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Structural and functional features of glycosyltransferases
Biochimie 83 (2001) 713−718 © 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. S0300908401012986/REV
Structural and functional features of glycosyltransferases C. Bretona*, J. Muchab, C. Jeanneaua a
Centre de Recherches sur les Macromolécules Végétales (affıliated to the University Joseph-Fourier), CNRS, BP 53, 38041 Grenoble cedex 9, France b Zentrum fur Angewandte Genetik, Universitat fur Bodenkultur Wien, Muthgasse 18, 1190 Vienna, Austria (Received 20 April 2001; accepted 19 June 2001) Abstract — Most of the glycosylation reactions that generate the great diversity of oligosaccharide structures of eukaryotic cells occur in the Golgi apparatus. This review deals with the most recent data that provide insight into the functional organization of Golgi-resident glycosyltransferases. We also focus on the recent successes in X-ray crystal structure determination of glycosyltransferases. These new structures begin to shed light on the molecular bases accounting for donor and acceptor substrate specificities as well as catalysis. © 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. glycosyltransferase / Golgi retention / domain structure / catalytic domain / stem
1. Introduction Glycosyltransferases (GTs) constitute a large group of enzymes that are involved in the biosynthesis of oligosaccharides and polysaccharides. These molecules of fascinating diversity mediate a wide range of functions, from structure and storage to specific signalling. Recent developments in molecular biology of GTs have revealed an unexpected diversity of these enzymes thus suggesting that glycosylation reactions probably require the participation of several hundreds of genes. The glycosyltransferases transfer sugar residues from an activated donor substrate, usually a nucleotide-sugar, to an acceptor that may be a lipid, a protein or a growing oligosaccharide. These enzymes are typically grouped into families based on the type of sugar they transfer (i.e., galactosyltransferases, sialyltransferases, etc.). Glycosylation reactions proceed with either an inversion or retention of stereochemistry at the C1 position of the donor sugar. Despite the fact that many GTs recognize identical donor or acceptor substrates, there is surprisingly limited sequence homology among different classes. Glycosyltransferases have been classified into 51 distinct sequencebased families (available at CAZy server, http://afmb.cnrs-mrs.fr/∼pedro/CAZY). Human sequences (approximately 200 sequences have already been listed) are spread over 33 families. *Correspondence and reprints. E-mail address: [email protected] (C. Breton). Abbreviations: CTS, cytoplasmic, transmembrane, stem; FucT, fucosyltransferase; GnT, N-acetylglucosaminyltransferase; GT, glycosyltransferase; ppGalNAcT, polypeptide: N-acetylgalactosaminyltransferase; TMD, transmembrane domain.
In eukaryotes, most of GTs are resident membrane proteins of the endoplasmic reticulum and the Golgi apparatus. Golgi resident GTs are those that received the most attention because they are responsible for the synthesis of complex glycans that play major roles in recognition or signalling events [1]. Carbohydrates represent major components of the outer surface of mammalian cells and there is now abundant evidence that terminal glycosylation sequences are differentially expressed in cells and are subject to change during development, differentiation and oncogenic transformation [2]. Glycosyltransferases are type II transmembrane proteins consisting of a short amino-terminal cytoplasmic domain followed by a transmembrane domain, a stem region, and a large globular catalytic domain facing the luminal side (figure 1). Soluble GT forms are also present in biological fluids. They arise from proteolytic cleavage in the stem region. Because major questions remain concerning the subcellular organization and the regulation of the glycosylation machinery within the cell, the aim of the present review is to focus on the most recent data that contribute to a better understanding of the in vivo mode of action of GTs in the Golgi, with special emphasis on the contribution of each domain in enzyme function. This review also examines insights into the 3D structures and mechanisms of action of these enzymes.
2. Roles of the cytoplasmic, transmembrane, and stem regions (CTS) in Golgi retention Although much progress has been made in recent years in understanding the organization and structure of the
714
Figure 1. Topology of Golgi resident glycosyltransferases.
