro the majority had enlarged bodies and abundant processes. The content of protein and DNA was essentially unchanged during the entire period of culture. The incorporation of radioactivity from [3H]glucosamine into gangliosides declined slightly, reaching about 65% of the initial values at the end of the culture period. The proliferating activity measured by the incorporation of [3H]thymidine into DNA decreased to 10% or less of the initial value after 3 days in vitro. Almost at the same chronological times as in ovo, the synthesis of GD3 and of a ganglioside partially identified as GT3 decreased from 70 and 19% of the total incorporation into gangliosides in the first 20 h of culture to about 7 and 5%, respectively, after 3 days in vitro. Conversely, the synthesis of GDI a increased from about 6% at the beginning to about 70% at the end of the culture times. Immunocytochemical analyses of the expression of gangliotetraosyl gangliosides in cultured cells showed that these gangliosides appeared in the bodies and processes of cells having neuronal morphology; very little immunostaining of the scarce flattened cells, probably Müller cells, was to and. The results indicate that the changes in ganglioside metabolism, which lead to decreased synthesis of gangliosides lacking the galactosyl‐N‐acetyl‐galactosaminyl disaccharide end and to increased synthesis of gangliotetraosyl gangliosides, occur in cells that in culture differentiate into neurons.

Understanding the stepwise synthesis of glycolipids.

Glycolipid expression is highly regulated during development and differeniation. The control relies mainly on transcriptional modulation of key glycosyltransferases acting at the branching points of the pathway of biosynthesis. Transferases are Golgi residents that depend on N-glycosylation and oligosaccharide processing for proper folding in the endoplasmic reticulum. The N-terminal domain bears information for their transport to the Golgi, retention in the organelle and differential concentration in sub-Golgi compartments. In the Golgi, some transferases associate forming functional multienzyme complexes. It is envisaged that the machinery for synthesis in the Golgi complex, and its dynamics, constitute a potential target for fine tuning of the control of glycolipid expression according to cell demands.

Glycosylation of glycolipids in the Golgi complex

Gangliosides are a family of glycolipids characterized by containing a variable number of sialic acid residues. Nearly, all animal cells contain at least some class of ganglioside in their membranes, but membranes from the CNS are characterized by their high content of these lipids. The synthesis of the oligosaccharide moiety of glycolipids is carried out in the Golgi complex. In this study, I will discuss the cellular and molecular basis of the organization of the glycosylating machinery in the Golgi complex, with particular attention to the mutual relationships, sub‐Golgi localization, and intracellular trafficking of glycolipid glycosyltransferases, and to their relationships with the corresponding glycolipid acceptors and sugar nucleotide donors. I will also discuss how the organization of the glycosylating machinery in the Golgi may adapt to events controlling glycolipid expression.

GA2/GM2/GD2 synthase localizes to the trans-golgi network of CHO-K1 cells.

UDP-GalNAc:lactosylceramide/GM3/GD3 beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T) transforms its acceptors into the gangliosides GA2, GM2 and GD2. It is well established that it is a Golgi-located glycosyltransferase, but its sub-Golgi localization is still unclear. We addressed this question in Chinese hamster ovary K1 cell clones stably transfected with a c-myc-tagged version of GalNAc-T which express the enzyme at different levels of activity. In these cell clones we examined the effect of brefeldin A (BFA) on the synthesis of glycolipids (in metabolic-labelling experiments) and on the sub-Golgi localization of the GalNAc-T (by immunocytochemistry). We found that in cell clones expressing moderate levels of activity, GalNAc-T immunoreactivity behaved as the trans-Golgi network (TGN) marker mannose-6-P receptor (M6PR) both in BFA-treated and untreated cells, and that BFA completely blocked the synthesis of GM2, GM1 and GD1a. On the other hand, in cell clones expressing high levels of activity and treated with BFA, most GalNAc-T immunoreactivity redistributed to the endoplasmic reticulum, as did the medial-Golgi marker mannosidase II, and the synthesis of GM2, GM1 and GD1a was not completely blocked. These results indicate that GalNAc-T is a TGN-located enzyme and that the mechanism that localizes it to this compartment involves steps that, when saturated, lead to its mislocalization to the cis-, medial- or trans-Golgi. Changes of Golgi membrane properties by modification of local glycolipid composition due to the activity of the expressed enzyme were not the main cause of mislocalization, since it persists when glycolipid synthesis is inhibited with d, l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCl.

