glycosylation biology

8/6/2019 Glycosylation Biology

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Leading Edge

Review

Cell 126, September 8, 2006 2006 Elsevier Inc. 855

Glycans are one o the our basic components o cells

and may also be the most abundant and diverse o

natures biopolymers. Existing as covalent linkages o

saccharides oten attached to proteins and lipids, gly-

cans constitute a signicant amount o the mass and

structural variation in biological systems. The eld o

glycobiology is ocused upon understanding the struc-

ture, chemistry, biosynthesis, and biological unction o

glycans and their derivatives. Glycobiology has a longhistory that began with investigations o the basic con-

stituents o cells and the nature o the polysaccharide

carbohydrate component. Clinical applicability arose

early with the discovery o the human blood groups,

although evidence that these were glycan antigens

came later (Landsteiner, 1931). In addition, the anti-

thrombotic glycan heparin is one o the most commonly

used drugs (Linhardt, 1991; Shriver et al., 2004), with

current estimates o a billion doses prescribed annu-

ally. As glycobiology is increasingly interrelated with

other disciplines, nomenclature and terminology within

the eld continue to evolve. The word glycan is now

oten used to encompass oligosaccharide, polysac-charide, and carbohydrate, as not all glycans are oligo-

mers and the term carbohydrate can be conused with

components o intermediary energy metabolism. More

recently, in parallel with approaches to dene genom-

ics, proteomics, and lipidomics, the term glycomics

has emerged, which reers to the study o the glycan

structures that compose an organisms glycome.

The mammalian glycome repertoire is estimated to be

between hundreds and thousands o glycan structures,

and could be larger than the proteome. Although the

diversity o glycan structures theoretically is vast, con-

straints are provided by the mechanisms o glycan syn-

thesis and regulation. Mammalian glycans are ormed

by an endogenous portolio o cellular enzymes and

substrates that have been retained in an evolutionary

investment encompassing millions o years and span-

ning 1%2% o the genome. Vertebrates, and especially

mammals, have evolved a highly complex glycan reper-

toire that is structurally distinct rom that o invertebrates,

lower eukaryotes, and prokaryotes. It is increasingly evi-

dent that the variation in glycomes among organisms is

a molecular basis or interspecies recognition systems.

Glycans o nonvertebrate organisms, or example, canmodulate the development and activation o the mamma-

lian immune system (Cobb and Kasper, 2005). Mamma-

lian glycans are remarkably well conserved, but species-

specic variations also exist, and these dierences may

be involved in the emergence o distinct traits including

susceptibilities to inectious pathogens (Gagneux and

Varki, 1999; also see the Essay by A. Varki, page 841 o

this issue). Engineering new chemical modications into

glycans o living cells may improve the ability to detect

glycan unction and contribute to uture diagnosis and

treatment o disease (Prescher et al., 2004; also see the

Minireview by J. Prescher and C. Bertozzi, page 851 o

this issue). Indeed, glycosylation deects in mice as wellas humans and their links to disease have shown that

the mammalian glycome contains a signicant amount

o biological inormation (Lowe and Marth, 2003; Freeze,

2006). This review ocuses upon the involvement o

mammalian glycans in the molecular and cellular mech-

anisms that control health and disease.

Structure and Topology of Mammalian Glycosylation

Nine monosaccharides are used in the enzymatic pro-

cess o glycosylation in mammals. Conserved biosyn-

thetic pathways provide all nine monosaccharides rom

sugars and precursors ubiquitously present in the diet.

Except in cases o rare genetic deects, dietary intake o

monosaccharides or mammalian glycans has not been

Glycylai i Cellular Mechaim

f Healh ad DieaeKazuaki Ohtsubo1 and Jamey D. Marth1,*1Howard Hughes Medical Institute and Department o Cellular and Molecular Medicine, 9500 Gilman Drive-MC0625, University

o Caliornia, San Diego, La Jolla, CA 92093, USA

*Contact: [email protected]

DOI 10.1016/j.cell.2006.08.019

Glycosylation produces an abundant, diverse, and highly regulated repertoire o cellular

glycans that are requently attached to proteins and lipids. The past decade o research on

glycan unction has revealed that the enzymes responsible or glycosylationthe glycosyl-

transerases and glycosidasesare essential in the development and physiology o living

organisms. Glycans participate in many key biological processes including cell adhesion,molecular trafcking and clearance, receptor activation, signal transduction, and endocytosis.

