Chapter 3 Cellular Organization of Glycosylation …eskolab.ucsd.edu/advGlyco2016/assigned_reading04-07.pdf3/22/2016 Cellular Organization of Glycosylation ... The focus is mainly

Chapter 3

Cellular Organization of Glycosylation

Essentials of Glycobiology 2nd

edition

3/22/2016 Cellular Organization of Glycosylation - Essentials of Glycobiology - NCBI Bookshelf

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Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold SpringHarbor Laboratory Press; 2009.

Chapter 3 Cellular Organization of GlycosylationAjit Varki, Jeffrey D Esko, and Karen J Colley.

This chapter provides an overview of glycosylation from the perspective of a single cell, taking into account thepatterns of expression, topology, and other features of the biosynthetic and degradative enzymes that are common tomost cell types. The focus is mainly on eukaryotic cells, for which more information is available.

GLYCOSYLATION IS UNIVERSAL IN LIVING ORGANISMS

It is a remarkable fact that every freeliving cell and every cell type within multicellular organisms is covered with adense and complex layer of glycans. Even enveloped viruses that bud from surfaces of infected cells carry with themthe glycosylation patterns of the host cell. Additionally, most secreted molecules are glycosylated and the extracellularmatrices of multicellular organisms are rich in glycans and glycoconjugates. The matrices secreted by unicellularorganisms when they congregate (e.g., bacterial biofilms; see Chapter 20) also contain glycans. The reason for theapparent universality of cellsurface and secreted glycosylation is not clear, but it suggests that evolution hasrepeatedly selected for glycans as being the most diverse and flexible molecules to position at the interface betweencells and the extracellular milieu. For example, the enormous diversity, complexity, and flexibility of glycans mayallow host cells to make changes to avoid pathogens, without causing major deleterious effects on cellular functions.

Like membrane proteins, secretory proteins in eukaryotic cells typically pass through an endoplasmic reticulum (ER)Golgi pathway, the cellular system in which many major glycosylation reactions occur (see below). Perhaps for thisreason, most proteins in the blood plasma of animals (with the exception of albumin) are also heavily glycosylated.Glycosylation of secreted proteins may provide solubility, hydrophilicity, and negative charge, thus reducingunwanted nonspecific intermolecular interactions in extracellular spaces and also protecting against proteolysis.Another, not mutually exclusive, hypothesis is that the glycans on secreted molecules act as decoys, bindingpathogens that seek to recognize cellsurface glycans to initiate invasion.

In bacteria, archaea, and fungi, glycans have critical structural roles in forming the cell wall and in resisting largedifferences in osmolarity between the cytoplasm and the environment. Glycans surrounding bacteria could also have arole in defense against bacteriophages or antibiotics generated by other microorganisms in the environment.

TOPOLOGICAL ISSUES RELEVANT TO GLYCAN BIOSYNTHESIS

The ERGolgi Pathway of Eukaryotes

Studies stemming from the classic work of George Palade and colleagues have indicated that most cellsurface andsecreted molecules in eukaryotic cells originate in the ER. They then make their way via an intermediate compartmentthrough multiple stacks of the Golgi apparatus, finally being distributed to various destinations from the transGolginetwork. Along the way, lipids and proteins are modified by a variety of glycosylation reactions mediated byglycosyltransferases (see Chapter 5). Figure 3.1 superficially depicts some steps in the synthesis of the major glycanclasses in the ERGogli pathway of animal cells. These pathways are discussed in the following sections and in otherchapters of this book. As mentioned earlier, the ERGolgi pathway is a universal feature of eukaryotic cells and alsoharbors other glycanmodifying enzymes (see below).

Not all glycans and glycoconjugates assemble within the ERGolgi pathway. For example, many cytoplasmic andnuclear proteins contain OGlcNAc, OGlc, or OFuc, and these modifications occur in the cytoplasm (see Chapters17 and 18). Hyaluronan and chitin assembly occurs at the plasma membrane, with direct extrusion into theextracellular matrix (see Chapter 15). In plant cells, cellulose synthesis also occurs at the plasma membrane (seeChapter 22).

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Regardless of their location, most glycosylation reactions use activated forms of monosaccharides (most oftennucleotide sugars) as donors for reactions that are catalyzed by enzymes called glycosyltransferases (see Chapter 4 fora listing of these enzymes and details about their biochemistry). A variety of glycan modifications are also found innature (see Chapter 5). Of these, the most common are generated by sulfotransferases, acetyltransferases, andmethyltransferases, which use activated forms of sulfate (3′phosphoadenyl5′phosphosulfate; PAPS), acetate (acetylCoA), and methyl groups (Sadenosylmethionine; AdoMet), respectively. Almost all the donors for glycosylationreactions and glycan modifications are synthesized within the cytoplasmic compartment from precursors ofendogenous origin. In eukaryotes, most of these donors must be actively transported across a membrane bilayer inorder to become available for reactions within the lumen of the ERGolgi pathway.

Much effort has gone into understanding the mechanisms of glycosylation and glycan modification within the ER andthe Golgi apparatus, and it is clear that a variety of interacting and competing factors determine the final outcome ofthe reactions. The glycosyltransferases, processing glycosidases, and sulfotransferases are well studied (see Chapter5), and their location has helped to define various functional compartments of the ERGolgi pathway. A popularmodel envisioned these enzymes as being physically lined up along this pathway in the precise sequence in whichthey actually work. In fact, there is considerable overlap of the enzymes across Golgi stacks and the actualdistribution of a given enzyme depends on the cell type.

Some glycan chains are made on the cytoplasmic face of intracellular membranes and flipped across to the other side,but most are added to the growing chain on the inside of the ER or the Golgi (see Figure 3.1). Regardless, the portionof a molecule that faces the inside of the lumen of the ER or Golgi will ultimately face the outside of the cell or theinside of a secretory granule or lysosome. To date there are no welldocumented exceptions to this topological rule. Aconsequence of this topological asymmetry is that many classes of glycans are optimized to be involved in cell–celland cell–matrix interactions. Of course, these topological considerations are reversed for nuclear and cytoplasmicglycosylation (see below), because the active sites of the relevant glycosyltransferases for these reactions face thecytoplasm. Perhaps not surprisingly, the types of glycans found on the two sides of the cell membrane are generallyquite distinct from each other.

Glycosylation Pathways in Eubacteriae and Archaea

Much less is known about the topology of glycoconjugate assembly in eubacteriae and Archaea (formerly grouped asprokaryotes). Bacterial cells perform most glycosylation reactions on the inner aspect of the cytoplasmic membrane,using precursors assembled in the cytoplasm (see Chapter 20). These glycan intermediates are then flipped across thecytoplasmic membrane and used to form polymeric (often large) structures in the periplasm (see Chapter 21). Ingramnegative organisms, which have an outer membrane, some of the glycans or glycoconjugates must betransferred between membranes or flipped across the outer membrane. The mechanisms underlying these processesare active areas of research. Even less is known about the topology of glycosylation pathways in Archaea.

GOLGI ENZYMES SHARE SECONDARY STRUCTURE

Despite the lack of sequence homology among different families of glycosyltransferases and sulfotransferases, almostall Golgi enzymes share some features. Early studies of the cell biology and biochemistry of vertebrateglycosyltransferases indicated that some of these activities could be found in soluble form in secretions and bodyfluids; others were identified as membranebound activities within cells, and some exhibited both properties. Cellfractionation studies generally found cellassociated transferase activities in membranerich microsomal fractions,which could be liberated in soluble form with the aid of detergents. These observations implied that some transferasesprobably represent membranespanning proteins, whereas others correspond to secreted proteins. However, followingthe initial molecular cloning efforts that defined the sequences of a β14 galactosyltransferase, an α26sialyltransferase, and the blood group A α13 Nacetylgalactosaminyltransferase, it became clear that Golgiglycosyltransferases share a common secondary structure that could account for all previous findings.

