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Keywords:

  • gangliosides;
  • glycosphingolipids;
  • glycosyltransferases;
  • Golgi;
  • Golgi retention motifs

Abstract

  1. Top of page
  2. Abstract
  3. Brief historical view
  4. The pathway of biosynthesis
  5. Domain organization of glycosyltransferases
  6. Folding requirements
  7. ER to Golgi transport
  8. Golgi complex retention
  9. Regulation of glycolipid synthesis
  10. Acknowledgements
  11. References

J. Neurochem. (2011) 117, 589–602.

Abstract

Brain tissue is characterized by its high glycosphingolipid content, particularly those containing sialic acid (gangliosides). As a result of this observation, brain tissue was a focus for studies leading to the characterization of the enzymes participating in ganglioside biosynthesis, and their participation in driving the compositional changes that occur in glycolipid expression during brain development. Later on, this focus shifted to the study of cellular aspects of the synthesis, which lead to the identification of the site of synthesis in the neuronal soma and their axonal transport toward the periphery. In this review article, we will focus in subcellular aspects of the biosynthesis of glycosphingolipid oligosaccharides, particularly the mechanisms underlying the trafficking of glycosphingolipid glycosyltransferases from the endoplasmic reticulum to the Golgi, those that promote their retention in the Golgi and those that participate in their topological organization as part of the complex membrane bound machinery for the synthesis of glycosphingolipids.


Abbreviations used
BFA

brefeldin A

CALP

calsenilin-like protein

CERT

ceramide transfer protein

CHO

Chinese-hamster ovary

COG

conserved oligomeric Golgi complex

COPI

coat protein complex I

COPII

coat protein complex II

ER

endoplasmic reticulum

FAPP2

four-phosphate adaptor protein

FRET

fluorescence resonance energy transfer

GalCer

galactosylceramide

GalNAcT

UDP-GalNAc : LacCer/GM3/GD3/GT3 N-acetylgalactosaminyltransferase

GalT

UDP-Gal:ceramide galactosyltransferase

GalT1

UDP-Gal : glucosylceramide galactosyltransferase

GalT2

UDP-Gal : GA2/GM2/GD2/GT2 galactosyltransferase

Gb3

globotriaosyl ceramide

GEM

glycolipid-enriched domains

GGT

glycolipid glycosyltransferases

GlcCer

glucosylceramide

GlcT

UDP-Glc : ceramide glucosyltransferase

Ntd

N-terminal domain

PM

plasma membrane

SialT1

CMP-NeuAc : lactosylceramide sialyltransferase

SialT2

CMP-NeuAc : GM3 sialyltransferase

SLs

sphingolipids

TGN

trans-Golgi network

TMD

transmembrane domain

Brief historical view

  1. Top of page
  2. Abstract
  3. Brief historical view
  4. The pathway of biosynthesis
  5. Domain organization of glycosyltransferases
  6. Folding requirements
  7. ER to Golgi transport
  8. Golgi complex retention
  9. Regulation of glycolipid synthesis
  10. Acknowledgements
  11. References

Looking back over the last half century, studies on the chemistry and metabolism of glycosphingolipids have frequently been in the center of the neurochemistry scene. The introduction in the 1960s of cell subfractionation and electron microscopy techniques allowed Palade and co-workers to postulate the Golgi complex as a key station in the process of vectorial intracellular transport of proteins in secretory cells (Palade 1975). The Golgi complex was established as the main site of glycoprotein glycosylation, by demonstrating the enrichment of galactosyltransferase (Fleischer et al. 1969; Morre et al. 1969) and the uptake of glucose and galactose (Neutra and Leblond 1966) into the Golgi. These concepts were soon adopted by neurochemists, and techniques were developed and applied to brain tissue for the separation and localization of biochemical functions in different subcellular fractions (Eichberg et al. 1964; Salganicoff and DeRobertis 1965). Cross-contamination of these fractions raised controversy with respect to the autonomy of nerve endings/synaptosomes for the synthesis of gangliosides [Gangliosides are named according to Svennerholm (Svennerholm 1963)]. It was initially reported that the bulk of ganglioside glycosyltransferase activity was located in synaptosomal rich fractions (Roseman 1970; Den et al. 1975). However, it was soon established that the activity for synthesis localized to the neuronal perikaryon (Maccioni et al. 1978). Upon subcellular fractionation, they localized to membranes of similar densities, but different from synaptic plasma membranes (PM); under the electron microscope, the separated membranes appeared enriched in membranous structures with Golgi element morphologies (Landa et al. 1977). Thus, neurons were not different from non-neural cell types in which many ganglioside glycosyltransferases were found concentrated in Golgi enriched subcellular fractions (Keenan et al. 1974). It was established that gangliosides and glycoproteins are transported from the neuronal soma to the nerve endings in the fast wave of axonal transport (Forman and Ledeen 1972; Landa et al. 1981), and also in a retrograde manner, from the periphery to the soma (Aquino et al. 1985).

The pathway of biosynthesis

  1. Top of page
  2. Abstract
  3. Brief historical view
  4. The pathway of biosynthesis
  5. Domain organization of glycosyltransferases
  6. Folding requirements
  7. ER to Golgi transport
  8. Golgi complex retention
  9. Regulation of glycolipid synthesis
  10. Acknowledgements
  11. References

The enzymology of ganglioside oligosaccharide synthesis in the Golgi complex constituted an important chapter in the biochemistry of glycolipids disclosed during the 1960s. The enzyme requirements for each transfer step, the specificities of the enzymes for the glycolipid acceptors and the sugar nucleotide donors, as well as the identities of the products formed were described during those years by various groups (see Fig. 1). The emergent, simplified pathway of glycolipid synthesis of the o-, a-, b-, and c- series starting from ceramide is the one depicted in Fig. 1.