This review discusses the increasingly sophisticated molecular mechanisms being discov-

ered by which mammalian glycosylation governs physiology and contributes to disease.


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856 Cell 126, September 8, 2006 2006 Elsevier Inc.

rigorously established to have a benecial eect on

human health or in the treatment o disease. Biosynthetic

pathways control the production and endogenous unc-

tions o dierent glycan structures. The structural diver-

sity o the mammalian glycome is produced predomi-

nantly in the secretory pathway o the cell. Moreover, it is

within the Golgi apparatus that glycans become increas-

ingly oligomeric and branched as they transit through

this latter portion o the secretory system bound mostly

or the cell surace and extracellular compartments.

Glycosylation produces dierent types o glycans

(or glycoconjugates) that are typically attached to cel-

lular proteins and lipids (Figure 1). Protein glycosylation

encompasses N-glycans, O-glycans, and glycosami-

noglycans (requently termed proteoglycans). N-gly-

cans are linked to asparagine residues o proteins, spe-

cically a subset residing in the Asn-X-Ser/Thr moti,

whereas O-glycans are attached to a subset o serines

and threonines (Schachter, 2000; Yan and Lennarz,

2005). Although glycosaminoglycans are also linked to

serine and threonine, they are linear, are produced by

dierent biosynthetic pathways, and are oten highly sul-

ated (Esko and Selleck, 2002). Lipid glycosylation in the

secretory pathway is also a prevalent modication and

creates glycolipids (glycosphingolipids) that include the

sialic acid-bearing gangliosides (Maccioni et al., 2002).

Glycosylphosphatidylinositol (GPI) -linked proteins share

a common membrane-bound glycolipid linkage struc-

ture that is attached to various proteins (Kinoshita et al.,

1997). Hyaluronan is a unique glycan type unattached

to either proteins or lipids that is secreted into extracel-

lular compartments (Weigel et al., 1997). Less common

types o protein glycosylation also occur, or example,

on lysine, tryptophan, and tyrosine residues o specic

proteins, such as glycogen, which was the rst identi-

ed glycoprotein. In addition, although technically not

glycosylation, acetyltranserase and sulotranserase

enzymes residing in the secretory pathway requently

attach acetyl and sulate groups to selected saccha-

rides residing on some oligosaccharide chains and can

thereby modulate glycan structure and unction (Klein

and Roussel, 1998; Fukuda et al., 2001).

Some orms o glycosylation occur outside o the

secretory pathway. Among most eukaryotic organisms,

N-acetylglucosamine has been ound linked to serine

and threonine residues (O-GlcNAc) on many cytoplas-

mic and nuclear proteins (Hart, 1997). Similar to protein

phosphorylation, GlcNAcylation is an enzymatic modi-

cation that typically has a shorter hal-lie than that o

the attached proteins. This refects the presence o a

regulated cytoplasmic N-acetylglucosaminidase, which

removes O-GlcNAc, leaving the serine or threonine resi-

due subsequently available or another round o GlcNAc-

ylation or sometimes phosphorylation. O-GlcNAc is a

highly regulated posttranslational modication required

or the viability o many mammalian cell types perhaps

by acting as a nutrient sensor, preventing protein phos-

phorylation, or regulating protein turnover (Zhang et al.,

2003; ODonnell et al., 2004; Zachara and Hart, 2004).

It is useul to distinguish secretory and cytoplasmic

glycosylation rom glucuronidation, the latter being an

enzymatic process linking a single glucuronic acid to

bile salts and xenobiotics (molecules that are oreign to

cells) (Tukey and Strassburg, 2000). In contrast to glyco-

sylation, glycation reers to the covalent linkage o sac-

charides such as glucose to proteins by a nonenzymatic

Figure 1. Mammalia Glyca Liage

Prduced by GlycylaiThere are nine nucleotide sugar donors and mul-

tiple protein and lipid acceptor motis or glycosyl-

transerases, which produce 14 dierent glycans in

stereoisomeric congurations ( or ) linked at thenumber 1 position o the donor sugar ring. The at-

tached monosaccharide requently then becomes

a saccharide acceptor in 1 o 49 other glycosyl-

transerase reactions. This results in glycosidic

bonds with or congurations o the donor sac-

charide linked through position 1 or 2 to position

2, 3, 4, or 6 o an acceptor saccharide. Glycan

diversication is dictated by the combinatorial and

regulated application o this enzymatic potential.