Golgi apparatus, there is not yet a clear picture of the glycosylation events occurring in this cellular compartment [3]. Many factors can affect glycosylation: i.e., the availability of sugar donors, the distribution of GTs in the various Golgi sub-compartments and availability of acceptor substrates. For many years, the general concept of a sequential organization was favored, stating that GTs acting early in protein-linked oligosaccharide biosynthesis are located in the medial/trans-Golgi (e.g., GnT I), whereas the late GTs that are involved in the terminal decoration of oligosaccharide motifs (α2,3/6sialyltransferases, α1,3/4-fucosyltransferases) are located within the trans-Golgi/TGN. There is now evidence that most GTs show an overlapping distribution throughout the Golgi apparatus and also that they demonstrate cell-type specific sub-compartmentation [4]. The transmembrane domain (TMD) was for a long time considered to be the key determinant for GT localization. Two hypotheses concerning the mechanism of Golgi protein retention have dominated the literature: i) the bilayer thickness hypothesis [5]; and ii) the oligomerization-kin recognition hypothesis [6]. The first model suggests that retention depends on the length of a membrane-spanning domain and thickness of the membrane along the Golgi complex. The second model postulates the retention through homo/hetero-oligomerization, which prevents enzymes from entering the transport vesicles. However, neither hypothesis alone is completely satisfaying. Three GTs, namely GnT I, β4-GalT1 and ST6GalI, have been extensively studied to identify the mechanisms of Golgi retention (reviewed in [4]). Results demonstrated that the TMD of these GTs are clearly important for their retention but also that frequently their cytoplasmic and/or flanking luminal sequences may play accessory or even indepen-
Breton et al. dent roles in the Golgi retention process. Two enzymes of the medial Golgi, GnT1 and α-mannosidase II, were found tightly associated in vivo through their luminal domains and not their TMD [7]. Similarly, a region in the stem of GnT V was recently shown to be responsible for Golgi localization, probably through homo-oligomer formation [8], and stable localization of ST6GalI was correlated with the formation of insoluble oligomers [9]. Modification of the cytoplasmic domain of α2fucosyltransferase was recently shown to alter its Golgi localization and therefore its temporal order of action [10]. Recent data provided evidence for the presence of targeting signals in the CTS regions of Golgi GTs which mediate, first, their Golgi retention; second, their targeting to specific in vivo functional areas; and third, susceptibility of the enzyme towards intracellular proteolysis, which in turn might regulate the GT intracellular turnover [11]. Altogether, these observations suggest that more than one retention mechanism may be at work with probably subtle differences between cell types. Another matter of debate concerns the organization of GTs within Golgi membranes. Previous reports demonstrated that GTs may form large hetero-oligomeric structures [6, 12]. However, differences exist between early and late acting Golgi GTs. The medial Golgi enzymes (GnT I and GnT II) exist as large molecular mass complexes, in contrast to the late-acting β4-GalT and α2-FucT that are present as monomers and dimers [13]. The formation of large complexes does not require the cytoplasmic tail nor the transmembrane domains of GnT I but rather is dependent on the luminal domain. However, the nature of these complexes is still obscure and may represent multi-enzyme complexes like those observed in the cis-Golgi of yeast [12]. The differences in behavior observed for these GTs may contribute to their differential localization and are probably critical for their action in the coordinated synthesis of N-glycans. Physical and functional association was also recently demonstrated for two Golgi-resident GTs, β4-GalNAcT and β3-GalT2, which act in succession in the synthesis of glycolipids [14]. Such association is thought to improve the efficiency of glycolipid synthesis and support the idea of cooperative sequential specificity in the synthesis of complex oligosaccharides. 3. Role of the stem region in enzyme function Within a group of homologous GT sequences, the length and amino acid composition of catalytic domains are relatively well conserved and variations in protein sizes are generally attributable to differences in the length of the stem region. The stem region can be defined as the peptide portion after the TMD that can be removed without altering the activity. It has long been considered as a flexible tether which may play a role in positioning the
Features of glycosyltransferases
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catalytic domain away from the lipid layer, thus facilitating access to the substrates. The longest stem region (∼ 400 aa) reported so far was found in the recently cloned ppGalNAc-T5, a region rich in hydroxyamino acids and lysine and which contains seven potential N-glycosylation sites [15]. In contrast, ST6GalNAc III enzyme displays a very short stem peptide, if any [16]. Sequence analysis of stem regions from various GT classes most often reveals high variability in amino acid composition and little secondary organization. Even though this region is predicted to be highly flexible, it often contains cysteine residues as well as several N- and/or O-glycosylation sites which can contribute to a local conformation. A recent work indicated that the stem region of ST6GalI could play a role in discriminating glycoprotein acceptors and that it may exert a steric control onto the catalytic domain [17]. Although the role of the stem region in enzyme activity is still unclear and much more experimental data are needed, it is tempting to speculate that it could modulate the in vivo acceptor specificity. 4. Influence of N-glycosylation on GT trafficking and activity Glycosyltransferases are very often themselves glycosylated. As it has been proposed for other proteins that N-glycosylation can serve as signal for protein transport to the cell surface, the influence of the glycosylation status on activity and cellular localization was examined for some GTs. It was shown for ST6GalI that N-glycosylation may stabilize the protein but is not required for activity of the full-length enzyme in vivo. In contrast, soluble forms of the protein are efficiently secreted and active in their fully glycosylated forms. If unglycosylated, they are
retained in RE and inactive [18]. In that case, N-glycans may play a secondary role in protein transport by influencing the conformational behavior of the protein. For other GTs, N-linked oligosaccharides were found necessary for both efficient RE to Golgi transport and/or catalytic activity in in vitro assays [19–21]. In fact, many examples in the literature show that the role of N-glycosylation varies from protein to protein. 5. Catalytic domain: structure and mechanism For many years, our knowledge of the mechanism of action of GTs was hampered by the lack of 3D-structures particularly of eukaryotic GTs. The first X-ray crystal structure to be solved was the β-glucosyltransferase of phage T4, in 1994 [22]. In the past 2 years, seven crystal structures of GTs from prokaryotes and eukaryotes have been determined (table I). Complete removal of CTS regions appears to be a key determinant to the successful crystallization of catalytic domains of eukaryotic enzymes. Therefore none of the current 3D-structures provide information on the possible structure and function of the stem domain. Intriguingly, comparison of crystal structures reveals that GTs are probably comprised of an unexpected small number of protein folds. Although belonging to different GT families showing no primary sequence identity, the various 3D structures reported to date share a similar class of fold, consisting in a threelayer α/β/α sandwich that resembles the ‘Rossmann fold’. On a structural basis, these GT structures have been classified into two distinct superfamilies (see table I) [23]. The BGT superfamily includes BGT and MurG, which both are α/β proteins comprising two similar Rossmanntype domains separated by a deep cleft. The SpsA super
Table I. Summary of currently known 3D structures of glycosyltransferases. GT familya
Enzyme
Origin
Function
DxD motif Mechanism References
phage T4 E. coli
DNA glucosylation Peptidoglycan biosynthesis
no no
inverting inverting
[22] [32]
Spore coat formation Lactose/N-glycan biosynthesis N-glycan biosynthesis Proteoglycan biosynthesis Lipo-oligosaccharide biosynthesis Biosynthesis of the xenoantigen
yes yes yes yes yes
inverting inverting inverting retaining
[33] [34] [35] [36] [25]
yes
retaining
[37]
BGT superfamily n.c. 28
β-GlcT (BGT) β4-GlcNAcT (MurG) SpsA superfamily
2 7 13 43 8
SpsAb β4-GalT1 β2-GlcNAcT (GnT I) β3-GlcAT I α4-GalT (LgtC)
B. subtilis Bovine Rabbit Human N. meningitidis
6
α3-GalT
Bovine
a
Number refers to the CAZy classification (see in the text). SpsA is thought to be an inverting enzyme but the donor and acceptor specificity of SpsA still have to be determined. n.c., non-classified.