image

Figure 1.  Simplified pathway of biosynthesis of o-, a-, b- and c- series gangliosides. GalT, UDP-Gal : ceramide galactosyltransferase; GlcT, UDP-Glc : ceramide glucosyltransferase; GalT1, UDP-Gal : glucosylceramide galactosyl transferase; GalNAcT, UDP-GalNAc : LacCer/GM3/GD3/GT3 N-acetylgalactosaminyltransferase; GalT2, UDP-Gal : GA2/GM2/GD2/GT2 galactosyltransferase; SialT4, CMP-NeuAc : GA1/ GM1/GD1b/GT1c sialyltransferase; SialT5, CMP-NeuAc : GM1b/GD1a/GT1b/GQ1c sialyltransferase; SialT1, CMP-NeuAc : lactosylceramide sialyltransferase; SialT2, CMP-NeuAc : GM3 sialyltransferase; SialT3, CMP-NeuAc : GD3 sialyltransferase. Competition experiments with rat liver Golgi membranes indicated that SialT5 also catalyzes the synthesis of GT3 in vitro (Iber et al. 1992; Kono et al. 1996). It is still unsettled whether SialT2 and SialT3 are the same (Nakayama et al. 1996; Watanabe et al. 1996), or different enzymes (Iber et al. 1992; Zeng et al. 1997). For simplicity, the nucleotide sugar donors that participate in each transfer step are not shown. Gangliosides are named according to Svennerholm (1963).

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The addition of the first sugar to ceramide is carried out by transferases of different localization and membrane insertion topology: the endoplasmic reticulum (ER) concentrated UDP-Gal : ceramide galactosyltransferase (GalT) that converts it into galactosylceramide (GalCer) or the UDP-Glc : ceramide glucosyltransferase (GlcT) that converts it into glucosylceramide (GlcCer). GalT has a type I topology (the C-terminus oriented to the cytoplasmic side and the N-terminus oriented toward the lumenal side), with the lumenal N-terminal domain (Ntd) bearing an ER retrieval signal and the catalytic site (Schulte and Stoffel 1993; Sprong et al. 1998). GlcT, in contrast, has a type III topology (an N-terminal uncleaved signal anchor sequence and a long cytoplasmic tail), with a cytoplasmically oriented catalytic site (Coste et al. 1986; Futerman and Pagano 1991; Jeckel et al. 1992). In cells active for the synthesis of GalCer (some epithelial cells, Schwann cells), a fraction of the UDPGal transporter, mostly present in the Golgi complex, associates to the ER-located ceramide galactosyltransferase and provides the necessary lumenal galactose (Sprong et al. 2003). GalCer is substrate for a Golgi-localized sulfotransferase that transfer the sulfate moiety from the donor 3′ phosphoadenyl-5′ phosphosulfate (PAPS) and converts it into sulfatide.

In liver cells, GlcT was reported to be widely distributed between Golgi and ER membranes (Futerman and Pagano 1991) but located in proximal and distal Golgi fractions by others (Jeckel et al. 1992). Immunocytochemical visualization of a tagged form of Drosophila melanogaster GlcT transfected to B19 melanoma cells lacking GlcT revealed its presence both in ER and Golgi membranes (Kohyama-Koganeya et al. 2004).

From the ER ceramide is transported to the proximal Golgi by the cytosolic ceramide transfer protein (CERT), where it is converted to GlcCer by GlcT. It has been suggested that CERT extracts ceramide from the ER through its FFAT and START domains and transfers it to the Golgi apparatus in a non-vesicular manner. This would occur at close apposition sites between the transitional ER and the Golgi apparatus membranes with participation of its pleckstrin homology domain (Hanada et al. 2009). CERT mutant mice embryos accumulate ceramide, but the most prominent phenotype is not the expected apoptosis; rather, severe organogenesis defects and mitochondrial and ER compromise by accumulated ceramide was suggested as the main cause of early death of mutant embryos (Wang et al. 2009). From its cytoplasmic site of synthesis by GlcT, GlcCer is transported to the late Golgi by four-phosphate adaptor protein (FAPP2). FAPP2 mediates the delivery of GlcCer from proximal to distal Golgi where it translocates to the lumen. GlcCer is converted into LacCer and higher glycolipid derivatives by Golgi resident glycosyltransferases in the lumen of the distal Golgi (D’Angelo et al. 2007). GlcCer is also transported by a non-vesicular, brefeldin A (BFA) insensitive, mechanism directly to the PM where it turns over rapidly but is poorly used for synthesis of more complex glycolipids (Warnock et al. 1994). It has been proposed that FAPP2 transports GlcCer from the cis Golgi to the ER where it translocates to the lumen; in that way it enters the secretory pathway and reaches the Golgi lumen for LacCer synthesis and further elongation to higher order derivatives (Halter et al. 2007). It will be interesting to know if a FAPP2 knock out mice accumulates GlcCer and show defective synthesis of GlcCer derived higher order glycosphingolipids.

Domain organization of glycosyltransferases

  1. Top of page
  2. Abstract
  3. Brief historical view
  4. The pathway of biosynthesis
  5. Domain organization of glycosyltransferases
  6. Folding requirements
  7. ER to Golgi transport
  8. Golgi complex retention
  9. Regulation of glycolipid synthesis
  10. Acknowledgements
  11. References

Most Golgi resident glycosyltransferases are synthesized and co-translationally inserted in the ER membranes with type II topology (the N-terminus oriented to the cytoplasm and the C-terminus oriented toward the lumen). Translocation is stopped by a signal anchor sequence, the transmembrane domain (TMD), located in the N-terminal region to which a voluminous C-terminus bearing the catalytic domain is appended (Paulson and Colley 1989) (Fig. 2). They are transported from the ER as integral membrane proteins toward the Golgi complex where they concentrate and cycle through the ER (Lippincott-Schwartz et al. 1989). An imbalance between the rates of anterograde and retrograde transport, favoring anterograde transport, determines their concentration as residents of the Golgi complex; the Golgi to ER ratio was calculated to be 90 : 10 at steady state (Rhee et al. 2005). The molecular basis of glycolipid glycosyltransferases (GGTs) folding, and particularly transport and retention in the Golgi, are only partially resolved issues at present.

image

Figure 2.  General topology and domain organization of glycosyltransferases. The enzymes are type II-transmembrane proteins, with an N-terminal domain (Ntd), constituted by a short cytoplasmic tail, a single transmembrane domain (TMD) and a lumenal stem region, and followed by a globular catalytic C-terminal domain (Paulson and Colley 1989).