This includes hyaluronan synthesis, which oc-

curs by copolymerization o two nucleotide sugar

donors. The ormation o iduronic acid by the

epimerization o glucuronic acid subsequent to

glycosylation is not depicted. Although some di-

saccharide sequences are ound on multiple types

o glycans, others are specic to one or ew glycan

types. Those potential mammalian disaccharidesequences that have not been observed in nature

are indicated (). N-glycosylation (*) is initiated by

transer en bloc o a presynthesized dolichol lipid-

linked oligosaccharide precursor. Ser/Thr, serine/

threonine; Asn, asparagine; hLys, hydroxylysine;

Trp, tryptophan; Tyr, tyrosine; Cer, ceramide; PI,

phosphatidylinositol.


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in vivo. Mechanisms altering the intracellular location o

glycosyltranserases and glycosidases can be an e ec-

tive means o regulating glycan ormation by controlling

access to acceptor substrates. Major changes in the gly-

come are induced by the loss o some chaperones and

multiprotein complexes that alter glycosyltranserase

tracking between the endoplasmic reticulum and Golgi

(Wu et al., 2004; Ju and Cummings, 2005; Foulquier et

al., 2006). A potential means by which glycosyltrans-

erases and glycosidases may be regulated is through

phosphorylation o their cytoplasmic tails, which might

modulate intermolecular interactions leading to dieren-

tial substrate access and intracellular tracking. There

is also evidence o competition among glycosyltrans-

erases in vivo or substrates in the secretory pathway,

which can modiy glycan ormation. Some glycosyl-

transerases that generate dierent saccharide linkages

have distinct specicities or nucleotide sugar donorsbut the same acceptor substrate specicity; whereas

others bear identical donor specicity but act on di-

erent acceptor substrates. Glycosyltranserases o the

ormer type can be mutually exclusive in the assembly

line o glycan ormation; whichever enzyme modies the

substrate rst can thereby redirect the synthetic path-

way and alter the structural outcome. This was observed

by the in vivo blockade o Core 2 GlcNAcT unction due

to endogenous expression o ST3Gal-I in T cells. Both

glycosyltranserases can act on the same acceptor sub-

strate, and loss o ST3Gal-I elevated Core 2 O-glycan

synthesis without a change in Core 2 GlcNAcT enzyme

activity (Priatel et al., 2000).Disengagement o glycosyltranserases and glyco-

sidases rom their membrane-anchored locations can

occur by proteolysis and would be expected to abolish

their activities in glycan ormation, although evidence o

this potential orm o glycan regulation currently awaits

urther experimentation. The enzymes o mammalian

glycosylation are predominantly type 2 transmembrane

glycoproteins that contain a luminal catalytic domain

linked to a luminal membrane-proximal stem domain.

Cleavage by secretory proteases within the stem

domain results in secretion o a catalytic domain rag-

ment. This ragment can be ound in most body fuids

and can be induced, or example, in response to infam-

mation (McCarey and Jamieson, 1993). The purpose o

this proteolysis is unknown, and the range o enzymes

aected is unclear. Although such glycosyltranserase

ragments retain enzymatic activity, and hence their abil-

ity to bind to available acceptor substrates, they are not

likely to be catalyzing glycan ormation among extracel-

lular compartments, as the concentration o nucleotide

sugar donors outside o the cells secretory pathway is

ar below enzyme substrate binding anities.

The hydrolysis o glycans on mammalian glycopro-

teins and glycolipids is associated with their degradation

in lysosomes. However, endogenous mechanisms that

cleave glycans at the cell surace may exist. Hydrolysis

o mammalian cell-surace glycans is in act a eature o

some pathogen inection strategies such as sialic acid

binding and cleavage by infuenza virus (Gagneux and

Varki, 1999; also see the Minireview by L. Comstock and

D. Kasper, page 847 o this issue). Proteolysis and tra-

cking to the cell surace would place mammalian glyco-

sidase enzymes in the region o the cell-surace glyco-

calyx, where some glycans might be hydrolyzed. At least

one mammalian glycosidase that cleaves sialic acids

rom glycans is a transmembrane protein that reaches

the plasma membrane (Wang et al., 2004). Although

examples o cell-surace glycoprotein alterations consis-

tent with removal o specic glycan linkages have been

describedsuch as the highly reproducible reduction in

some sialic acid linkages ollowing immune activation

o mammalian lymphocytesthis may be explained by

endocytosis and turnover in which newly synthesized

glycoproteins bear dierent glycans due to modulation

o glycosyltranserase or glycosidase unction.The biosynthesis and availability o nucleotide sugar

donor substrates can exert broad control over mam-

malian glycan ormation. Blockade o donor biosyn-

thesis or unctional loss o donor-specic transporters

normally residing the endoplasmic reticulum and Golgi

membranes can abolish cellular glycans that contain,

or example, ucose or sialic acid linkages (Lubke et

al., 2001; Smith et al., 2002; Schwarzkop et al., 2002).