Almost all Golgi glycosyltransferases and sulfotransferases described to date have a single transmembrane domainflanked by a short aminoterminal domain and a longer carboxyterminal domain. This structure is characteristic of









socalled type II transmembrane proteins, whose single aminoterminal membranespanning domain functions as asignalanchor sequence, placing the short aminoterminal segment within the cytoplasm while directing the largercarboxyterminal domain to the other side of the biological membrane into which the signal anchor has been inserted(Figure 3.2). For plasmamembraneassociated type II proteins, the “other side” is the extracellular surface. Forglycosyltransferases, the “other side” is the lumen of the membranedelimited compartments that constitute the ERGolgi pathway. These include vesicles that transit from the ER to the cis cisterna of the Golgi, the cisternae of theGolgi apparatus itself, the vesiculotubular network of the transGolgi network, and membranedelimited structuresdistal to the transGolgi network. This arrangement predicts that the larger carboxyterminal domain contains thecatalytic activity of the transferase, and this supposition has extensive experimental support. The intralumenallocation of this domain allows it to participate in the synthesis of the growing glycans displayed by glycoproteins andglycolipids during their transit through the secretory pathway.

The type II transmembrane topology predicted by initial sequences of vertebrate glycosyltransferases has been widelyconfirmed experimentally. The topology may also explain reports of the expression of glycosyltransferases at thesurface of mammalian cells. Interestingly, several other types of Golgi enzymes (e.g., processing glycosidases) thathave been cloned share a similar topological arrangement. There are a few clear exceptions, such the UDPGlcNAc:lysosomal enzyme Nacetylglucosamine1phosphotransferase (GlcNAcphosphotransferase) and theGlcNAc1phosphodiester αNacetylglucosaminidase that are both involved in the synthesis of the Man6P targetingsignal of newly synthesized lysosomal hydrolases (see Chapter 30). The former is a multisubunit complex, and thelatter is a type I membranespanning glycoprotein with its amino terminus in the lumen of the Golgi apparatus. One ofthe sulfotransferases involved in heparan sulfate synthesis (GlcNAc 3Osulfotransferase 1) is a resident solubleenzyme in the Golgi. Likewise, the protein Ofucosyltransferase (Notch FucT) appears to be a soluble enzyme in theER.

All of the above considerations do not apply to the glycosyltransferases involved in nuclear and cytoplasmic glycansynthesis. For example, the soluble GlcNAc transferase responsible for synthesizing the Olinked GlcNAc of nuclearand cytoplasmic proteins (see Chapter 18) has no detectable homology with the Golgi GlcNAc transferases. Anothervariation is presented by the hyaluronan and cellulose synthases, which are multipass membrane proteins present inthe plasma membrane, extruding their products directly into the extracellular space. Similarly, all of the enzymes thatuse dolichollinked precursors (and, in bacteria, bactoprenollinked precursors) have more complex multimembranespanning structures and contain a motif thought to bind to the isoprenoid chain.

GLYCOSYLTRANSFERASES CAN ALSO BE GLYCOSYLATED

Many Golgi glycosyltransferases have consensus Nglycosylation sequences as well as serine and threonine residuesthat could be modified by glycosylation processes. Biochemical analyses indicate that many mammalianglycosyltransferases are indeed posttranslationally modified by glycosylation, especially Nglycosylation.Glycosylation is, in some instances, required for proper folding and/or activity, and a few studies indicate thatglycosyltransferases are subject to “autoglycosylation.” There is also limited evidence that glycosyltransferases maybe modified by phosphorylation. The functional relevance of such posttranslational modifications remains unknown.

LOCALIZATION OF GLYCOSYLTRANSFERASES IN GOLGI COMPARTMENTS

Biochemical and ultrastructural studies indicate that glycosyltransferases partially segregate into distinctcompartments within the secretory pathway. Generally speaking, enzymes acting early in glycan biosyntheticpathways have been localized to cis and medial compartments of the Golgi, whereas enzymes acting later in thebiosynthetic pathway tend to colocalize in the transGolgi cisternae and the transGolgi network. These observationshave prompted extensive exploration of the mechanisms whereby glycosyltransferases achieve this compartmentalsegregation. An effort was made to find Golgiretention sequences, by analogy with the KDEL tetrapeptide implicatedin retention/retrieval of ERassociated proteins. Although some general conclusions arise from these studies, thereader should consider the following caveats:

Observations made with one enzyme are not necessarily applicable to others.






The Golgiretention properties of any given glycosyltransferase may vary depending on the cell type in whichlocalization is examined.

Variations in the expression level of a glycosyltransferase in an experimental system can have a major influenceon retention/localization properties.

Many studies used chimeric proteins composed of segments of a glycosyltransferase fused to a reporter protein,but conclusions from such experiments have not always been verified using intact glycosyltransferases orchimeras with a different reporter protein.

In vitro studies using intact Golgi compartments indicate some spatial and functional overlap among enzymesthat were previously thought to be segregated on the basis of data from other less sensitive techniques.

Most information relevant to the retention of glycosyltransferases within specific Golgi compartments derives fromexperiments done with an α26 sialyltransferase (ST6GalI), a β 14 galactosyltransferase (GalTI), and an Nacetylglucosaminyltransferase I (GlcNAcTI). The former pair of enzymes tends to concentrate in the transGolgicompartments and the transGolgi network, whereas GlcNAcTI localizes mostly to the medialGolgi compartment.With respect to ST6GalI, multiple signals and mechanisms may be involved in its Golgi localization. Thetransmembrane domain and flanking sequences are sufficient to direct heterologous proteins to the Golgi, and itappears that it is the length of the transmembrane segment (provided it is hydrophobic in nature) and not the precisesequence of this domain that is critical. Replacement of this region with a longer unrelated hydrophobic sequencedoes not compromise the Golgi localization of the intact enzyme, suggesting that other sequences are involved inlocalization. Recent evidence suggests that the cytoplasmic tail of this protein may mediate an additional localizationmechanism and that the enzyme’s lumenal sequences are involved in a secondary oligomerization event that stabilizesGolgi retention.

In contrast, an examination of the Golgiretention determinants of GalTI points mainly to an important role for thetransmembrane domain. The sequences that flank the membranespanning domain seem less important in Golgiretention for this enzyme. Retention of GlcNAcTI is also dictated largely by its transmembrane domain, although itslumenal sequences are also involved in an oligomerization mechanism that has a role in its localization. Consideredtogether, the available experimental observations suggest that the localization of glycosyltransferases within specificregions of the Golgi apparatus is probably not determined by simple primary sequence motifs. Rather, this process islikely determined by several different regions of each enzyme that mediate multiple redundant localizationmechanisms.

Golgi localization may also be mediated by retention (in the context of the vesicular transport model) and continuousretrieval to earlier Golgi cisternae (in the context of the cisternal maturation model). Three models have beenproposed to account for the localization of glycosyltransferases to specific Golgi subcompartments. In theoligomerization/kinrecognition model, glycosylation enzymes form homooligomers or heterooligomers throughinteractions between their transmembrane and lumenal sequences after arriving at the proper Golgi compartment.Heterooligomerization or kin recognition among enzymes in the same pathway would also presumably enhance theefficiency of the sequential glycosylation reactions. Some glycosylation enzymes have been found to formhomooligomers (e.g., ST6GalI, GlcNAcT1 and GlcNAcT2), and oligomerization appears to have a role in theirstable localization in the correct Golgi compartment. Experimental support for kin recognition comes from theobservation that some pairs of glycosyltransferases known to catalyze sequential reactions in the same pathwaycolocalize to a specific Golgi compartment and are coimmunoprecipitated from cell extracts. However, this type ofassociation has not been demonstrated for most of the enzymes.