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Folding requirements

  1. Top of page
  2. Abstract
  3. Brief historical view
  4. The pathway of biosynthesis
  5. Domain organization of glycosyltransferases
  6. Folding requirements
  7. ER to Golgi transport
  8. Golgi complex retention
  9. Regulation of glycolipid synthesis
  10. Acknowledgements
  11. References

When examined, N-glycosylation motifs (Asp-X-Ser/Thr) of GGTs have been found occupied with N-linked oligosaccharides. In GalNAcT (Haraguchi et al. 1995), GalT2 (Martina et al. 2000), and SialT2 (Martina et al. 1998; Daniotti et al. 2000), N-glycosylation was found critical for their activity and subcellular localization, probably reflecting known oligosaccharide-mediated quality controls of folding in the ER (D’Alessio et al. 2010). Inhibition of the only N-glycosylation site (Asn 143) of GalT2 either by tunicamycin or by site directed mutagenesis results in the loss of enzyme activity and retention in the ER. Castanospermine (an inhibitor of ER glucosidase I and II) partially impairs the ER exit of unglycosylated GalT2, which has reduced Km and Vmax values (Martina et al. 2000). All three N-glycosylation motifs of SialT2 were found glycosylated and necessary to attain and maintain a catalytically functional folding, and for exiting the ER. N-glycan trimming in the ER, while not required for enzyme activity, is necessary for proper trafficking of SialT2 to the Golgi (Martina et al. 1998). In F-11 cells, ER retention and a four-fold increase in the turnover of SialT2, but not of GalNAcT, was observed upon castanospermine inhibition of N-glycan processing, which lead to link processing and transport of SialT2 with its function in ganglioside biosynthesis (Bieberich et al. 2000; Yu et al. 2004).

ER to Golgi transport

  1. Top of page
  2. Abstract
  3. Brief historical view
  4. The pathway of biosynthesis
  5. Domain organization of glycosyltransferases
  6. Folding requirements
  7. ER to Golgi transport
  8. Golgi complex retention
  9. Regulation of glycolipid synthesis
  10. Acknowledgements
  11. References

Proteins synthesized in the ER are transported along the secretory pathway as cargo in coat protein complex II (COPII)-coated vesicles formed in ER exit sites. Selective concentration of cargo occurs by direct or indirect interactions between COPII coat proteins (Sec23 and Sec24) and amino acid signals present in the cytoplasmic tails of cargo (Bonifacino and Glick 2004). For the particular case of GGTs, it is well established that the Ntd of glycosyltransferases with type II topology is able to promote the exit from the ER and concentration in the Golgi complex of reporter proteins substituting the catalytic domain at the C-terminus (Cole et al. 1998; Martina et al. 2000; Giraudo et al. 2001).

A dibasic motif ([RK]-x-[RK]) has been described in our laboratory that facilitates the export of type II integral proteins with short cytoplasmic tails to the Golgi complex through direct interaction with Sar1 and subsequent recruitment of COPII coat components (Giraudo and Maccioni 2003a). This has been shown both in mammalian cells (Giraudo and Maccioni 2003a; Guo and Linstedt 2006; Kizuka et al. 2006; Quintero et al. 2010) and plant cells (Yuasa et al. 2005; Strasser et al. 2006). Additionally, a single basic amino acid was reported as sufficient to achieve efficient export of glycosyltransferases in plants (Schoberer et al. 2009), leading to the suggestion that basic amino acids in cytoplasmic tails could simply ensure correct insertion into the membrane and/or determine the edge of the TMD (Nilsson et al. 2009). However, the ER-exit impairing mutation of the TMD-proximal KSRGR motif for QSTGT (Yuasa et al. 2005), makes alteration of TMD borders unlikely, because of the hydrophilic nature of the residues used. Additionally, in vitro binding experiments were conclusive in demonstrating a specific and direct interaction of Sar1 with basic residues present in peptides containing the sequence of cytoplasmic tails of GGTs (Giraudo and Maccioni 2003a; Guo and Linstedt 2006).

Also, computational docking identified a putative binding pocket in Sar1 involved in the interaction with the [R/K](X)[R/K] motif. Sar1 mutants with alanine replacement of amino acids predicted to be involved in the interaction were tested in vitro and in cells. In vitro, mutant versions showed reduced ability to bind immobilized peptides with the cytoplasmic tail sequence of GalT2. In cells, Sar1 mutants affect the exiting of GGTs from the ER, resulting in an ER/Golgi concentration ratio favoring the ER. The effect was specific as neither the typical Golgi localization of GM130 nor the exiting and transport of the G protein of the Vesicular Stomatitis Virus were affected (Quintero et al. 2010).

Other types of proteins have been described to interact directly with different isoforms of Sec24 through a di-acidic signal (DxE), a LxxLE motif, a YNNSNPF motif, and the [IL]xM motif (Miller et al. 2003; Mossessova et al. 2003; Mancias and Goldberg 2008). The DxE motif has been demonstrated to be functional in type I, II, and III transmembrane proteins (Hanton et al. 2005). For the remaining signals, it is unclear if any of them is unique to a certain transmembrane topology.