In contrast, glucosamine supplementation to the hex-

osamine biosynthetic pathway can elevate synthesis

o some donor substrates and increase production o

various glycans in mammalian cells (Zachara and Hart,

2004; Lau et al., 2005). Precisely how this occurs mayrefect multiple actors including increased catalysis and

changes in gene expression. The impact o such aug-

mented glycosylation upon mammalian physiology is

not yet known, although this matter is worthy o careul

investigation. With a number o regulatory mechanisms

available, several and perhaps all o those discussed

above are involved in modulating mammalian glycan

expression.

Determinants of Mammalian Glycan Function

Few biological roles or mammalian glycosylation had

been established even a decade ago. The rapid pace o

discovery since then refects the application o genetic

tools and approaches to expand upon the existing oun-

dation o enzymatic, biochemical, and structural knowl-

edge. Glycosylation, like phosphorylation, produces

numerous structural modications, each o which may

be capable o signaling. Likewise, absence o a single

kinase or glycosyltranserase aects the modication

o multiple proteins and lipids. In studies o phosphory-

lation, this is commonly interpreted as disruption o a

signal transduction cascade. In glycosylation, the speci-

city o most glycosyltranserases and glycosidases or

substrates is dened by glycan structure instead o pro-

tein and lipid determinants. Thereore, single enzymes

can glycosylate multiple, seemingly unrelated, proteins

and lipids. How then does glycan ormation achieve a


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high level o specicity in cellular unction? The answer

may come rom combining knowledge o glycan syn-

thesis and regulation with the phenotypes observed in

intact organisms bearing deects in glycan ormation.

Cultured cells bearing various enzymatic deects in

the pathways o glycosylation typically lack signicant

phenotypes, yet a high degree o evolutionary conser-

vation is typical among mammalian glycosyltranserase

and glycosidase orthologs (Amado et al., 1999; Kikuchi

and Narimatsu, 2006). Glycosylation in the secretory

pathway positions most glycans at the cell surace andextracellular compartments where cell-cell communi-

cation is occurring among various cell types in intact

organisms. Such intercellular physiology is not typi-

cally replicated in immortalized cell-line monoculture

systems. GlcNAcT-1 glycosyltranserase deciency, or

example, which was well tolerated among cell lines in

vitro, resulted in severe embryonic deects with situs

inversus o heart loop ormation, aberrant vasculariza-

tion, and other morphogenic abnormalities in mouse

ontogeny, indicating the need to use intact organisms

to study mammalian glycosylation (Ioe and Stanley,

1994; Metzler et al., 1994). Since these ndings, doz-

ens o mouse lines have been created bearing germlinedeects in specic steps o the various glycosylation

pathways. Remarkably, most o these inherited glycan

deciencies result in discrete phenotypes that refect

the dysunction o specic cell types and diverse bio-

logical systems (Lowe and Marth, 2003).

Glycans possess distinct structural elements that

govern interactions with other molecules. Glycans can

promote or inhibit intra- and intermolecular binding that

includes both homotypic and heterotypic interactions

(Figure 3). Furthermore, mammalian glycans can be so

substantial in size and requency o attachment that they

contribute the majority o mass and charge comprising

some glycoproteins and glycolipids. For example, the

neural cell adhesion molecule NCAM has a uniquely

large negatively charged and devel-

opmentally regulated glycan structure

known as polysialic acid that inhib-

its homotypic NCAM protein-protein

binding (Homan and Edelman, 1983).

It has been shown that polysialic acid

on NCAM must be regulated in mouse

development or selective axonal tra-

cking, emotional and cognitive memory, and brain mor-

phogenesis. Comparative studies o phenotypes have

indicated that the polysialic acid glycan component is

required or the proper execution o almost all o the

physiological unctions attributed to NCAM (Angata et

al., 2004; Weinhold et al., 2006). Furthermore, mucins

represent an example o a class o glycoproteins bear-

ing a large number o O-glycan linkages that can induce

steric eects that extend the conormation o a peptide

backbone and may thereby serve to prominently dis-

play a large number o glycan decoys or pathogen lec-tin receptors in human resistance to oral and mucosal

inection (Tabak, 1995).