A second model depends on partitioning the glycosyltransferases into lipid bilayers of different thicknesses. Thisbilayer thickness or lipidpartitioning model was proposed on the basis of observations that a cholesterolconcentration gradient in the secretory pathway yields lipid bilayers of increasing thickness in the direction of cis totrans across the Golgi stack and that the transmembrane regions of glycosyltransferases are generally shorter thanthose of plasma membrane proteins. The model predicts that each glycosyltransferase sorts itself into the proper Golgi



location by virtue of the length of its transmembrane segment, which will retain the enzyme once it reaches the propercompartment during the enzyme’s transit through the secretory pathway. This model was formulated largely on thebasis of experiments involving ST6GalI, where the length of the membranespanning domain appears to play animportant part in Golgi retention. However, the general applicability of this model is not apparent because there is noconsistent relationship between the length of the transmembrane segment and retention in a specific Golgicompartment for a variety of glycosyltransferases. This model may therefore help to explain the overall phenomenonof retention of glycosyltransferases in the Golgi apparatus versus the delivery of other membrane proteins to theplasma membrane.

The third mechanism is based largely on the cisternal maturation model of transport through the secretory pathway. Inthis model, a new Golgi cisterna containing cargo molecules forms at the cis face of the stack and it progressively“matures” as Golgi glycosylation enzymes that define each subcompartment are transported into the new cisternafrom the old cisterna to modify the cisternal cargo proteins. In this model, the steadystate localization of the Golgienzymes is maintained by continuous retrograde transport. The cytoplasmic tails of enzymes may mediate interactionswith coat proteins that select the enzymes for transport in vesicles or tubules. In support of this mechanism, thecytoplasmic tails of some glycosylation enzymes, such as ST6GalI and FucTI, have been shown to have a role intheir Golgi localization.

Taken together, evidence suggests that glycosylation enzymes use multiple mechanisms to maintain their localizationin the Golgi. The number of signals and mechanisms used by an enzyme could determine how stable its Golgilocalization actually is, whether it is able to move to a later compartment, and whether it can be cleaved and secretedinto the extracellular space (see below).

PROTEOLYTIC CLEAVAGE AND SECRETION OF GOLGI GLYCOSYLTRANSFERASES

Many Golgi enzymes are secreted by cells, sometimes in large quantities, and can be found in cell culturesupernatants and various body fluids. The nature of the secreted forms of glycosyltransferases first became clear fromanalysis of aminoterminal peptide sequences of purified mammalian glycosyltransferases that had been isolated assoluble forms without the aid of detergents. These studies showed that the soluble forms were actually derived fromtheir membraneassociated forms by virtue of one or more proteolytic cleavage events that occurred a short distanceaway from the transmembrane segment within the stem region (Figure 3.2). These proteolytic cleavage events releasea catalytically active fragment of the glycosyltransferase from its transmembrane tether and allow the cell to exportthis fragment to the extracellular space. The existence of catalytically active fragments of glycosyltransferases that arealso deficient in various portions of the stem region together with mutational analyses imply that the stem regioncontributes little to the catalytic function of a glycosyltransferase. Nevertheless, some experimental analyses suggestthat peptide sequences within the stem region can contribute to acceptor substrate preference.

The signals within the glycosyltransferase sequence that direct proteolysis are not defined, but it appears that theproteolytic cleavages are relatively specific and are generated by proteases functioning in the trans regions of theGolgi apparatus and beyond. The production of these soluble enzymes from cell types such as hepatocytes andendothelium can also be dramatically upregulated under certain inflammatory conditions. Because these circulatingenzymes do not have access to adequate concentrations of donor nucleotide sugars (primarily located inside cells),they should be functionally incapable of performing a transfer reaction in the extracellular spaces. The biologicalsignificance of these soluble transferases therefore remains a mystery. Possibilities to consider include a lectinlikeactivity recognizing their acceptor substrates and/or a role in scavenging small amounts of circulating sugarnucleotides that might otherwise be available to certain microbes, such as gonococci.

TURNOVER AND RECYCLING OF GLYCANS

Like all components of living cells, glycans turn over constantly. Some glycoconjugates, such as transmembraneheparan sulfate proteoglycans, turn over by shedding from the cell surface through limited proteolysis. Mostglycoconjugate turnover occurs by endocytosis and subsequent degradation in lysosomes (see Chapter 41).Endoglycosidases can initially cleave glycans internally, producing substrates for exoglycosidases in the lysosome.





Once broken down, individual monosaccharides are then typically exported from the lysosome into the cytoplasm, sothat they can be reused (see Figure 1.8, Chapter 1). In contrast to the relatively slow turnover of glycans derived fromthe ERGolgi pathway, glycans of the nucleus and cytoplasm may be more dynamic and rapidly turned over (seeChapters 17 and 18). Glycans in bacterial cells (especially those in the cell wall) also turn over during cell divisionwhen the cell wall undergoes cleavage and remodeling.

NUCLEAR AND CYTOPLASMIC GLYCOSYLATION IS VERY COMMON

Until the mid1980s, a commonly stated dogma was that glycoconjugates such as glycoprotein and glycolipids occurexclusively on the outer surface of cells, on the internal (lumenal) surface of intracellular organelles, and on secretedmolecules. As discussed above, this was in accord with information about the topology of the biosynthesis of theclasses of glycans known at the time, which took place within the lumen of the GolgiER pathway. Thus, despitesome clues to the contrary, the cytoplasm and nucleus (which are topologically semicontinuous because of theexistence of nuclear pores) were assumed to be devoid of glycosylation capacity. However, it has now become clearthat certain types of glycoconjugates are synthesized and reside within the cytoplasm and nucleus. Indeed, one ofthem (Olinked GlcNAc; see Chapter 18) may well be numerically the most common type of glycoconjugate in manycells.

GLYCOSYLATION REACTIONS AT THE PLASMA MEMBRANE

Because prokaryotic cells do not have an ERGolgi pathway, they typically generate their cellsurface glycans at theinterface of the cytoplasmic membrane and the cytoplasm or in the periplasm (see Chapter 20). Other glycansassembled at the plasma membrane include hyaluronan in vertebrate cells, chitin in invertebrate cells, and cellulose inplant cells. The topological difficulties of using cytoplasmic hydrophilic sugar nucleotides to manufacture glycansthat are found on the opposite side of the cellsurface membrane are obvious. In eukaryotes, the process appears toinvolve enzyme molecules with multiple passes through the membrane that also act as a pore. In bacteria, membraneflippases may exist to aid in the transfer of intermediates across the various membranes. On the other hand, typicalGolgi enzymes have also been claimed to be present at the cell surface in some animal cell types, with their activesites facing the extracellular space. It is not known how they could function at this location or how the nucleotidesugar donors would be delivered to this location. On the other hand, there are examples of remodeling of cellsurfaceglycans in animal cells, e.g., the sulf enzymes that modify heparan sulfate glycosaminoglycan (see Chapter 16) andsialidases that may remove cellsurface sialic acids (see Chapter 14).

GLYCOSYLATION IN UNEXPECTED SUBCELLULAR LOCATIONS

There are scattered reports of glycosylation in unexpected subcellular locations, for example, gangliosides inmitochondria and Nglycans in the nucleus. Many of these claims are based on incomplete evidence (see Chapter 17for some discussion of these issues). One possibility is that there are indeed glycans in these unexpected cellularlocations, but that their true structures are actually novel. Conversely, there are instances where the structural evidenceis strong, but there is inadequate evidence to be certain about the topology of the claimed structures. Regardless, pastexperience tells us that the cell biology of glycosylation can hold many surprises, and dogmatic positions about suchcontroversial issues are not warranted.