b
716
Breton et al.
Figure 2. Illustration of the presence of conserved peptide regions in GnTI and GnTII protein families, suggesting a common genetic origin and a similar overall 3D structure. A. Schematic representation of GnTI and GnTII showing the location of the three best conserved regions in the catalytic domain. B. Multiple sequence alignment of the conserved regions. The invariant or highly similar residues are in white letters on a black background and other conserved positions have a grey background. Residues involved in UDP-GlcNAc/Mn2+ binding are indicated by asterisks [35]. The proposed catalytic base in region 3 is designated by an arrow. h, human; Ce, Caenorhabditis elegans; Nt, Nicotiana tabacum; At, Arabidopsis thaliana. Accession numbers (from GenBankTM/EBI Databank) for the selected peptide sequences are: M55621 (h GnTI), AF082012 (Ce GnTI), Y16832 (Nt GnTI), U15128 (h GnTII), AF251126 (Ce GnTII), AJ249274 (At GnTII).
Features of glycosyltransferases family comprises all six other GT structures. They are α/β proteins and adopt a fold highly similar to SpsA, whose topology consists of two closely associated domains forming a conical shape with a large pocket on one face capable to accommodate both the donor and acceptor substrates. Almost all of these proteins have been crystallized in the presence of their donor substrate product UDP or the complete donor sugar (i.e., GnT I). Two of these structures, the human β3-GlcAT-I and LgtC from N. meningitidis, have acceptor substrate bound. Similarities in the spatial arrangement of secondary structure elements involved in UDP binding have been observed in all members of the SpsA superfamily, irrespective of the stereochemistry of the reaction. The presence of a common nucleotide binding domain (NBD) in both inverting and retaining GTs is in favor of a common origin. These structures have provided considerable insight into the structural bases for catalysis, particularly on the role played by the conserved DxD motif. This motif has been identified in many different GT families that use various nucleotide sugars and acceptors [24], and it was shown, in several crystal structures, to interact mainly with the phosphate groups of nucleotide donor through the coordination of a metal cation. It is found at equivalent positions in all members of the SpsA superfamily and is probably shared by all GTs having a strict requirement for divalent cations to be active. We anticipate that the NBD identified in SpsA and structural homologues will be common to all GTs sharing a DxD motif. Our current estimation is that at least 17 distinct families (out of a total of 51) of the CAZy database may be concerned. For BGT and presumably MurG, which do not share the DxD motif, basic residues of the active site make direct contacts with the diphosphate moiety of UDP. Threading analyses currently suggest that at least 12 GT families may share the BGT/MurG fold (unpublished data). These studies have undoubdtedly begin to shed light on the glycosyl transfer reaction of inverting and retaining GTs. Although it has been assumed that similar mechanisms are employed by glycosidases and glycosyltransferases, one cannot yet exclude the existence of unique catalytic process for GTs [25]. In absence of crystal structure, structural information can be extracted from the primary peptide sequences using computational tools. For instance, conserved structural features as well as consensus peptide motifs have been evidenced in the catalytic domains of all α1,2-FucTs and α1,6-FucTs from prokaryotic and eukaryotic origin, thus suggesting a common ancestor [26]. Threading analyses suggest that fucosyltransferases adopt a similar topology as BGT ([27], and unpublished data). Despite a lack of sequence identity, we herein demonstrate that GnT I and GnT II are related proteins. Using the sensitive hydrophobic cluster analysis method [28], we identified three conserved regions in the catalytic domains of all GnT I and GnT II enzymes (figure 2). These conserved peptide motifs are spread over a large portion of the catalytic
717 domain and since catalytic amino acids are submitted to intense conservation pressure, they are generally located in these regions. Region 2, which comprises a DxD motif (EDD or EED), is remarkably conserved in all GnT I and GnT II proteins. Therefore, from these results, we can postulate that GnT II, like GnT I, shares a similar fold as SpsA. Except the presence of a common DxD motif in GnT III and GnT IV (absent in GnT V), we failed to identify similar conserved regions in these enzymes which, like GnT I and GnT II, participate to the synthesis of N-glycans. 6. Other domains A characteristic feature of ppGalNAcTs is the presence of an additional C-terminal domain that is present in all enzymes cloned to date. This domain comprises about 130 amino acids and was shown to adopt a fold similar to the lectin domain of ricin and abrin [29]. None of the mutations done in this domain does alter the catalytic properties of ppGalNAcT1 [30]. However, recently it was shown to function as a GalNAc lectin which confers glycopeptide specificity to ppGalNAcT4 isoform [31]. The function, if any, of this extra C-terminal domain in other ppGalNAcT isoforms still remains to be elucidated. 7. Conclusion The last decade has witnessed the fact that complex carbohydrates are mediators of biospecific information and their associations to diseases has also been demonstrated. Because they are central to all biosynthetic processes involving sugars, GTs are important targets for the development of new drugs with applications in cancer and in many microbial infections. A better knowledge of these enzymes and of their mechanism of action in vivo and in vitro is essential for the rational design of specific and effective inhibitors and for our understanding of the intracellular glycosylation machinery. Progress in this field will also benefit the development of improved catalysts through the engineering of GTs and will extend their potential use in the chemoenzymatic synthesis of complex carbohydrate-based therapeutic and diagnostic compounds.