By studying the universality of these motifs in transmembrane proteins which localize to the three major subcellular localizations (ER, Golgi, and PM), we found the most abundant motif which could explain ER-export is the dibasic motif ([RK]x[RK]). When localized within 20 amino acids of the TMD, this motif is present in 19.2% of ER proteins, but in 51.0% of Golgi proteins and 43.3% of PM proteins that necessarily exit the ER upon entering the secretory pathway (R. Quiroga and H. J. F. Maccioni, in preparation). Similar results were obtained for type I proteins with short cytoplasmic tails, although a small number of proteins were analyzed. Keeping in mind that the presence of this motif in ER-localized proteins could be owing to ER–Golgi cycling, this evidence supports the claim that a dibasic motif, when located close to the TMD, is involved in specific ER-exit mechanisms, and not simply determining TMD borders.

Golgi complex retention

  1. Top of page
  2. Abstract
  3. Brief historical view
  4. The pathway of biosynthesis
  5. Domain organization of glycosyltransferases
  6. Folding requirements
  7. ER to Golgi transport
  8. Golgi complex retention
  9. Regulation of glycolipid synthesis
  10. Acknowledgements
  11. References

Cytoplasmic tail interactions with other proteins

Why GGTs are retained in the Golgi and do not continue their journey along the secretory pathway is still a matter of study. The only motif described so far that mediates the retention of Golgi resident proteins is the FLxK motif (more precisely [FL]-[LIV]-x(0-2)-[RK]; Tu et al. 2008), which is conserved in the cytoplasmic tails of 16 yeast glycosyltransferases; mutation of its residues results in loss of Golgi localization. The Vps74 protein, which is reported to interact with this motif, is conserved from yeast to metazoans, and its deletion from the genome of Saccharomyces cerevisiae results in mislocalization of glycosyltransferases (Tu et al. 2008).

In silico analysis of the prevalence of the FLxK motif in our collection of mammalian type II proteins shows that it is present in the cytoplasmic tails of approximately 26% of either ER, Golgi, or PM proteins, suggesting that other requirements, like the context and/or distance from the TMD, may be of relevance to the efficiency of the motif in promoting Golgi retention. A careful analysis of the 16 Golgi resident enzymes analyzed in Tu et al. (2008), showed that the FLxK motif most frequently occurred in the vicinity of di-basic motifs, and in proximity to TMDs. Because of this, we analyzed the presence of a di-hydrophobic motif [FLIVM] [FLIVM] in the vicinity of the di-basic motif [RK] x [RK] (Giraudo and Maccioni 2003a), and this in turn close to the cytoplasmic border of the TMD (maximum of 10 amino acids away from the edge of the TMD). For simplicity, we will call this motif LLxRR.

The analysis of this new LLxRR motif (see Table 1) suggests that its presence in the vicinity of a TMD border could mediate retention in the Golgi in mammalian cells, or perhaps regulate intra-Golgi localization through interaction with coatomer protein I (COPI) coat proteins, since it is enriched approximately fourfold in the cytoplasmic tail of Golgi-localized proteins when compared with PM- and ER-localized proteins. Nevertheless, this motif is present in a reduced number (12%) of the total Golgi-localized proteins, because of which we propose the main mechanism driving Golgi retention is TMD-mediated retention (see next).

Table 1.   Abundance of proteins which localize to the ER, the Golgi complex, and the plasma membrane compartments, and bear FLxK or LLxRR motifs in their cytoplasmic tails
CompartmentTotal sequences analyzedFLxK motifLLxRR motif
n%n%
  1. A dataset was constructed as follows: 254 Homo sapiens bitopic proteins with annotated subcellular location and type II transmembrane topology were gathered from the Swissprot database. These sequences were used to search the ORTHOMCL database. 2447 mammalian orthologues were gathered (448 proteins predicted to localize to the ER, 1433 to the Golgi apparatus and 566 to the plasma membrane). Motif searches were performed using PERL scripts written in the lab. Data in table includes those proteins that in addition to localizing to the ER, the Golgi complex, or the plasma membrane compartments, bear consensus motifs FLxK or LLxRR in their cytoplasmic tails. Total stands for the number of sequences analyzed for each compartment. Shown in table are the number (n) and percentage (%) of these sequences which contain a FLxK motif ([FL][LIV]x[RK]) (Tu et al. 2008) or a LLxRR motif ([VLIFM]-[VLIFM]-x(0,2)-[RK]-x(0,1)-[RK] at no more than 10 amino acids from the TMD border).

Endoplasmic reticulum44813430164
Golgi complex14333712622115
Plasma membrane56613223173

A yeast two-hybrid screening using elements of the cytoplasmic tail of GalT2 as bait identified calsenilin and its close homolog calsenilin-like protein (CALP), both members of the recoverin-neuronal calcium sensor family of calcium-binding proteins, as a binding partner of the GalT2 tail. In vitro, GalT2 binds to immobilized recombinant CALP, and CALP binds to immobilized peptides with the GalT2 cytoplasmic tail sequence. GalT2 and calsenilin interact physically when co-expressed in Chinese-hamster ovary (CHO)-K1 cells. The expression of CALP or calsenilin affects Golgi localization of GalT2, and of two other glycosyltransferases, SialT2 and GalNAcT, by redistributing them from the Golgi to the ER, whereas the localization of the G protein of the vesicular stomatitis virus or the Golgin GM130 was essentially unaffected. Conversely, the expression of GalT2 affects the localization of calsenilin and CALP by shifting a fraction of the molecules from being mostly diffuse in the cytosol, to clustered structures in the perinuclear region. These combined in vivo and in vitro results suggest that CALP and calsenilin are involved in the trafficking of Golgi glycosyltransferases, perhaps dynamically regulating the amount of GGTs that reach the Golgi (Quintero et al. 2008).