FURTHER READING

1. Paulson JC, Colley KJ. Glycosyltransferases. Structure, localization, and control of cell typespecificglycosylation. J. Biol. Chem. 1989;264:17615–17618. [PubMed: 2681181]

2. Bretscher MS, Munro S. Cholesterol and the Golgi apparatus. Science. 1993;261:1280–1281. [PubMed:8362242]

3. Baenziger JU. Proteinspecific glycosyltransferases: How and why they do it. FASEB J. 1994;8:1019–1025.[PubMed: 7926366]

4. Colley KJ. Golgi localization of glycosyltransferases: More questions than answers. Glycobiology. 1997;7:1–13. [PubMed: 9061359]

http://www.ncbi.nlm.nih.gov/books/n/glyco2/ch1/figure/ch1.f8/?report=objectonly









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5. Glick BS, Elston T, Oster G. A cisternal maturation mechanism can explain the asymmetry of the Golgi stack.FEBS Lett. 1997;414:177–181. [PubMed: 9315681]

6. Varki A. Factors controlling the glycosylation potential of the Golgi apparatus. Trends Cell Biol. 1998;8:34–40.[PubMed: 9695806]

7. Munro S. Localization of proteins to the Golgi apparatus. Trends Cell Biol. 1998;8:11–15. [PubMed: 9695801]8. Opat AS, van Vliet C, Gleeson PA. Trafficking and localization of resident Golgi glycosylation enzymes.Biochimie. 2001;83:763–773. [PubMed: 11530209]

9. Mironov AA, Beznoussenko GV, Polishchuk RS, Trucco A. IntraGolgi transport: A way to a new paradigm?Biochim Biophys Acta. 2005;1744:340–350. [PubMed: 15979506]

10. Czlapinski JL, Bertozzi CR. Synthetic glycobiology: Exploits in the Golgi compartment. Curr. Opin. Chem.Biol. 2006;10:645–651. [PubMed: 17056297]









Figures

FIGURE 3.1

Initiation and maturation of the major types of eukaryotic glycoconjugates in relation to subcellular trafficking inthe ERGolgi–plasma membrane pathway. This illustration outlines the different mechanisms and topology forinitiation, trimming, and elongation of the major glycan classes in animal cells. Asterisks represent the addition ofouter sugars to glycans in the Golgi apparatus. Nglycans and glycosylphosphatidylinositol (GPI) anchors areinitiated by the enbloc transfer of a large preformed precursor glycan to a newly synthesized glycoprotein. Oglycans and sulfated glycosaminoglycans are initiated by the addition of a single monosaccharide, followed byextension. The most common glycosphingolipids are initiated by the addition of glucose to ceramide on the outerface of the ERGolgi compartments, and the glycan is then flipped into the lumen to be extended. For a betterunderstanding of the events depicted in this figure, see details in other chapters of this book: Nglycans (Chapter8); Oglycans (Chapter 9); glycosphingolipids (Chapter 10); GPI anchors (Chapter 11); and sulfatedglycosaminoglycans (Chapter 16).








FIGURE 3.2

Typical transmembrane topology and proteolytic processing of Golgi glycosyltransferases. Golgiglycosyltransferases and sulfotransferases generally have a single hydrophobic segment (TM) that functions as asignalanchor sequence. This segment spans the lipid bilayer of the tubular and vesicular structures of thesecretory pathway, including the membrane of the Golgi apparatus. This topology places the catalytic domain of aglycosyltransferase within the lumen of the Golgi apparatus and other membranedelimited structures of thesecretory pathway. The membranetethered form of a glycosyltransferase is susceptible to one or more proteolyticcleavage events that transect the enzyme within its “stem” region. Proteolysis can liberate a catalytically active,soluble form of the enzyme that may be released from the cell. With few exceptions, vertebrateglycosyltransferases have one or more potential asparaginelinked glycosylation sites (forked symbols). Whereexamined experimentally, one or more of these sites are used, indicating that most glycosyltransferases areglycoproteins.

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Chapter 6

Glycosyltransferases and Glycan-processing Enzymes

Essentials of Glycobiology 3rd

edition

Chapter6GlycosyltransferasesandGlycan-processing

Enzymes

Authors:JamesM.RiniandJeffreyD.Esko

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Glycosyltransferasesandglycosidasesareresponsiblefortheassembly,processing,andturnoverofglycans.Inaddition,thereareanumberoftransferasesthatmodifythechainsbytheadditionofacetyl,methyl,phosphate,sulfateandothergroups.Thischaptercoversthegeneralcharacteristicsofenzymesinvolvedinglycaninitiation,assemblyandprocessing,includingaspectsofsubstratespecificity,primarysequencerelationships,structures,andenzymemechanisms.

GENERALPROPERTIES

Thebiosynthesisofglycansisprimarilydeterminedbyglycosyltransferasesthatassemblemonosaccharidemoietiesintolinearandbranchedglycanchains.Asmightbeexpectedfromthecomplexarrayofglycanstructuresfoundinnature,glycosyltransferasesconstituteaverylargefamilyofenzymes.Inmanycasestheycatalyzeagroup-transferreactioninwhichthemonosaccharidemoietyofasimplenucleotidesugardonorsubstrate(e.g.,UDP-Gal,GDP-Fuc,orCMP-Sia;Chapter5)istransferredtotheacceptorsubstrate.Insomeinstances,thedonorsubstratescontainalipidmoiety,suchasdolichol-phosphate,linkedtomannoseorglucose.Forotherglycosyltransferases,thedonorsubstrateisdolichol-pyrophosphatelinkedtoanoligosaccharide,andinthesecasestheentireoligosaccharideistransferredtotheacceptorsubstrate(Chapter9).Otherlipid-linkedsugarsserveasdonorsubstratesforbacterialglycosyltransferasesinvolvedintheassemblyofpeptidoglycan(e.g.,undecaprenyl-pyrophosphoryl-N-acetylmuramicacid-pentapeptide-N-acetylglucosamine),lipopolysaccharide,andcapsules(Chapters21and22).Glycosyltransferasesthatusemonosaccharides,oligosaccharides,proteins,lipids,smallorganicmoleculesandDNAasacceptorsubstrateshavebeencharacterized,butonlyglycosyltransferasesinvolvedinthebiosynthesisofglycoproteins,proteoglycans,andglycolipidsarediscussedinthischapter.Amongtheseenzymes,thevastmajorityareresponsibleforelongatingtheglycanmoietyoftheiracceptorsubstrates;theremaindercatalyzetheinitialtransferofsugartoapolypeptideorlipidacceptor.Generallyspeaking,theenzymesthatelongateglycanchainsactsequentiallysothattheproductofoneenzymeyieldsapreferredacceptorsubstrateforthesubsequentactionofanother.Theendresultisalinearand/orbranchedstructurecomposedofmonosaccharideslinkedtooneanother.Generally,acceptorrecognitiondoesnotinvolvethepolypeptideorglycolipidmoietyoftheacceptorsubstrateifpresent,althoughseveralnotableexceptionsexistasdescribedbelow.Glycosidasesthatremovemonosaccharidestoformintermediatesthatareactedonbyglycosyltransferasesarealsoinvolvedinthebiosynthesisofglycans.Otherglycosidasesareinvolvedindegradationofglycans,e.g.inlysosomes(Chapter44).Inaddition,glycanscanbemodifiedbymanyotherenzymetypesincluding

sulfotransferases,phosphotransferases,O-acetyl-transferases,O-methyltransferases,pyruvyltransferases,andphosphoethanolaminetransferases.