References [1] Varki A., Biological roles of oligosaccharides: all of the theories are correct, Glycobiology 3 (1993) 97–130. [2] Montreuil J., Vliegenthart J.F.G., Schachter H. (Eds.), New Comprehensive Biochemistry, Vol 30, Glycoproteins and disease, Elsevier Science, Amsterdam, 1996.
718 [3] Varki A., Factors controlling the glycosylation potential of the Golgi apparatus, Trends Cell Biol. 8 (1998) 34–40. [4] Colley K.J., Golgi localization of glycosyltransferases: more questions than answers, Glycobiology 7 (1997) 1–13. [5] Munro S., Localization of proteins to the Golgi apparatus, Trends Cell Biol. 8 (1998) 11–15. [6] Nilsson T., Hoe M.H., Slusarewicz P., Rabouille C., Watson R., Hunte F., Watzele G., Berger E.G., Warren G., Kin recognition between medial Golgi enzymes in HeLa cells, EMBO J. 13 (1994) 562–574. [7] Nilsson T., Rabouille C., Hui N., Watson R., Warren G., The role of the membrane-spanning domain and stalk region of N-acetylglucosaminyltransferase I in retention, kin recognition and structural maintenance of the Golgi apparatus in HeLa cells, J. Cell Sci. 109 (1996) 1975–1989. [8] Sasai K., Ikeda Y., Tsuda T., Ihara H., Korekane H., Shiota K., Taniguchi N., The critical role of the stem region as a functional domain responsible for the oligomerization and Golgi localization of N-acetylglucosaminyltransferase V. The involvement of a domain homophilic interaction, J. Biol. Chem. 276 (2001) 759–765. [9] Chen C., Ma J., Lazic A., Backovic M., Colley K.J., Formation of insoluble oligomers correlates with ST6Gal I stable localization in the Golgi, J. Biol. Chem. 275 (2000) 13819–13826. [10] Milland J., Taylor S.G., Dodson H.C., McKenzie I.F., Sandrin M.S., The cytoplasmic tail of α1,2fucosyltransferase contains a sequence gor Golgi localisation, J. Biol. Chem. 276 (2001) 12012–12018. [11] Grabenhorst E., Conradt H.S., The cytoplasmic, transmembrane, and stem regions of glycosyltransferases specify their in vivo functional sublocalization and stability in the Golgi, J. Biol. Chem. 274 (1999) 36107–36116. [12] Jungmann J., Munro S., Multi-protein complexes in the cis Golgi of Saccharomyces cerevisiae with α-1,6-mannosyltransferase activity, EMBO J. 17 (1998) 423–434. [13] Opat A.S., Houghton F., Gleeson P.A., Medial Golgi but not late Golgi glycosyltransferases exist as high molecular weight complexes. Role of luminal domain in complex formation and localization, J. Biol. Chem. 275 (2000) 11836–11845. [14] Giraudo C.G., Daniotti J.L., Maccioni H.J., Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus, Proc. Natl. Acad. Sci., USA 98 (2001) 1625–1630. [15] Ten Hagen K.G., Hagen F.K., Balys M.M., Beres T.M., Van Wuyckhuyse B., Tabak L.A., Cloning and expression of a novel, tissue specifically expressed member of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family, J. Biol. Chem. 273 (1998) 27749–27754. [16] Sjoberg E.R., Kitagawa H., Glushka J., van Halbeek H., Paulson J.C., Molecular cloning of a developmentally regulated N-acetylgalactosamine α-2,6-sialyltransferase specific for sialylated glycoconjugates, J. Biol. Chem. 271 (1996) 7450–7459. [17] Legaigneur P., Breton C., El Battari A., Guillemot J.C., Auge C., Malissard M., Berger E., Ronin C., Exploring the acceptor recognition of the human β-galactoside -α2,6-sialyltransferase, J. Biol. Chem. (2001) in press. [18] Chen C., Colley K.J., Minimal structural and glycosylation requirements for ST6Gal I activity and trafficking, Glycobiology 10 (2000) 531–583. [19] Nagai K., Ihara Y., Wada Y., Taniguchi N., N-glycosylation is requisite for the enzyme activity and Golgi retention of N-acetylglucosaminyltransferase III, Glycobiology 7 (1997) 769–776. [20] Martina J.A., Daniotti J.L., Maccioni H.J., GM1 synthase depends on N-glycosylation for enzyme activity and trafficking to the Golgi complex, Neurochem. Res. 25 (2000) 725–731.