Retention by events involving the transmembrane domain

Numerous studies have demonstrated the importance of the TMD in determining the localization of Golgi resident proteins (Tang et al. 1992; Teasdale et al. 1992; Opat et al. 2001). In other studies, this capacity for Golgi retention (or ER retention) has been attributed to specific amino acids present in the TMD (Aoki et al. 1992; Sousa et al. 2003). However, more recently, it has been proposed that the main force that directs anterograde and retrograde transport of proteins between the ER, the Golgi, and the PM is the trafficking of lipids, more specifically, glycerophospholipids and sphingolipids (SLs) (Patterson et al. 2008; Jackson 2009). Enrichment of cargo proteins in SL-enriched domains and resident proteins in glycerophospholipid-enriched domains has been proposed. This is supported by the observation of SL delivery to the PM in Golgi-derived vesicles (Van Meer et al. 2008), an enrichment of sterols and SLs in these vesicles (Klemm et al. 2009), and an exclusion of sterols and SLs from COPI-coated vesicles (Schneiter et al. 1999). Differences in affinity of TMDs for lipid domains could condition lateral diffusion on the membrane, resulting in posterior sorting and trafficking (Lundbak et al. 2003; Ronchi et al. 2008).

Transmembrane length has been proposed as the major force driving protein concentration in ER and Golgi export and retention domains (Munro 1995; Ronchi et al. 2008; Dukhovny et al. 2009; Sharpe et al. 2010). Using a similar algorithm to the one used in Sharpe et al. (2010), to study TMD length of proteins, we determined that TMD length of type II proteins does not correlate linearly with distal endomembrane localization in our dataset. Rather, we found that Golgi-localized proteins have significantly shorter TMDs than ER-localized proteins, and both have significantly shorter TMDs than PM-localized proteins (R. Quiroga and H. J. F. Maccioni, in preparation). This suggests that TMD length mediates enrichment or exclusion of proteins containing said domains in highly curved, sphingomyelin enriched Golgi export domains. A consequence of these observations is that TMD-length-based mechanisms would not be important in determining ER-export, since either Golgi-localized proteins (with shorter TMDs than ER-localized proteins) or PM-localized proteins (with longer TMDs than ER-localized proteins) exit the ER. Conversely, these observations lend support to the notion that ER-export is mainly mediated by motif-mediated interactions with COPII components.

The sub-Golgi localization of ganglioside glycosyltransferases

Glycosyltransferases that transfer sugars to glycolipids and those acting on the terminal glycosylation of glycoproteins, are concentrated in the Golgi complex (Kornfeld and Kornfeld 1985). However, there are clear differences between glycosylation of these glycoconjugates. Synthesis and processing of N-linked glycoprotein oligosaccharides begins in the ER; processing continues in the Golgi complex by Golgi resident glycosidases and glycosyltransferases that concentrate along the cis to trans axis following the order in which they alternate in the processing pathway. It is now accepted that there is considerable overlapping of these enzymes along sub-Golgi compartments (Rabouille et al. 1995). In contrast, ceramide-linked oligosaccharides are synthesized by glycosyltransferases that act in succession on, or compete at branching points for common specific acceptors. So, they may not necessarily require the ordered spatial disposition along the Golgi cisternae of glycoprotein processing enzymes. Rather, their order of action depends heavily on their specificities for the nature and the anomeric configuration of the linkage of the terminal sugar of the glycolipid acceptor (Fig. 1).

Different approaches locate the site of ganglioside glycosylation to distal elements of the Golgi complex. However, a proximo-distal gradient of concentration of transferases is still detectable. Working with endogenous acceptors, Maxzúd et al. (1995) concluded that the common compartment supporting coupled conversion of LacCer to GD1a is distally located in the trans-Golgi network (TGN), whereas GalT1, SialT1 and SialT2 are present in proximal Golgi compartments but also extend their presence to the TGN. Conversely, the TGN concentrated GalNAcT (Giraudo et al. 1999) was virtually absent in proximal Golgi compartments. Later on, determination of enzyme activities in subfractionated Golgi complex membranes (Lannert et al. 1998; Allende et al. 2000) confirmed that the enzymes concentrate in late Golgi membranes. This picture is compatible with the results of metabolic labeling in the presence of pharmacological agents, such as BFA and monensin. BFA dissects the Golgi complex in vivo causing mixing of proximal Golgi elements with ER membranes and leaving the TGN fused with late endosomes; monensin is a sodium ionophore that affects distal Golgi function because of swelling of the cisternae. Both agents block the synthesis of complex gangliosides GM2, GM1, and GD1a and cause accumulation of the simple intermediates LacCer, GM3, and GD3 in the different cell systems assayed (reviewed in Maccioni et al. 1999). It should be taken into consideration that tubular and/or vesicular connections among cisternae (Marsh et al. 2004; Trucco et al. 2004) may allow the intermediate acceptors to diffuse rapidly across the stack, allowing coupling of different transfer steps even when the participating enzymes concentrate in different cisternae (Patterson et al. 2008; Pfeffer 2010).

Studies with endogenous acceptors also demonstrated that at branching points, the flow of common intermediates to one or another direction is determined by the ratio of the activities of the competing enzymes. This in turn is determined by the relative efficiency of transcription and translation of the respective genes, to variations in the lumenal environment affecting, that is, the fractional saturation with the respective sugar nucleotide donors, with the activating divalent cation, or even by modification of the intralumenal pH (Maxzúd et al. 1995; reviewed in Maccioni 2007). These conclusions were substantive in understanding the compositional changes of cell surface glycolipids that occurs during, for example, CNS development, or in directing the glycolipid composition of cells by over-expression of key glycosyltransferases (see below Crespo et al. 2010).

The influence of the relative saturation with the sugar nucleotide donor was a concurrent epigenetic factor contributing to the control of glycolipid expression in the developing rat retina. Most CNS structures of birds and mammals shift the expression of gangliosides from GD3 at early stages to GD1a at late developmental stages, which correlates with the up and down transcriptional regulation of, respectively, GalNAcT and SialT2 during development (Panzetta et al. 1980; Yu et al. 1988). However, the adult rat retina is characterized by expressing a low proportion of ganglio-series gangliosides relative to the expression of GD3 (Daniotti et al. 1991). This particular pattern of expression does not only correlate with the maintenance in the adult stage of an activity of SialT2 higher than that of GalNAcT and GalT2, but also with the presence of a developmentally up-regulated, Golgi-concentrated UDP-sugar pyrophosphatase activity (Martina et al. 1995; Martina and Maccioni 1996). This activity limits the availability of UDP-GalNAc and UDP-Gal, but not of CMP-NeuAc to the Golgi lumen, which contributes to direct the flow of LacCer toward formation of GM3 and GD3 instead of to a-series gangliosides (Bieberich and Yu 1999; Yu et al. 2004).