GLYCOSYLTRANSFERASESPECIFICITY

Mostglycosyltransferasesshowahighdegreeofspecificityforboththeirdonorandacceptorsubstrates,andthisleadSaulRosemanandcoworkerstoadvancethe“oneenzyme–onelinkage”hypothesis.ThehumanBbloodgroupα1–3galactosyltransferaseexemplifiesthisrule.ThisenzymecatalyzesaglycosylationreactioninwhichgalactoseisaddedinαlinkagetotheC-3hydroxylgroupofagalactoseresidueontheacceptorsubstrate(Figure6.1).However,theenzymeonlyactsongalactosemodifiedbyfucoseinα1–2linkage.Priormodificationbyothermonosaccharides,suchasα2–6-linkedsialicacid,yieldsaglycanthatisnotasubstrate(Figure6.1).Wenowknowthatthereareinstancesinwhichmorethanoneglycosyltransferasecanusethesameacceptortomakethesamelinkage.ThehumanfucosyltransferasesIII–VII,forexample,allattachfucoseinα1–3linkagetoN-acetyllactosaminemoietiesonglycans(Chapter14).Otherexamplesincludetheα2–3sialyltransferasesthatactongalactoseandtheβ1–4galactosyltransferasesthatactonN-acetylglucosamine.Insomerarecases,asingleenzymecancatalyzemorethanonereaction.HumanfucosyltransferaseIIIcanattachfucoseineitherα1–3orα1–4linkage,andanenzymecalledEXTL2canattacheitherN-acetylgalactosamineorN-acetylglucosamineinαlinkagetoglucuronicacid.Theβ1–4galactosyltransferaseinvolvedinN-acetyllactosamineformationexhibitsanunusualflexibilityinspecificity.Whenβ1-4galactosyltransferasebindsα-lactalbumin(thecomplexiscalledlactosesynthase),itswitchesitsacceptorspecificityfromN-acetylglucosaminetoglucose,whichenableslactosesynthesisduringmilkformation.Finally,someglycosyltransferaseshavetwoseparateactivesiteswithdifferentsubstratespecificities.Thosethatsynthesizethebackbonesofheparansulfate(EXT1/EXT2),forexample,haveoneactivesitethatcatalyzestheattachmentofN-acetylglucosaminetoglucuronicacidandanotherthatattachesglucuronicacidtoN-acetylglucosamine(Chapters16and17).However,theexamplesdescribedaboveareallexceptionstothegenerallystrictdonor,acceptorandlinkagespecificityexhibitedbymostglycosyltransferases,apropertythatservestodefineandlimitthenumberandtypeofglycanstructuresobservedinagivencelltypeororganism.Incontrasttothoseglycosyltransferasesthatelongatetheglycanmoietyoftheiracceptorsubstrates,initiationofthebiosynthesisofglycoproteinsandglycolipidsinvolvesglycosyltransferasesthatattachsugarstoeitherapolypeptidesidechainorasphingolipidbaseorglycerolipid.Asmightbeexpected,theseenzymesalsoshowahighdegreeofspecificityfortheirsubstratesaswillbediscussedinmoredetailbelowforthosethattransfertopolypeptide.Glycosyltransferasesthatinitiatethe

synthesisofglycosphingolipidstransferamonosaccharidemoietytowhatwasoriginallyaserineresidueintheceramidelipidprecursorofsphingolipids(seeChapter11).Becausedifferentglycolipidshavedifferentceramidemoieties,itappearsthatsomeglycosyltransferases,suchasthesialyltransferases,differentiallyrecognizetheirsubstratesbasedonthenatureoftheceramidemoiety.

GLYCOSYLTRANSFERASESTHATRECOGNIZETHEPROTEIN

MOIETYOFTHEIRACCEPTORSUBSTRATES

Theglycosyltransferasesthattransfersugardirectlytothepolypeptidechainofaproteinorglycoproteinrecognizetheiracceptorsubstratesinanumberofdifferentways.AlleukaryoticN-glycansareinitiatedbyoligosaccharyltransferase,anER-residentmulti-subunitenzymecomplexthatcotranslationallytransferstheN-glycanprecursorGlc3Man9GlcNAc2toasparagineresiduesinthesequencemotifAsn-X-Ser/Thr(whereXcanbeanyaminoacidexceptproline;Chapter9).Incontrast,thepolypeptideGalNActransferasesresponsiblefortheinitiationofmucin-typeO-glycansactaftertheproteinhasbeenfoldedandtransportedtotheGolgi(Chapter10).ThepolypeptideGalNActransferasesdonotrecognizeaspecificsequencemotifbutsomeisoformspecificityhasbeennoted.Ingeneral,theytransferGalNActothesidechainsofserineandthreonineresiduesfoundinrelativelyunstructuredregionsofthefoldedprotein.SomepolypeptideO-GalNActransferasespossessalectindomainthatservestodirecttheglycosyltransferasetoregionsofthepolypeptidethatalreadypossessglycanchains.Inthisway,regionsofpolypeptidethathaveahighdegreeofcarbohydratesubstitution,typicalofmucinstructures,canbesynthesized.InadditiontotheO-GalNAclinkageformedbythepolypeptideGalNActransferases,anumberofotherglycosyltransferasescanglycosylatethesidechainsofserineandthreonineresiduestogenerateO-GlcNAc,O-fucose,O-glucose,O-mannose,andO-xyloselinkages(Chapters13,17and19).Specificityforaparticularserineorthreonineresidueisachievedindifferentways.ThexylosyltransferasethatO-xylosylatesaserineresidueinchondroitinandheparansulfateproteoglycans,forexample,hasanabsoluterequirementforaglycineresiduelocatedcarboxyterminallytotheserineresidueandthepresenceofacidicresiduesinthevicinityoftheglycosylationsite.Incontrast,theO-GlcNActransferase,OGT,responsibleforaddingGlcNActoserineandthreonineresiduesonhundredsofnuclearandcytoplasmicproteins(Chapter19)lacksanyobviousconsensussequenceassociatedwithacceptorsubstratebindingspecificity.Foraminoacid–consensussequencesorglycosylationmotifsusedintheformationofglycopeptidebonds,seeTable6.1.TheER-residentO-fucosyltransferases,POFUT1andPOFUT2,thatspecificallyfucosylateepidermalgrowthfactor-like(EGF)domainandthrombospondintype1repeats(TSR),respectively(Chapter13),differfundamentallyfrommostotherglycosyltransferases.Inadditiontorecognizingaspecificsequencemotifcontaining

thetargetserineorthreonineresidue(Table6.1),theseenzymesonlyactonEGFdomainsandTSRsthatareproperlyfoldedanddisulfide-bonded.GlycosyltransferasesthataddO-glucoseandO-GlcNActootherserineandthreonineresiduesintheEGFdomainalsoexistandtheseenzymesalsorecognizeaspecificsequencemotifandrequireafoldedEGFdomain.Anumberofglycanelongatingglycosyltransferasesthatactonglycoproteinsalsorecognizethepolypeptidemoietyoftheiracceptorsubstrates.TheglycoproteinhormoneGalNActransferaseprovidesaninterestingexampleinwhichmodificationofanN-glycanisdependentonthepresenceoftheproteinsequencemotifPro-X-Arg/LyspositionedseveralaminoacidsaminoterminallytotheN-glycanbeingmodified.Themotifistypicallyfollowedcloselybyadditionalpositivelychargedresidues.TheX-raycrystalstructureofitsacceptorsubstrate,humanchorionicgonadotropin,showsthatthePro-X-Arg/Lysmotifisatthebeginningofashortsurface-exposedhelixthatalsocontainstheadditionalpositivelychargedresidues(Figure6.2).TheN-acetylgalactosamineresiduetransferredbythisenzymesubsequentlyundergoesabiologicallyimportant4-O-sulfationreactionthat,inthecaseoflutenizinghormoneandfollicle-stimulatinghormone,generatesadeterminantrecognizedbyspecificliverclearancereceptorsthatremovethemfromtheblood(Chapter34).GlycosyltransferasesthatonlyelongatethefucosemoietyaddedbyPOFUT1orPOFUT2alsoexist(Chapter13).ThespecificityshownbytheseenzymesstemsfromtheirabilitytorecognizeboththefucosemoietyandtheEGFandTSRcomponentsoftheiracceptorsubstrates.Additionalexamplesofenzymesthatelongateglycansonspecificglycoproteinsubstratesincludethepolysialyltransferasesspecificforneuralcelladhesionmolecule(N-CAM)(Chapter15)andbyEXTL3,anN-acetylglucosaminyltransferasethataddsGlcNAcinα1–4linkagetoglucuronicacidinthefirstcommittedsteptoheparansulfatebiosynthesisonproteoglycans(Chapter17).Inavariationonthistheme,GlcNAc-1-phosphotransferaseisabletoselectivelymodifytheN-glycansfoundonalargefamilyoflysosomalenzymesthatdifferinthree-dimensionalstructureandthatlackanyobviousandcommonproteinsequencemotif.Inthiscase,modificationbythisenzymehasbeenshowntobedependentonlysineresiduesappropriatelyspacedandpositionedrelativetotheN-glycansite(Chapter33).TheER-residentglucosyltransferase,UGGT,isalsoabletotransfersugartotheN-glycansonadiverserangeofglycoproteinsubstrates(Chapter39).Inthiscase,theenzymeaddsaglucosemoietytotheN-glycansonmisfoldedglycoproteinsrenderingthemsubstratesforthechaperonescalnexinandcalreticulin,whichinturnrecruitERp57,anenzymeinvolvedinpromotingproperdisulfidebondformation.