Breton et al. [21] Baboval T., Koul O., Smith F.I., N-glycosylation site occupancy of rat α-1,3 fucosyltransferase IV and the effect of glycosylation on enzymatic activity, Biochim. Biophys. Acta 1475 (2000) 383–389. [22] Vrielink A., Ruger W., Driessen H.P., Freemont P.S., Crystal structure of the DNA modifying enzyme beta-glucosyltransferase in the presence and absence of the substrate uridine diphosphoglucose, EMBO J. 13 (1994) 3413–3422. [23] Unligil U.M., Rini J.M., Glycosyltransferase structure and mechanism, Curr. Opin. Struct. Biol. 10 (2000) 510–517. [24] Breton C., Imberty A., Structure/function studies of glycosyltransferases, Curr. Opin. Struct. Biol. 9 (1999) 563–571. [25] Persson K., Ly H.D., Dieckelmann M., Wakarchuk W.W., Withers S.G., Strynadka N.C., Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs, Nat. Struct. Biol. 8 (2001) 166–175. [26] Oriol R., Mollicone R., Cailleau A., Balanzino L., Breton C., Divergent evolution of fucosyltransferase genes from vertebrates, invertebrates, and bacteria, Glycobiology 9 (1999) 323–334. [27] Breton C., Oriol R., Imberty A., Sequence alignment and fold recognition of fucosyltransferases, Glycobiology 6 (1996) vii–xii. [28] Gaboriaud C., Bissery V., Benchetrit T., Mornon J.P., Hydrophobic cluster analysis: an efficient new way to compare and analyse amino acid sequences, FEBS Lett. 224 (1987) 149–155. [29] Imberty A., Piller V., Piller F., Breton C., Fold recognition and molecular modeling of a lectin-like domain in UDPGalNAc:polypeptide N-acetylgalactosaminyltransferases, Prot. Engin. 10 (1997) 1353–1356. [30] Hagen F.K., Hazes B., Raffo R., deSa D., Tabak L.A., Structurefunction analysis of the UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase. Essential residues lie in a predicted active site cleft resembling a lactose repressor fold, J. Biol. Chem. 274 (1999) 6797–6803. [31] Hassan H., Reis C.A., Bennett E.P., Mirgorodskaya E., Roepstorff P., Hollingsworth M.A., Burchell J., Taylor-Papadimitriou J., Clausen H., The lectin domain of UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-T4 directs its glycopeptide specificities, J. Biol. Chem. 275 (2000) 38197–38205. [32] Ha S., Walker D., Shi Y., Walker S., The 1.9 A crystal structure of Escherichia coli MurG, a membrane-associated glycosyltransferase involved in peptidoglycan biosynthesis, Prot. Sci. 9 (2000) 1045–1052. [33] Charnock S.J., Davies G.J., Structure of the nucleotide-diphosphosugar transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed forms, Biochemistry 38 (1999) 6380–6385. [34] Gastinel L.N., Cambillau C., Bourne Y., Crystal structures of the bovine ß4-galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose, EMBO J. 18 (1999) 3546–3557. [35] Unligil U.M., Zhou S., Yuwaraj S., Sarkar M., Schachter H., Rini J.M., X-ray crystal structure of rabbit N-acetylglucosaminyltransferase I: catalytic mechanism and a new protein superfamily, EMBO J. 19 (2000) 5269–5280. [36] Pedersen L.C., Tsuchida K., Kitagawa H., Sugahara K., Darden T.A., Negishi M., Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I, J. Biol. Chem. 275 (2000) 34580–34585. [37] Gastinel L.N., Bignon C., Misra A.K., Hindsgaul O., Shaper J.H., Joziasse D.H., Bovine α1,3-galactosyltransferase catalytic domain structure and its relationship with ABO histo-blood group and glycosphingolipid glycosyltransferases, EMBO J. 20 (2001) 638–649.