Chinese hamster ovary cells lacking GM1 and GD1a synthesize gangliosides upon transfection with human GM2 synthase

GM3-positive Chinese hamster ovary cells (CHO-K1 cells) lack the ability to synthesize GM2 and the complex gangliosides GM1 and GD1a from [3H]Gal added to the culture medium. However, they acquire the ability to synthesize GM2 and to synthesize and immunoexpress complex gangliosides upon transient transfection with a cDNA encoding the human GM3:N-acetylgalactosaminyl transferase (GM2 synthase). The activities of endogenous GM1- and GD1a-synthases in the parental cell line and in cells transfected with the plasmid with or without the GM2 synthase cDNA were essentially identical and comparable in terms of specific activity with the endogenous GM3 synthase. Results indicate that glycosyltransferases acting on GM2 to produce GM1 and GD1a are constitutively present in CHO-K1 cells, and that the expression of their activities depend on the supply of the acceptor GM2. In addition, these results lend support to the notion that GM2 synthase is a key regulatory enzyme influencing the balance between simple and complex gangliosides.

Cytoplasmic Tails of SialT2 and GalNAcT Impose Their Respective Proximal and Distal Golgi Localization

Complex glycolipid synthesis is catalyzed by different glycosyltransferases resident of the Golgi complex. Most of them are type II membrane proteins comprising a lumenal, C‐terminal domain linked to an N‐terminal domain (Ntd) constituted by a short cytoplasmic tail (ct), a transmembrane, and a lumenal stem regions. They concentrate selectively in different sub‐Golgi compartments, in an overlapped manner, acting in succession in the addition of sugars to acceptor glycolipids. The Ntds are sufficient to localize glycosyltransferases in the Golgi complex, but it is not clear whether they also confer selective concentration in sub‐Golgi compartments. Here, we studied whether the Ntd of SialT2, localized in the proximal Golgi, and the one of GalNAcT, a trans/TGN Golgi‐concentrated enzyme, concentrate reporter proteins in the corresponding sub‐Golgi compartment. The sub‐Golgi concentration of the Ntds fused to spectral variants of the GFP was determined in CHO‐K1 cells from their behavior upon addition of brefeldin A. Fluorescence microscopy and subcellular fractionation showed that the SialT2 Ntd concentrates in a proximal sub‐Golgi compartment – and that of GalNAcT in TGN elements. Exchanging the transmembrane region and the cts of SialT2 and GalNAcT indicates that information for proximal or distal Golgi concentration is associated with the cts.

Disposition of Gangliosides and Sialosylglycoproteins in Neuronal Membranes

Abstract Labeled gangliosides and glycoproteins were obtained by incubation of homogenized neuronal perikarya from rat brain with CMP‐[3H]N‐acetyl neuraminic acid. The highest degree of labelling was observed in a subcellular fraction that also showed the highest specific activities for several ganglioside glycosyltransferases. The [3H] sialosylglycoconjugates of this fraction remained associated with the membranes after treatment with 1 m‐KCl, 125 mm‐EDTA, repeated freezing and thawing, or controlled sonication, but were solubilized by sodium deoxycholate (DOC) at a concentration high enough to solubilize the choline phospholipids. About 75% of the neuraminidase‐labile sialosyl residues of these labeled endogenous gangliosides and glycoproteins were protected from the action of added neuraminidase or pronase or both enzymes added together. The protection was not abolished by pretreatment of the membranes with high ionic strength or with EDTA but was abolished by sonication or low concentration of DOC. Between 50 and 80% of the neuraminidase‐labile sialosyl residues of the gangliosides of the neuronal perikaryon membrane fraction labeled in vivo by an intracerebral injection of N‐[3H]acetylmannos‐amine were, at 3 h after the injection, also protected from the action of added neuraminidase. The protection was abolished by the addition of DOC. In contrast with the behavior of the labeled glycoconjugates of this neuronal perikaryon fraction, the gangliosides and sialosylglycoproteins from intact synaptosomes were accessible to neuraminidase. It is suggested that most gangliosides and sialosylglycoproteins are sialosylated as intrinsic components of the neuronal perikaryon membrane fraction and that at some stage of the process of transport through the axon and incorporation into the synaptic plasma membrane they change their accessibility to added enzymes.