GLYCOSYLTRANSFERASESEQUENCEFAMILIESANDFOLDTYPES

Approximately1%ofgenesinthemammaliangenomeareinvolvedintheproductionormodificationofglycans.Almost200,000glycosyltransferasesequencesareknownacrossallkingdoms,andtheycompriseapproximately97glycosyltransferasefamiliesasdefinedbyprimarysequence.Thesearedescribedinthecarbohydrate-activeenzymes(CAZy)database(Chapter8).Althoughtheabsenceofsignificantsequencesimilaritybetweenmembersofonefamilyandanotherconstitutesthebasisforthisclassification,shortsequencemotifscommontothemembersofmorethanonefamilyhavebeenidentified.Thesesequenceelementsaretypicallyfoundamongglycosyltransferaseswithagivendonorsubstratespecificity;thesialylmotifscommontoeukaryoticsialyltransferasesareagoodexample(Figure6.3).Sequencemotifscommontogalactosyltransferases,fucosyltransferases,andN-acetylglucosaminyltransferaseshavealsobeenidentified.Incontrast,theso-called“DXD”motif(Asp–anyresidue–Asp)isnotassociatedwithanyparticularsubstratespecificity;thismotifisinvolvedinmetalionbindingandcatalysisasdiscussedinmoredetailbelow.Despitethelargenumberofsequencefamiliesthathavebeendefined,structuralanalysishasshownthatglycosyltransferasespossessalimitednumberoffoldtypes.Todate,structuresfor41membersofthe97familieshavebeendeterminedbyX-raycrystallography,andoftheseallbutafewpossesswhathavebeentermedtheGT-AorGT-Bfolds(Figure6.4).Thefirstapproximately120aminoacidsoftheGT-AfoldcatalyticdomainformastructuresimilartothattermedtheRossmannfold,foundinproteinsthatbindnucleotides,andintheGT-Aglycosyltransferasesthisregioninteractswiththenucleotidesugardonorsubstrate.Withonlyafewexceptions,theGT-AenzymeshavebeenfoundtopossessaDXDmotifandaremetal-ion-dependentglycosyltransferases.TheenzymescontainingaGT-B-foldpossesstwodistinctdomains,andalthoughthecarboxy-terminaldomainisprimarilyresponsibleforbindingthenucleotidesugardonorsubstrate,bothdomainspossesselementssimilartotheRossmannfold.UnlikeenzymesthatcontaintheGT-Afold,theGT-Bglycosyltransferasesaremetal-ionindependentanddonotpossessaDXDmotif.AlloftheGT-AandGT-Bglycosyltransferasesusenucleotidesugardonorsubstratesand,todate,thestructuresofonlyafewenzymesthatuselipid-linkedsugardonorshavebeendetermined.Ofthese,twoarebacterialpeptidoglycanglycosyltransferasesshowntopossessalambdalysozyme-likefoldandtwoareprokaryoticN-glycanoligosaccharyltransferases(PglBandAglB)thatpossessbothanα-helicaltransmembraneregionandamixedα/β-foldperiplasmicdomain(Figure6.4).PglBandAglBhavebeenclassifiedasGT-Cglycosyltransferasesandbecausetheyareevolutionarilyrelatedtothecoresubunitoftheeukaryoticoligosaccharyltransferase,theyprovideamodelforhowanenzymeco-translationallymediatesN-glycosylationineukaryotes.

CATALYTICANDKINETICMECHANISMS

Glycosyltransferasescatalyzetheirreactionswitheitherinversionorretentionofstereochemistryattheanomericcarbonatomofthedonorsubstrate(Figure6.5).Forexample,β1–4galactosyltransferase,aninvertingglycosyltransferase,transfersgalactosefromUDP-α-Galtogenerateaβ1-4-linkedgalactose-containingproduct.InversionofstereochemistryfollowsfromthefactthattheenzymeusesanSN2(substitutionnucleophilicbimolecular)reactionmechanismwhereanacceptorhydroxylgroupattackstheanomericcarbonatomofUDP-GalfromonesideandUDPleavesfromtheother(Figure6.5a).Typically,enzymesofthistypepossessanaspartateorglutamateresiduewhosesidechainservestopartiallydeprotonatetheincomingacceptorhydroxylgroup,renderingitabetternucleophile(asshowninFigure6.6fortheβ1–4galactosyltransferase).Inaddition,theseenzymespossessfeaturesthathelptopromoteleaving-groupdeparture.IntheGT-Aenzymes,ametalion,boundbytheDXDmotif,istypicallypositionedtointeractwiththediphosphatemoiety.ThepositivelychargedmetalionservestoelectrostaticallystabilizetheadditionalnegativechargethatdevelopsontheterminalphosphatemoietyoftheUDPleavinggroupduringbreakageofthesugar-phosphatebondofthedonorsubstrate(Figure6.6).InoneoftheGT-Aenzymesthatisnotmetaliondependent,positivelychargedsidechainsstabilizetheleavinggroup,astrategyalsousedbysomeoftheGT-B-foldenzymes.Althoughthemechanismusedbyretainingglycosyltransferasesisnotwellunderstood,insightintohowtheymightworkisprovidedbyourknowledgeofglycosidasemechanism.Onthebasisofmuchstructuralandenzymekineticanalysis,itiswellestablishedthatinvertingglycosidasesproceedviaasingleSN2displacementmechanism(Figure6.5a),whereasretainingglycosidasesuseadoubledisplacementmechanisminvolvingacovalentglycosyl-enzymeintermediate(Figure6.5b).Inthedoubledisplacementmechanism,anasparticacidorglutamicacidsidechainintheenzymeactivesitemakesthefirstattackandinversion,followedbyasecondwater-mediatedattack(ontheglycosyl-enzymeintermediate)andinversion,togiveoverallretentionofstereochemistry.Infact,usingmechanism-basedinhibitors,theglycosyl-enzymeintermediatehasbeentrappedandstudiedbyX-raycrystallographyforanumberofglycosidases.However,similarattemptstotrapandstudyglycosyltransferasereactionintermediateshavenotyetbeensuccessful.Analternatemechanism,alsoproposedforglycogenphosphorylase,isthatoftheSNimechanism.Inthiscase,theincomingnucleophileattacksfromthesamesideastheleavinggroup,leadingtoretentionofconfiguration.ManyglycosyltransferaseshavebeenshowntopossessaBiBisequentialkineticmechanisminwhichthedonorsubstratebindsbeforetheacceptorsubstrate,andtheglycosylatedacceptorisreleasedbeforethenucleosidemonosphosphateordisphosphate,dependingonthereaction.Suchkineticsarereadilyexplainedbyastructuralmodelinwhichtheactivesiterepresentsadeeppocket,withthe