Glycosylation determines ligand abundance or

endogenous mammalian lectins. Lectins are glycan

binding proteins that are typically highly selective or

specic glycan structures and have thereore been

extremely useul in studying glycan variation (Goldstein,

2002; Sharon and Lis, 2004). An expanding number o

mammalian lectins have been identied and are classi-

ed by sequence motis such as those that dene the

C-type lectins, S-type lectins, P-type lectins, and the

Siglecs (Crocker and Varki, 2001; Drickamer and Taylor,

2003). It is likely that other lectin domains exist, as someproteins with apparent lectin activity do not contain

canonical glycan binding motis. Both lectin binding by

chaperones and steric eects o glycans contribute sub-

stantially to protein olding prior to tracking to the Golgi

and beyond by reducing protein aggregation and retain-

ing nascent unolded glycoproteins in the endoplasmic

reticulum (Parodi, 2000; Helenius and Aebi, 2004).

The interaction between glycans and lectins typically

occurs with lower anity than protein-protein interac-

tions but with signicant avidity given that most lectins

can bind multiple glycan moieties and do so with high

specicity. Glycans in the region o the glycocalyx can

reach millimolar concentrations, and, when bound by

endogenous lectin receptors, such interactions can be

Figure 3. Glyca Mdulae Mlecular

IeraciLectin binding and steric mechanisms involving

glycan structures in the control o protein-protein

interactions are depicted. Glycans can modulate

intramolecular and intermolecular binding com-prising both homotypic and heterotypic interac-

tions. The participation o protein conormation

and protein-protein binding in concert with lectin

binding is also denoted. Most o these interac-

tions depicted refect various degrees o experi-

mental support among the current literature.


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dicult to disrupt by lower concentrations o glycan

ligand mimetics. Moreover, some mammalian lectins at

the cell surace can appear ully occupied, or masked,

by binding to endogenous glycan ligands (Crocker and

Varki, 2001). Yet even in the presence o glycan ligands

residing on numerous glycoproteins, recent studieshave observed that endogenous lectin binding in situ

on primary cell suraces can be surprisingly selective

or ligands presented by distinct glycoproteins, implying

that protein sequence and conormation contribute to

mammalian lectin binding selectivity (Collins et al., 2004;

Han et al., 2005; Ohtsubo et al., 2005). This may be ur-

ther understood when glycoprotein ligands are visual-

ized at the atomic level. Technical limitations at present,

however, restrict acquisition o three-dimensional struc-

tures o glycoproteins to those produced by expression

systems that lack most mammalian glycans.

Regulation of Cellular Mechanisms by GlycansBy binding to lectins and sterically modulating molecu-

lar interactions, mammalian glycans participate in mul-

tiple cellular mechanisms that contribute to health and

disease. These basic structural paradigms have been

ound to regulate protein olding, cell adhesion, molec-

ular tracking and clearance, receptor activation, sig-

nal transduction, and endocytosis (Figure 4). Numer-

ous unctions o mammalian glycans are now evident,

and some may have arisen ear ly in the evolution o mul-

ticellular and vertebrate organisms.

Cell Adhesion

Lectin binding can evoke cell-cell adhesion and aggrega-

tion among primitive eukaryotic multicellular organisms

(such as sponges) and may have contributed to cell-

based kin recognition in the evolution

o the earliest metazoans. Mammalian

lectin involvement in cell-cell adhesion

is best characterized or the selectins

and their glycan ligands that include a

key ucose linkage on the sialyl-Lewis

X oligosaccharide (Lowe, 2003). This

cell adhesion system is highly regu-

lated on specic cell suraces including

the endothelium o the vasculature and

on most leukocytes, thereby contribut-

ing to leukocyte tracking responses

that are essential in immune-system

homeostasis, hematopoiesis, and infammation (Rosen,

2004). Glycosylation can also modulate cell-cell adhe-

sion in early mammalian embryos, and loss o some

glycans disrupts ertilization, by mechanisms that are

less well resolved. However, these results indicate

that other lectin-ligand binding interactions governingcell-cell adhesion likely exist (Surani, 1979; Akama et

al., 2002; Shur et al., 2004). When cellular portolios

o glycosyltranserases and glycosidases are altered

in embryogenesis and disease and act upon a dier-

ent assemblage o protein and lipid substrates, the

production o rare glycoprotein and glycolipid epitopes

can occur. Such unusual glycan-dependent epitopes

oten dene todays known oncoetal and stem cell

biomarkers that refect the various binding specicities

o dierent monoclonal antibodies. Glycan biomarkers

o biologic and pathogenic processes urther include

selectin-dependent cell adhesion that is associated in

some contexts with tumorigenic activity (Varki and Varki2001; Chen et al., 2005).