Structural and functional features of glycosyltransferases C. Bretona*, J. Muchab, C. Jeanneaua a
Centre de Recherches sur les Macromolécules Végétales (affıliated to the University Joseph-Fourier), CNRS, BP 53, 38041 Grenoble cedex 9, France b Zentrum fur Angewandte Genetik, Universitat fur Bodenkultur Wien, Muthgasse 18, 1190 Vienna, Austria (Received 20 April 2001; accepted 19 June 2001) Abstract — Most of the glycosylation reactions that generate the great diversity of oligosaccharide structures of eukaryotic cells occur in the Golgi apparatus. This review deals with the most recent data that provide insight into the functional organization of Golgi-resident glycosyltransferases. We also focus on the recent successes in X-ray crystal structure determination of glycosyltransferases. These new structures begin to shed light on the molecular bases accounting for donor and acceptor substrate specificities as well as catalysis. © 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. glycosyltransferase / Golgi retention / domain structure / catalytic domain / stem
1. Introduction Glycosyltransferases (GTs) constitute a large group of enzymes that are involved in the biosynthesis of oligosaccharides and polysaccharides. These molecules of fascinating diversity mediate a wide range of functions, from structure and storage to specific signalling. Recent developments in molecular biology of GTs have revealed an unexpected diversity of these enzymes thus suggesting that glycosylation reactions probably require the participation of several hundreds of genes. The glycosyltransferases transfer sugar residues from an activated donor substrate, usually a nucleotide-sugar, to an acceptor that may be a lipid, a protein or a growing oligosaccharide. These enzymes are typically grouped into families based on the type of sugar they transfer (i.e., galactosyltransferases, sialyltransferases, etc.). Glycosylation reactions proceed with either an inversion or retention of stereochemistry at the C1 position of the donor sugar. Despite the fact that many GTs recognize identical donor or acceptor substrates, there is surprisingly limited sequence homology among different classes. Glycosyltransferases have been classified into 51 distinct sequencebased families (available at CAZy server, http://afmb.cnrs-mrs.fr/∼pedro/CAZY). Human sequences (approximately 200 sequences have already been listed) are spread over 33 families. *Correspondence and reprints. E-mail address: [email protected] (C. Breton). Abbreviations: CTS, cytoplasmic, transmembrane, stem; FucT, fucosyltransferase; GnT, N-acetylglucosaminyltransferase; GT, glycosyltransferase; ppGalNAcT, polypeptide: N-acetylgalactosaminyltransferase; TMD, transmembrane domain.
In eukaryotes, most of GTs are resident membrane proteins of the endoplasmic reticulum and the Golgi apparatus. Golgi resident GTs are those that received the most attention because they are responsible for the synthesis of complex glycans that play major roles in recognition or signalling events [1]. Carbohydrates represent major components of the outer surface of mammalian cells and there is now abundant evidence that terminal glycosylation sequences are differentially expressed in cells and are subject to change during development, differentiation and oncogenic transformation [2]. Glycosyltransferases are type II transmembrane proteins consisting of a short amino-terminal cytoplasmic domain followed by a transmembrane domain, a stem region, and a large globular catalytic domain facing the luminal side (figure 1). Soluble GT forms are also present in biological fluids. They arise from proteolytic cleavage in the stem region. Because major questions remain concerning the subcellular organization and the regulation of the glycosylation machinery within the cell, the aim of the present review is to focus on the most recent data that contribute to a better understanding of the in vivo mode of action of GTs in the Golgi, with special emphasis on the contribution of each domain in enzyme function. This review also examines insights into the 3D structures and mechanisms of action of these enzymes.
2. Roles of the cytoplasmic, transmembrane, and stem regions (CTS) in Golgi retention Although much progress has been made in recent years in understanding the organization and structure of the
714
Figure 1. Topology of Golgi resident glycosyltransferases.
Golgi apparatus, there is not yet a clear picture of the glycosylation events occurring in this cellular compartment [3]. Many factors can affect glycosylation: i.e., the availability of sugar donors, the distribution of GTs in the various Golgi sub-compartments and availability of acceptor substrates. For many years, the general concept of a sequential organization was favored, stating that GTs acting early in protein-linked oligosaccharide biosynthesis are located in the medial/trans-Golgi (e.g., GnT I), whereas the late GTs that are involved in the terminal decoration of oligosaccharide motifs (α2,3/6sialyltransferases, α1,3/4-fucosyltransferases) are located within the trans-Golgi/TGN. There is now evidence that most GTs show an overlapping distribution throughout the Golgi apparatus and also that they demonstrate cell-type specific sub-compartmentation [4]. The transmembrane domain (TMD) was for a long time considered to be the key determinant for GT localization. Two hypotheses concerning the mechanism of Golgi protein retention have dominated the literature: i) the bilayer thickness hypothesis [5]; and ii) the oligomerization-kin recognition hypothesis [6]. The first model suggests that retention depends on the length of a membrane-spanning domain and thickness of the membrane along the Golgi complex. The second model postulates the retention through homo/hetero-oligomerization, which prevents enzymes from entering the transport vesicles. However, neither hypothesis alone is completely satisfaying. Three GTs, namely GnT I, β4-GalT1 and ST6GalI, have been extensively studied to identify the mechanisms of Golgi retention (reviewed in [4]). Results demonstrated that the TMD of these GTs are clearly important for their retention but also that frequently their cytoplasmic and/or flanking luminal sequences may play accessory or even indepen-