nucleotidesugarsubstrateatthebottomandtheacceptorsubstratestackedontop.Iftheacceptorsubstratewastobindfirst,itwouldstericallyprecludedonorsubstratebindingand,assuch,catalysiswouldnotoccur.Owingtothestackedarrangement,italsofollowsthatreleaseoftheglycosylatedproductmustprecedereleaseofthenucleosidephosphate.Althoughlargelyconsistentwithsuchamodel,theX-raycrystalstructuresofglycosyltransferase-substratecomplexesalsoshowthatsubstrate-dependentorderingofflexibleloopsisafeaturecommontoglycosyltransferases.Typically,donorsubstratebindingordersaloop(s)thatinturnfacilitatesacceptorsubstratebinding.Moreover,looporderingalmostcertainlyservestoexcludebulkwaterfromtheactivesite,astrategyusedbymostenzymestocreateanactivesiteenvironmentthatreducestheenergyofthetransitionstateandpromotescatalysis.GLYCOSIDASESGlycosidesareaverylargefamilyofenzymeswith135memberscurrentlylistedintheCAZydatabase.Unliketheglycosyltransferases,however,membersofthisfamilyhaveevolvedindependentlymanytimes,afactreflectedinthediversearrayofthree-dimensionalstructuresobservedfortheseenzymes.Glycosidasesplayimportantrolesinthedegradationofglycanstructuresforuptakeandmetabolismofsugarsandfortheturnoverofglycoconjugatesinvariouscellularprocesses.Glycosidasesarealsoinvolvedintheformationofintermediatesthatareusedassubstratesforglycosyltransferasesinthebiosynthesisofglycans.TheuseofglycosidasesinthiswayisparticularlyimportantinthebiosynthesisofN-glycancontainingglycoproteinsinhighereukaryotesandisthoughttobeassociatedwiththeacquisitionofcomplexN-glycansduringtheevolutionofmulticellularorganisms.Inthiscase,thenascentglycoproteinglycan,Glc3Man9GlcNAc2-Asn,istrimmedbyglucosidasesandmannosidasesintheERandGolgitogeneratesubstratesfortheglycosyltransferasesthatleadtocomplexandhybrid-typeN-glycans(Chapter9).GlucosidaseII,oneofthetwoER-residentglucosidasesinvolved,alsoworksinconjunctionwithUGGTduringglycoproteinfoldingtoallowrepeateddeglucosylation/reglucosylationduringwhathasbeentermedthecalnexin/calreticulinqualitycontrolcycle(Chapter39).AninterplaybetweenglycosylationanddeglycosylationisalsofoundtooccurwiththeO-GlcNAcmoietyfoundonmanynuclearandcytoplasmicproteins(transferredbyO-GlcNActransferase;Chapter19).Inthiscase,removaloftheGlcNAcmoietybytheglycosidase,O-GlcNAcase,providesameansofdynamicallyregulatingtheextentofO-GlcNAcylationandthediverseprocessesthatitmediates.

SULFATIONANDOTHERMODIFICATIONS

ThesulfotransferasesarealargefamilyofenzymesfoundinboththecytoplasmandtheGolgi.Sulfotransferasesplayaparticularlyimportantroleintheproductionofglycosaminoglycans(Chapter17)andtheformationofL-selectinligands,glycansrequiredforthetraffickingoflymphocytesacrosshighendothelialvenulesinlymph

nodes(Chapter34).Allsulfotransferasesuse3′-phospho-adenosine-5′-phosphosulfate(PAPS)asthesulfatedonor(Chapter5).Althoughthesequencesimilarityamongsulfotransferasescanbeverylow,theyallpossessconservedsequencemotifsresponsibleforbindingthe5′and3′phosphategroupsofPAPS.Moreover,structuralanalysishasshownthatallofthesulfotransferasessolved,todate,possessthesamebasicstructure.Mechanistically,theenzymeproceedsviaanSN2-likereaction,withthehydroxylgroupoftheacceptormakinganin-lineattackonthesulfategroup.StructuralstudiesandmutagenesishaveshownthatahistidineresidueservestoactivatethehydroxylnucleophileandalysineassistsinstabilizingthePAPleavinggroup.Interestingly,aconservedserineresidueseemstobeinvolvedinmodulatingtheactivityoftheseenzymestopreventPAPShydrolysisintheabsenceofacceptorsubstrate.PhosphorylationofsugarresiduesbyATPdependentkinasesalsooccurs,e.g.atC-2ofO-xyloseinproteoglycans(Chapter17)andattheC-6positionofO-mannosein α-dystroglycan,afterthemannosehasbeenfurtherglycosylated(Chapter13).Phosphoglycosylation,aprocessinwhichasugarphosphateistransferredfromanucleotidesugardonordirectlytoaserineresidueonaprotein(e.g.,GlcNAc-P-serine),occursinDictyostelium.Ineukaryoticcells,mannose-6-phosphateontheN-glycansoflysosomalenzymesoccursbyatwo-stepprocessinvolvingUDP-GlcNAcasthephosphatedonor.Inthefirststep,GlcNAc-1-phosphotransferaseusesUDP-GlcNActogenerateGlcNAcα1-P-6-Manα1-(N-glycan),andinthesecondsteptheGlcNAcmoietyisremovedbyasecondenzymetogiveP-6-Manα1-(N-glycan)(Chapter33).O-Acetylationoccursinbacteriaandinthemodificationofsialicacids,butlittleinformationisavailableonthechemistryofthesereactions.N-DeacetylationofGlcNAcresiduesoccursduringheparin/heparansulfateformation(Chapter17),lipopolysaccharideassembly(Chapters21and22),andGPI-anchorsynthesis(Chapter12).Thebacterialenzymeiszincdependent,butin-depthstudiesofthevertebrateN-deacetylaseshavenotbeenperformed.N-DeacetylationofN-acetylneuraminicacid(themostcommonsialicacid)hasalsobeenreported(Chapter15).Finally,glycanscanbemodifiedinmanyotherways,includingpyruvylation(e.g.,intheformationofN-acetylmuramicacid;Chapter21and22),theadditionofethanolaminephosphate(e.g.,duringGPI-anchorsynthesis;Chapter12),andalkylation,deoxygenation,andhalogenationinmicrobialglycans.Allofthesereactionsarecatalyzedbyuniquetransferasesoroxidoreductasesandrepresentareasofactiveresearch.

ACKNOWLEDGEMENTS

TheauthorsappreciatehelpfulcommentsandsuggestionsfromLeiFeng,TetsuyaHirataandChristopherSaeul.

FURTHERREADING

Albesa-JoveD,GigantiD,JacksonM,AlzariPM,GuerinME.2014.Structure-functionrelationshipsofmembrane-associatedGT-Bglycosyltransferases.Glycobiology24:108-124.

BennettEP,MandelU,ClausenH,GerkenTA,FritzTA,TabakLA.2012.Controlofmucin-typeO-glycosylation:AclassificationofthepolypeptideGalNAc-transferasegenefamily.Glycobiology22:736-756.

FushinobuS,AlvesVD,CoutinhoPM.2013.Multiplerewardsfromatreasuretroveofnovelglycosidehydrolaseandpolysaccharidelyasestructures:newfolds,mechanisticdetails,andevolutionaryrelationships.CurrOpinStructBiol23:652-659.

HebertDN,LamribenL,PowersET,KellyJW.2014.TheintrinsicandextrinsiceffectsofN-linkedglycansonglycoproteostasis.NatChemBiol10:902-910.

Hurtado-GuerreroR,DaviesGJ.2012.Recentstructuralandmechanisticinsightsintopost-translationalenzymaticglycosylation.CurrOpinChemBiol16:479-487.

JanetzkoJ,WalkerS.2014.Themakingofasweetmodification:structureandfunctionofO-GlcNActransferase.JBiolChem289:34424-34432.

LairsonLL,HenrissatB,DaviesGJ,WithersSG.2008.Glycosyltransferases:structures,functions,andmechanisms.AnnuRevBiochem77:521-555.