Relevance of cytoplasmic tails in conferring sub-Golgi localization

Studies in CHO-K1 cells that co-express fusions of spectral variants of green fluorescent protein (GFP) and the Ntds of SialT2 and GalNAcT showed SialT2 spread along the proximal and distal Golgi (Daniotti et al. 2000) and GalNAcT as a typical distal Golgi resident enzyme (Giraudo et al. 1999). In the presence of BFA, SialT2 showed preferential redistribution into the ER, in comparison with GalNAcT that mostly remains concentrated in the post-BFA compartment (Uliana et al. 2006a). These results indicate that the Ntds of these enzymes carry information for such localization. Swapping the cytoplasmic tails of the two enzymes results in localization trends similar to the donors of these tails (Schaub et al. 2006; Uliana et al. 2006b). It is unlikely that a conserved binding motif participates in the differential sub-Golgi concentration, because of the lack of homology in cytoplasmic tail sequences (apart from the conserved ER exiting [RK(X)RK] motif described before); among several possibilities, SialT2 may be actively retained in proximal Golgi elements, that is, by interaction of its cytoplasmic tail with other cytoplasmically exposed Golgi protein partners. GalNAcT, with a shorter tail would bind with less affinity to these partners or interact with different, more distally located ones. GalT1 and SialT1, which in CHO-K1 cells associate with SialT2 (Giraudo and Maccioni 2003b) could passively follow the behavior and dynamics of SialT2.

For the case of SialT1, three mouse isoforms with distinct intracellular dynamics, having cytoplasmic tail lengths of 69, 42, and 14 amino acids, are translated by leaky scanning of a single mRNA. It has been suggested that this may modulate the synthesis of GM3 under pathological or physiological conditions (Uemura et al. 2009). It is expected that future work will clarify how information encoded in Ntds determines sub-Golgi localization.

Functional associations of glycosyltransferases

Many years ago the existence of a multiglycosyltransferase system/organization acting in the synthesis of glycolipids and glycoproteins was postulated (Roseman 1970; Caputto et al. 1974). It was in the last 10 years that experimental evidence for the existence of glycosyltransferase associations began to accumulate. Giraudo et al. (2001) demonstrated the association of two glycolipid glycosyltransferases, GalNAcT and GalT2. Epitope tagged GalNAcT and GalT2 heterologously expressed in CHO-K1 cells mutually co-immunoprecipitate, and immunocomplexes are able to couple the two transfer steps leading to the synthesis of GM1 from exogenous GM3 (Fig. 3). The Ntd of the transferases participate in the physical interaction, as shown by competition experiments and by determination of molecular proximity in living cells by fluorescence resonance energy transfer (FRET). FRET was detected in the Golgi area for the Ntds of GalNAcT and GalT2 fused to cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) (Fig. 4). Enzyme complex formation improves the efficiency of glycolipid synthesis by channeling the intermediates from the position of product to the position of acceptor along the transfer steps (Giraudo et al. 2001). Kin oligomerization may participate in conferring Golgi residence to these proteins, as was suggested for the glycoprotein processing enzymes GlcNAcT1 and Mannosidase II (Nilsson et al. 1996; Füllekrug and Nilsson 1998).

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Figure 3.  Schematic representation of the GalNAcT–GalT2 enzyme complex that functions in the coupling of the sugar transfer steps leading to conversion of GM3 into GM1. GalNAcT and GalT2 are depicted as non-covalently associated and interacting through their Ntds. GM3 is converted to GM2 by GalNAcT, which without leaving the complex would be taken by GalT2 that converts it to GM1. The mechanism of GM1 dissociation from GalT2 is still poorly understood.

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Figure 4.  Glycolipid glycosyltransferase hetero- and homocomplexes evidenced by FRET microscopy in living cells. CHO-K1 cells were transfected with the Ntds of GalNAcT fused to CFP and GalT2 fused to YFP (a) or GalT2 fused to CFP and GalT2 fused to YFP (b). The last column corresponds to corrected FRET images, pseudo colored according to energy transfer levels. FRET signal is evidenced in Golgi regions were the co-expressing enzymes co-localize. Although SialT2CFP and GalT2YFP also co-localize in the Golgi complex, no FRET between their Ntds was observed (c).

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GalT1, SialT1, and SialT2 were also found physically associated in a SialT1-dependent manner, with participation of the Ntds. Co-immunoprecipitation and FRET experiments showed that these transferases interact poorly, if at all, with GalNAcT and GalT2, suggesting that ganglioside synthesis is organized in distinct units formed by the association of particular GGTs (Giraudo and Maccioni 2003b). In F-11A cells, a sub-strain of neuroblastoma F-11 cells, SialT2 was found physically interacting with GalNAcT and with pyrene-labeled GM3, but not with SialT1 (Bieberich et al. 2002). This result may seem at first glance at variance with those observed in CHO-K1 cells. However, since most higher eukaryotic cells express at least GM3 at the cell surface, and higher order gangliosides appear, that is, during differentiation, it is possible that a GalT1 and SialT1 basic complex is present in the majority of cells, and that either other complexes are formed (as in CHO-K1 cells), or that the basic complex adjusts its complexity according to the differentiation-dependent up- or down-regulation of the participating transferases, as may be the case of F-11A cells.