Breton et al. dent roles in the Golgi retention process. Two enzymes of the medial Golgi, GnT1 and α-mannosidase II, were found tightly associated in vivo through their luminal domains and not their TMD [7]. Similarly, a region in the stem of GnT V was recently shown to be responsible for Golgi localization, probably through homo-oligomer formation [8], and stable localization of ST6GalI was correlated with the formation of insoluble oligomers [9]. Modification of the cytoplasmic domain of α2fucosyltransferase was recently shown to alter its Golgi localization and therefore its temporal order of action [10]. Recent data provided evidence for the presence of targeting signals in the CTS regions of Golgi GTs which mediate, first, their Golgi retention; second, their targeting to specific in vivo functional areas; and third, susceptibility of the enzyme towards intracellular proteolysis, which in turn might regulate the GT intracellular turnover [11]. Altogether, these observations suggest that more than one retention mechanism may be at work with probably subtle differences between cell types. Another matter of debate concerns the organization of GTs within Golgi membranes. Previous reports demonstrated that GTs may form large hetero-oligomeric structures [6, 12]. However, differences exist between early and late acting Golgi GTs. The medial Golgi enzymes (GnT I and GnT II) exist as large molecular mass complexes, in contrast to the late-acting β4-GalT and α2-FucT that are present as monomers and dimers [13]. The formation of large complexes does not require the cytoplasmic tail nor the transmembrane domains of GnT I but rather is dependent on the luminal domain. However, the nature of these complexes is still obscure and may represent multi-enzyme complexes like those observed in the cis-Golgi of yeast [12]. The differences in behavior observed for these GTs may contribute to their differential localization and are probably critical for their action in the coordinated synthesis of N-glycans. Physical and functional association was also recently demonstrated for two Golgi-resident GTs, β4-GalNAcT and β3-GalT2, which act in succession in the synthesis of glycolipids [14]. Such association is thought to improve the efficiency of glycolipid synthesis and support the idea of cooperative sequential specificity in the synthesis of complex oligosaccharides. 3. Role of the stem region in enzyme function Within a group of homologous GT sequences, the length and amino acid composition of catalytic domains are relatively well conserved and variations in protein sizes are generally attributable to differences in the length of the stem region. The stem region can be defined as the peptide portion after the TMD that can be removed without altering the activity. It has long been considered as a flexible tether which may play a role in positioning the
Features of glycosyltransferases
715
catalytic domain away from the lipid layer, thus facilitating access to the substrates. The longest stem region (∼ 400 aa) reported so far was found in the recently cloned ppGalNAc-T5, a region rich in hydroxyamino acids and lysine and which contains seven potential N-glycosylation sites [15]. In contrast, ST6GalNAc III enzyme displays a very short stem peptide, if any [16]. Sequence analysis of stem regions from various GT classes most often reveals high variability in amino acid composition and little secondary organization. Even though this region is predicted to be highly flexible, it often contains cysteine residues as well as several N- and/or O-glycosylation sites which can contribute to a local conformation. A recent work indicated that the stem region of ST6GalI could play a role in discriminating glycoprotein acceptors and that it may exert a steric control onto the catalytic domain [17]. Although the role of the stem region in enzyme activity is still unclear and much more experimental data are needed, it is tempting to speculate that it could modulate the in vivo acceptor specificity. 4. Influence of N-glycosylation on GT trafficking and activity Glycosyltransferases are very often themselves glycosylated. As it has been proposed for other proteins that N-glycosylation can serve as signal for protein transport to the cell surface, the influence of the glycosylation status on activity and cellular localization was examined for some GTs. It was shown for ST6GalI that N-glycosylation may stabilize the protein but is not required for activity of the full-length enzyme in vivo. In contrast, soluble forms of the protein are efficiently secreted and active in their fully glycosylated forms. If unglycosylated, they are
retained in RE and inactive [18]. In that case, N-glycans may play a secondary role in protein transport by influencing the conformational behavior of the protein. For other GTs, N-linked oligosaccharides were found necessary for both efficient RE to Golgi transport and/or catalytic activity in in vitro assays [19–21]. In fact, many examples in the literature show that the role of N-glycosylation varies from protein to protein. 5. Catalytic domain: structure and mechanism For many years, our knowledge of the mechanism of action of GTs was hampered by the lack of 3D-structures particularly of eukaryotic GTs. The first X-ray crystal structure to be solved was the β-glucosyltransferase of phage T4, in 1994 [22]. In the past 2 years, seven crystal structures of GTs from prokaryotes and eukaryotes have been determined (table I). Complete removal of CTS regions appears to be a key determinant to the successful crystallization of catalytic domains of eukaryotic enzymes. Therefore none of the current 3D-structures provide information on the possible structure and function of the stem domain. Intriguingly, comparison of crystal structures reveals that GTs are probably comprised of an unexpected small number of protein folds. Although belonging to different GT families showing no primary sequence identity, the various 3D structures reported to date share a similar class of fold, consisting in a threelayer α/β/α sandwich that resembles the ‘Rossmann fold’. On a structural basis, these GT structures have been classified into two distinct superfamilies (see table I) [23]. The BGT superfamily includes BGT and MurG, which both are α/β proteins comprising two similar Rossmanntype domains separated by a deep cleft. The SpsA super
Table I. Summary of currently known 3D structures of glycosyltransferases. GT familya
Enzyme
Origin
Function
DxD motif Mechanism References
phage T4 E. coli
DNA glucosylation Peptidoglycan biosynthesis
no no
inverting inverting
[22] [32]
Spore coat formation Lactose/N-glycan biosynthesis N-glycan biosynthesis Proteoglycan biosynthesis Lipo-oligosaccharide biosynthesis Biosynthesis of the xenoantigen
yes yes yes yes yes
inverting inverting inverting retaining
[33] [34] [35] [36] [25]
yes
retaining
[37]
BGT superfamily n.c. 28
β-GlcT (BGT) β4-GlcNAcT (MurG) SpsA superfamily
2 7 13 43 8
SpsAb β4-GalT1 β2-GlcNAcT (GnT I) β3-GlcAT I α4-GalT (LgtC)
B. subtilis Bovine Rabbit Human N. meningitidis
6
α3-GalT
Bovine
a
Number refers to the CAZy classification (see in the text). SpsA is thought to be an inverting enzyme but the donor and acceptor specificity of SpsA still have to be determined. n.c., non-classified.
b
716
Breton et al.