LombardV,GolacondaRamuluH,DrulaE,CoutinhoPM,HenrissatB.2014.Thecarbohydrate-activeenzymesdatabase(CAZy)in2013.NucleicAcidsRes42:D490-495.

SchwarzF,AebiM.2011.MechanismsandprinciplesofN-linkedproteinglycosylation.CurrOpinStructBiol21:576-582.

SpecialeG,ThompsonAJ,DaviesGJ,WilliamsSJ.2014.Dissectingconformationalcontributionstoglycosidasecatalysisandinhibition.CurrOpinStructBiol28:1-13.

VasudevanD,HaltiwangerRS.2014.NovelrolesforO-linkedglycansinproteinfolding.GlycoconjJ31:417-426.

Figure Legends

FIGURE6.1.ThestrictacceptorsubstratespecificityofglycosyltransferasesisillustratedbythehumanBbloodgroupα1–3galactosyltransferase.TheBtransferaseaddsgalactoseinα1–3linkagetotheHantigen(top).Thisenzymerequirestheα1–2-linkedfucosemodificationoftheHantigenforactivitybecausetheBtransferasedoesnotaddtoanunmodifiedtype-2precursor(middle),orprecursorsmodifiedbysialylresidues(bottom)orothermonosaccharides(notshown).(Forthemonosaccharidesymbolcode,seeFigure1.5,whichisreproducedontheinsidefrontcover.)

FIGURE6.2.HumanchorionicgonadotropinshowingthedeterminantsofrecognitionusedbyglycoproteinhormoneN-acetylgalactosaminyltransferase.Ribbondiagramofafragment(residues34–58)ofhumanchorionicgonadotropin(PDB[proteindatabank]ID1HRP).ThePro40-Leu41-Arg42tripeptideandresiduesLys44andLys45correspondtoresiduesessentialforrecognitionbyglycoproteinhormoneN-acetylgalactosaminyltransferase.ResiduesAsn52-Val53-Thr54correspondtotheN-glycosylationsequencemotifoftheN-glycan(atAsn52)modifiedbytheglycoproteinhormoneN-acetylgalactosaminyltransferase.Onlythechitobiosecore(GlcNAcβ1–4GlcNAc)oftheacceptorN-glycanisshown.

FIGURE6.3.Sialylmotifs.Domainstructureofatypicalsialyltransferase,showingthesialylmotifssharedbythisfamilyofenzymes.ThesialylLmotifof48–49aminoacidssharessignificantsimilarityamongmembersandmaybeupto65%identicalinaminoacidsequence.ThesialylSmotifissmaller(~23aminoacids)anddivergesmoreamongmembersofthefamily,withonlytwoabsolutelyconservedresidues.Inbothcases,identicalresiduesareindicatedandresiduesshowingsimilarityare

denotedbyparentheses.(Asterisk)PositionofahighlyconservedsequenceH-X4-E.Additionalconservedmotifsmayexistaswell.

FIGURE6.4.RibbondiagramsofrepresentativeGT-A,GT-B,GT-Candlysozyme-typefoldglycosyltransferases.TheGT-AandGT-Bstructurescorrespondtothoseofrabbitβ1-2N-acetylglucosaminyltransferaseI(PDBID1FOA)andT4phageβ-glucosyltransferase(PDBID1J39),respectively.Inbothcases,theboundnucleotidesugardonorsubstrateisshowninstickrepresentation.TheGT-CstructureisthatofCampylobacterlarioligosaccharyltransferase,PglB(PDBID3RCE),andthelysozyme-typestructureisthatoftheStaphylococcusaureusbacterialpeptidoglycanglycosyltransferase(PDBID2OLV)incomplexwithmoenomycin(stickrepresentation).

FIGURE6.5.Schematicrepresentationof(a)invertingand(b)retainingcatalyticmechanisms.(a)SN2-likeattackoftheacceptorleadstoinversionofstereochemistryatC1.Foraglycosidasereaction,R2wouldcorrespondtoaprotonandR1wouldbetheremainderoftheglycan.AandBlabelgeneralacidandbasegroupsinthecatalyticsiteoftheenzyme.Foraglycosyltransferasereaction,R2wouldcorrespondtotheremainderoftheacceptorsubstrateandR1wouldtypicallybethenucleosidemonophosphateordiphosphatemoietyofthedonorsubstrate.(b)Thismechanismhasonlybeenestablishedforglycosidases.TwosuccessiveSN2-likereactionsseparatedbyaglycosyl-enzymeintermediateleadtoretentionoftheconfigurationatC1.R1correspondstotheremainderoftheglycanandR2toaproton.

FIGURE6.6.Catalyticsiteofbovineβ1–4galactosyltransferase.Compositefigureshowsselectedresidues/atomsofthesuperimpositionofthedonorcomplex(PDBID1TW1)ontheacceptorcomplex(PDBID1TW5).O4designatestheC4hydroxylgroupoftheGlcNAcβ1–4GlcNAcacceptorsubstratepositionedforin-lineSN2attack

(arrow)onC1oftheUDP-Galdonorsubstrate.ThebaseformofD318servestopartiallydeprotonatetheC4hydroxylgrouprenderingitabetternucleophile.ThepositivelychargedMg++ioncoordinatesthetwophosphatesoftheUDPleavinggroup,promotingcleavageoftheC1-PβbondbystabilizingtheadditionalnegativechargethatdevelopsonPβoftheleavinggroup.D252-V253-D254correspondstotheDXDmotifinbovineβ1–4galactosyltransferase.

TABLE6.1Aminoacid–consensussequencesorglycosylationmotifsfortheformationofglycopeptidebondsGlycopeptidebond ConsensussequenceorpeptidemotifGlcNAc-β-Asn Asn-X-Ser/Thr(X=anyaminoacidexceptPro)Glc-β-Asn Asn-X-Ser/ThrGalNAc-α-Ser/Thr repeatdomainsrichinSer,Thr,Pro,Gly,Alawithno

specificsequenceGlcNAc-α-Thr Thr-richdomainnearProresiduesGlcNAc-β-Ser/Thr Ser/Thr-richdomainsnearPro,Val,Ala,Gly

EGFmodules(Cys-X-X-G-X-Ser/Thr-G-X-X-Cys)Man-α-Ser/Thrα Ser/Thr-richdomainsFuc-α-Ser/Thr EGFmodules(Cys-X-X-X-X-Ser/Thr-Cys)

TSRmodules(Cys-X-X-Ser/Thr-Cys-X-X-Gly)Glc-β-Ser EGFmodules(Cys-X-Ser-X-Pro/Ala-Cys)Xyl-β-Ser Ser-Gly(inthevicinityofoneormoreacidicresidues)Glc/GlcNAc-Thr Rho:Thr-37;Ras,Rac;Cdc42:Thr-35Gal-Thr Gly-X-Thr(X=Ala,Arg,Pro,Hyp,Ser)(ventworm)Gal-β-Hyl collagenrepeats(X-Hyl-Gly)Ara-α-Hyp repetitiveHyp-richdomains(e.g.,Lys-Pro-Hyp-Hyp-

Val)GlcNAc-Hyp Skp1:Hyp-143Glc-α-Tyr glycogenin:Tyr-194GlcNAc-α-1-P-Ser Ser-richdomains(e.g.,Ala-Ser-Ser-Ala)Man-α-1-P-Ser Ser-richrepeatdomainsMan-α-C-Trp Trp-X-X-TrpMan-6-P-ethanolamine-protein

GPIattachedaftercleavageofcarboxy-terminalpeptide

BasedonSpiroR.G.2002.Glycobiology12:43R–56R;seealsoTable1.7.(EGF)Epidermalgrowthfactor;(TSR)thrombospondinrepeats;(Hyp)hydroxyproline;(Hyl)hydroxylysine;(GPI)glycosylphosphatidylinositol;(Ara)arabinose.

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