We have recently observed, by FRET microscopy experiments in living cells, that GalT2 Ntds are able to form homocomplexes, as they do in heterocomplexes (Fig. 4) (Ferrari 2010). Also, the existence of N-glycosylation enzyme homocomplexes of exclusive Golgi localization was documented (Young 2004) and recently evidenced by bimolecular fluorescence complementation (Hassinen et al. 2010). Complex formation might be also considered as an enzyme activity regulation mechanism, by eliminating the enzyme excess in a specific glycosylation process in absence of the other functional component. However, the stoichiometric relationship between enzymes in a complex could regulate ganglioside biosynthesis. Up to now, the stoichiometry in enzyme complexes and their activities have not been connected. Preliminary experiments in our laboratory performed by FRET microscopy in live cells indicate that the stoichiometric relationship in the enzymatic complex between the enzymes involved in complex ganglioside synthesis, GalNAcT and GalT2, is 1 : 2. If this ratio affects the activities of the enzymes, it would favor the displacement of intermediates to a galactosylated final product (i.e. GM1 or GD1a). The variation in this ratio along time and/or space could be important in ganglioside biosynthetic regulation (Ferrari 2010).

The possibility that GGT associations involve segregation in specialized membrane domains of the Golgi complex, similar to the glycolipid-enriched domains (GEM) at the cell surface (Simons and van Meer 1988; Simons and Ikonen 1997; Sonnino and Prinetti 2010) was investigated in CHO-K1 cells (Crespo et al. 2004). While PM GM3 and most GD3 and GT3 behave as GEM constituents, their newly synthesized counterparts still remaining in the Golgi do not. This suggests that glycolipid products enter GEM after their synthesis in the Golgi cisternae, along the secretory pathway and/or at the cell surface. Interestingly, most of the GGT complexes in Golgi membranes do not behave as GEM constituents either. In agreement with this observation, it was recently proposed that GGTs are enriched in glycerophospholipid enriched domains (Patterson et al. 2008). Triton X-100 insoluble complexes of GlcNAcTI and GlcNAcTII interacting through their lumenal domains have also been described in CHO cells (Opat et al. 2000), but these complexes do not fit in classic ‘raft’ characteristics since they were not affected by cholesterol depletion.

The transmembrane domains are relevant to the associations

As mentioned earlier, when both tail-deleted or tail-mutated Ntd of GalT2 and GalNAcT were co-expressed, they both stayed in the ER as expected, but in an association close enough to undergo FRET (Giraudo and Maccioni 2003b). This indicates that complex formation between these two enzymes occurs in the ER, and importantly, that complex formation depends more on the TMD than on the cytoplasmic tail. In line with this observation, co-transfection of tail-deleted GalT2 with normal GalNAcT leads to the movement of an important fraction of tail-deleted GalT2 toward the Golgi complex, where it undergoes FRET with GalNAcT. Fluorescence Recovery After Photobleaching experiments indicate that the rate of transport is essentially the same for normal GalT2/GalNAcT pair than for pairs in which one member bears a deleted tail (Giraudo and Maccioni 2003b). This indicates that for moving a complex from the ER to the Golgi, it is sufficient that one member of the complex interacts with the export system to move the whole complex to the Golgi. So, complex formation complicates the interpretation of results of experiments in which the localization of a given GGT is evaluated after parts of it have been modified or deleted, since the association to endogenous partners may passively influence the localization of the mutated version. Disulfide-bonded homodimers of some GGTs also occur in the ER; their relationships with enzyme activity and complex formation may vary for each protein and is an unsettled issue at this moment (for review, see Young, 2004).

Non-glycolipid glycosyltransferase complexes

Complexes between glycosyltransferases acting on other glycosylation pathways have been described, which seem to assemble in the ER and exert their function in the Golgi apparatus. Examples are the uronosyl 5-epimerase/2-O-sulfotransferase complex (Pinhal et al. 2001) and the EXT1/EXT2 complex (McCormick et al. 2000), which are involved in the synthesis and co-polymerization of glucuronic acid and N-acetylgalactosamine during heparan sulfate biosynthesis, respectively. In yeast, two different types of Golgi mannosyltransferase complexes have been described (Jungmann and Munro 1998; Jungmann et al. 1999), which actively recycle through the ER (Todorow et al. 2000), a behavior shared by ganglioside glycosyltransferase complexes (Giraudo and Maccioni 2003b). It has been proposed that these mannosyltransferase complexes regulate the expressed glycan diversity by altering their composition in mannan backbone (Stolz and Munro 2002). Conversely to the case of the EXT1/EXT2 complex, that upon formation shifts from the ER to the Golgi (McCormick et al. 2000), the association of the ER-located UDP-galactose : ceramide galactosyltransferase with the Golgi UDP-galactose transporter promotes a shift of the transporter toward the ER (Sprong et al. 2003). However, this is not the case for the Golgi-located CMP-NeuAc transporter and the sialyl transferases, since redistribution of ST6Gal I STtyr isoform or ST8Sia IV polysialyltransferase to the ER is not accompanied by the redistribution of the Golgi CMP-NeuAc transporter (Zhao et al. 2006). Two glucuronyltransferases (GlcAT-P and GlcAT-S) and a sulfotransferase (HNK-1ST) involved in HNK1 biosynthesis were found physically and functionally associated in the Golgi through their C-terminal catalytic domains (Kizuka et al. 2006).

Regulation of glycolipid synthesis

  1. Top of page
  2. Abstract
  3. Brief historical view
  4. The pathway of biosynthesis
  5. Domain organization of glycosyltransferases
  6. Folding requirements
  7. ER to Golgi transport
  8. Golgi complex retention
  9. Regulation of glycolipid synthesis
  10. Acknowledgements
  11. References

As mentioned earlier, genetic and epigenetic factors affecting the activity balance of enzymes acting at branching points in the pathway of synthesis from LacCer on, are crucial for determination of the pattern of glycolipid expression at the cell surface. Transcriptional regulation of genes coding for key glycolipid glycosyltrasferases is determinant for this control (Zeng and Yu 2008).