Figure 2. Illustration of the presence of conserved peptide regions in GnTI and GnTII protein families, suggesting a common genetic origin and a similar overall 3D structure. A. Schematic representation of GnTI and GnTII showing the location of the three best conserved regions in the catalytic domain. B. Multiple sequence alignment of the conserved regions. The invariant or highly similar residues are in white letters on a black background and other conserved positions have a grey background. Residues involved in UDP-GlcNAc/Mn2+ binding are indicated by asterisks [35]. The proposed catalytic base in region 3 is designated by an arrow. h, human; Ce, Caenorhabditis elegans; Nt, Nicotiana tabacum; At, Arabidopsis thaliana. Accession numbers (from GenBankTM/EBI Databank) for the selected peptide sequences are: M55621 (h GnTI), AF082012 (Ce GnTI), Y16832 (Nt GnTI), U15128 (h GnTII), AF251126 (Ce GnTII), AJ249274 (At GnTII).
Features of glycosyltransferases family comprises all six other GT structures. They are α/β proteins and adopt a fold highly similar to SpsA, whose topology consists of two closely associated domains forming a conical shape with a large pocket on one face capable to accommodate both the donor and acceptor substrates. Almost all of these proteins have been crystallized in the presence of their donor substrate product UDP or the complete donor sugar (i.e., GnT I). Two of these structures, the human β3-GlcAT-I and LgtC from N. meningitidis, have acceptor substrate bound. Similarities in the spatial arrangement of secondary structure elements involved in UDP binding have been observed in all members of the SpsA superfamily, irrespective of the stereochemistry of the reaction. The presence of a common nucleotide binding domain (NBD) in both inverting and retaining GTs is in favor of a common origin. These structures have provided considerable insight into the structural bases for catalysis, particularly on the role played by the conserved DxD motif. This motif has been identified in many different GT families that use various nucleotide sugars and acceptors [24], and it was shown, in several crystal structures, to interact mainly with the phosphate groups of nucleotide donor through the coordination of a metal cation. It is found at equivalent positions in all members of the SpsA superfamily and is probably shared by all GTs having a strict requirement for divalent cations to be active. We anticipate that the NBD identified in SpsA and structural homologues will be common to all GTs sharing a DxD motif. Our current estimation is that at least 17 distinct families (out of a total of 51) of the CAZy database may be concerned. For BGT and presumably MurG, which do not share the DxD motif, basic residues of the active site make direct contacts with the diphosphate moiety of UDP. Threading analyses currently suggest that at least 12 GT families may share the BGT/MurG fold (unpublished data). These studies have undoubdtedly begin to shed light on the glycosyl transfer reaction of inverting and retaining GTs. Although it has been assumed that similar mechanisms are employed by glycosidases and glycosyltransferases, one cannot yet exclude the existence of unique catalytic process for GTs [25]. In absence of crystal structure, structural information can be extracted from the primary peptide sequences using computational tools. For instance, conserved structural features as well as consensus peptide motifs have been evidenced in the catalytic domains of all α1,2-FucTs and α1,6-FucTs from prokaryotic and eukaryotic origin, thus suggesting a common ancestor [26]. Threading analyses suggest that fucosyltransferases adopt a similar topology as BGT ([27], and unpublished data). Despite a lack of sequence identity, we herein demonstrate that GnT I and GnT II are related proteins. Using the sensitive hydrophobic cluster analysis method [28], we identified three conserved regions in the catalytic domains of all GnT I and GnT II enzymes (figure 2). These conserved peptide motifs are spread over a large portion of the catalytic
717 domain and since catalytic amino acids are submitted to intense conservation pressure, they are generally located in these regions. Region 2, which comprises a DxD motif (EDD or EED), is remarkably conserved in all GnT I and GnT II proteins. Therefore, from these results, we can postulate that GnT II, like GnT I, shares a similar fold as SpsA. Except the presence of a common DxD motif in GnT III and GnT IV (absent in GnT V), we failed to identify similar conserved regions in these enzymes which, like GnT I and GnT II, participate to the synthesis of N-glycans. 6. Other domains A characteristic feature of ppGalNAcTs is the presence of an additional C-terminal domain that is present in all enzymes cloned to date. This domain comprises about 130 amino acids and was shown to adopt a fold similar to the lectin domain of ricin and abrin [29]. None of the mutations done in this domain does alter the catalytic properties of ppGalNAcT1 [30]. However, recently it was shown to function as a GalNAc lectin which confers glycopeptide specificity to ppGalNAcT4 isoform [31]. The function, if any, of this extra C-terminal domain in other ppGalNAcT isoforms still remains to be elucidated. 7. Conclusion The last decade has witnessed the fact that complex carbohydrates are mediators of biospecific information and their associations to diseases has also been demonstrated. Because they are central to all biosynthetic processes involving sugars, GTs are important targets for the development of new drugs with applications in cancer and in many microbial infections. A better knowledge of these enzymes and of their mechanism of action in vivo and in vitro is essential for the rational design of specific and effective inhibitors and for our understanding of the intracellular glycosylation machinery. Progress in this field will also benefit the development of improved catalysts through the engineering of GTs and will extend their potential use in the chemoenzymatic synthesis of complex carbohydrate-based therapeutic and diagnostic compounds.
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