Additionally, the amount of glycolipids synthesized by cells must also be under regulatory control to encompass fluctuations in membrane biogenesis activity accompanying cell growth and/or differentiation. In this respect, the issue of synthesis and transport of GlcCer mentioned before merits particular comments. GlcCer synthesis is the first step in the pathway of ganglioseries glycolipid biosynthesis, so provision of ceramide by CERT for the synthesis of GlcCer, regulation of the activity of GlcT, and/or the subsequent transport of GlcCer to translocation sites constitute potential targets for regulating the amount of ganglioseries glycolipids that a cell synthesizes (De Matteis et al. 2007). In this respect, it is interesting to note that c-fos, a transcription factor that is being revealed as an activator of phospholipid synthesis in growing cells (Gil et al. 2004), also activates GlcCer synthesis (Crespo et al. 2008). Modulation of this key step of glycolipid synthesis by non-vesicular events may enhance the possibility of regulation independently of vesicular transport.

The Golgi complex dynamics, the compartmental organization that supports its multiple functions, particularly glycosylation, and the topological relationships between elements of the machinery for glycosylation add complexity to the understanding of glycolipid synthesis regulation. Glycosyltransferases and other Golgi residents cycle between distal and proximal compartments, and also between the Golgi and the ER, with participation of COPI vesicles that use the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein machinery for fusion. A previous tethering process has been described in which monomeric or oligomeric tethering proteins approximate and give specificity to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated fusion process (Whyte and Munro 2002). It was recently reported that the synthesis of globotriaosyl ceramide (Gb3), the receptor of shiga toxin, was defective in cells over-expressing a truncated form of glutamate receptor, ionotropic, N-methyl-d-aspartate associated protein 1; glutamate receptor, ionotropic, N-methyl-d-aspartate associated protein 1 was found associated with the α-galactosyltransferase acting on LacCer (Gb3 synthase) and the cells show accumulation of LacCer. Compositional changes were attributed to mislocalization and enhanced degradation of the TGN concentrated Gb3 synthase (Yamaji et al. 2010).

Cells lacking the expression of COG2, a member of the conserved oligomeric Golgi (COG) tethering complex (ldlC CHO cells) have pleiotropic defects in processes associated to trans and medial Golgi leading to abnormal synthesis of N-, O- and ceramide-linked oligosaccharides (Kingsley et al. 1986; Krieger et al. 1989). In humans, mutations in members of the COG tethering complex lead to congenital disorders of glycosylation of Type II. The ldlC cells characterize by a prolonged block of vesicle tethering that causes an extensive Golgi ribbon fragmentation. Spessott et al. (2010b) found that the reduced GM3 level in these cells, and the accompanying LacCer accumulation, was not attributable to decreased activity of the GT that synthesizes it from LacCer (SialT1). Rather, a mislocalization of SialT1, which is reverted upon COG2 transfection, appeared as responsible for the defect. In the same cell line, Spessott et al. (2010a) found that sphingomyelin synthesis was also defective, and ceramide accumulates. Here, again the defect appears associated to a mislocalization of sphingomyelin synthase 1, a normally TGN-localized enzyme that transfer the phosphorylcholine moiety of phosphatidycholine to acceptor ceramide. In ldlC cells, sphingomyelin synthase 1 was found in small vesicular structures dispersed in the cytoplasm suggesting uncoupling between sub-Golgi compartments bearing enzymes and substrates (Spessott et al. 2010a,b).

It is interesting to note that co-expression of interacting GGTs promoted their mutual activation, as was the case of SialT2 and GalNAcT in F-11A cells (Bieberich et al. 2002), and of SialT2 and GalT1 and SialT1 in CHO-K1 cells (Uliana et al. 2006a). Also, it was reported that the recombinant soluble C-terminal catalytic domains of the β1,3GlcNAcT8 and β1,3GlcNAcT2 participating in the elongation of multiatennary N-glycans are able to associate and that the association enhances their enzymatic activities (Seko and Yamashita 2005). It is not known whether these activation events are of genomic or epigenomic nature.

Cell surface associated glycosphingolipid degrading and synthesizing activities have been well documented in non-neural cells. Cell surface degradation of GM3 by over-expression of a membrane-associated neuraminidase is a source of ceramide that modulates cell proliferation and apoptosis, and also promotes an increase of GM3 synthase mRNA and GM3 synthase activity in human fibroblasts (Valaperta et al. 2006). Recently, the presence of SialT2 activity in the PM of CHO-K1 and SK-Mel-28 human cells, capable of using extracellular CMP-NeuAc to convert PM GM3 or exogenous GM3 into GD3 has been shown (Crespo et al. 2010). Further investigations will unravel the effects of cell surface activities of glycosphingolipid remodeling on the modulation of the activities of intracellular synthesizing enzymes, and on the phenotypic changes affecting cell-cell interaction or cell signaling events. Also, the organization of the assembly line in multienzyme complexes containing different sets of functionally coupled glycosyltransferases may constitute a supramolecular organization capable of finely tuning glycoconjugate expression in response to regulatory cues. These control levels may superimpose to those exerted through the transcriptional control of individual glycosyltransferases.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Brief historical view
  4. The pathway of biosynthesis
  5. Domain organization of glycosyltransferases
  6. Folding requirements
  7. ER to Golgi transport
  8. Golgi complex retention
  9. Regulation of glycolipid synthesis
  10. Acknowledgements
  11. References

This work was supported in part by Grants from the Secretaría de Ciencia y Tecnología, Universidad Nacional de Córdoba, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina, Agencia Nacional de Promoción Científica y Tecnológica, Argentina. Authors apologize for the omission of relevant references, which was because of the limitation of the available space. The useful suggestions of three anonymous referees are also acknowledged.

References

  1. Top of page
  2. Abstract
  3. Brief historical view
  4. The pathway of biosynthesis
  5. Domain organization of glycosyltransferases
  6. Folding requirements
  7. ER to Golgi transport
  8. Golgi complex retention
  9. Regulation of glycolipid synthesis
  10. Acknowledgements
  11. References