Sel/Nonsel Recognition

The ability o mammalian lectins to recognize glycans

rom divergent organisms such as bacteria, yeast, and

invertebrates underlies a mechanism o sel/nonsel rec-

ognition. This is exemplied by Toll-like receptor activa-

tion o the innate immune system rom binding to bacte-

rial glycan ligands (Bar ton and Medzhitov, 2003). Many

lectins are expressed on cells o the mammalian innate

immune system, and several bind to glycans specically

expressed among phylogenetically older organisms.

Perhaps deects in mammalian glycosylation can infu-

ence sel/nonsel recognition and in some contexts lead

to autoimmune disease. In this regard, the absence in

Figure 4. Cellular Mechaim f Glyca

FuciGlycans produced in the secretory pathway

participate in multiple mechanisms o cellular

regulation. The infuence o glycans on protein-

protein interactions encompasses a number ocellular unctions that span rom nascent protein

olding and intracellular tracking to roles in

extracellular compartments where cell-cell com-

munication is modulated by adhesion, molecular

and cellular homeostasis, receptor activation

and signal transduction, and endocytosis.


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actor receptors by mechanisms that also modulate

ligand binding and receptor dimerization (Miljan and

Bremer, 2002). More unusual and rare glycan linkages

are also regulatory. For example, Notch receptor tra-

cking, ligand binding, and activation is regulated by an

O-ucose linkage produced in the endoplasmic reticu-

lum and elaborated urther in the Golgi by the Fringe gly-

cosyltranserase (Haines and Irvine, 2003). In contrast,

it can be dicult to identiy the molecular constituents

involved when a genetic deect in glycan ormation alters

the glycosylation o multiple proteins and/or lipids. This

provides the impetus or developing glycoproteomics

and glycolipidomics technologies that can extend the

identication o the cellular proteome and lipidome to

more precisely characterize biologically relevant mol-

ecules (Dell and Morris, 2001).

The phenotypes associated with glycan linkage

deects have oten revealed the roles o glycans inimportant cellular processes that were not previously

thought to be regulated by protein glycosylation. Loss

o the GlcNAcT-V glycosyltranserase, or example, was

ound to induce coclustering o T cell receptors at the

cell surace, reducing the threshold or immune activa-

tion and causing autoimmune disease (Demetriou et al.,

2001). This was attributed to the loss o T cell receptor

binding to galectin-3, one o a amily o lectins implicated

in maintaining a spatially conned arrangement o cell-

surace glycoproteins (Morris et al., 2004). In addition,

receptors or EGF and TGF- on epithelial cells, the IgM B

cell antigen receptor (BCR), and the glucose transporter

2 glycoprotein on pancreatic cells are all prooundlymodulated by mammalian glycosylation, indicating that

dierent glycans produced in the Golgi maintain these

receptors at the cell surace by reducing their rates o

endocytosis (Partridge et al., 2004; Ohtsubo et al., 2005;

Collins et al., 2006; Grewal et al., 2006) .

Endocytosis

Endocytosis plays a critical role in cell biology by pro-

viding access to material rom extracellular compart-

ments, directing molecular cargo to distinct organelles,

terminating or modiying signals emanating rom the cell

surace, and inducing the turnover o cell-surace mol-

ecules. Recent studies have ound that mammalian gly-

cans produced in the Golgi modulate the endocytosis o

cell-surace glycoproteins and thereby control receptor

expression and hence thresholds or cell signaling. The

glycan linkage produced by GlcNAcT-V, or example,

retards EGF and TGF- receptor endocytosis, thereby

altering receptor activation and signaling among epi-

thelial carcinoma cells (Partridge et al., 2004). Dierent

cell types appear to use distinct glycans to alter rates o

endocytosis, and selectivity can urther exist among the

cell-surace glycoproteins o a given cell type. Pancre-

atic cells, but not hepatocytes, or example, appear

to use a lectin mechanism to decrease the rate o endo-

cytosis involving glucose transporter 2but not other

similarly glycosylated proteinsin preventing the onset

o type 2 diabetes (Ohtsubo et al., 2005).

Lectins and their ligands can modulate cell-surace

receptor activation coincident with the regulation o

receptor endocytosis. This is perhaps best character-

ized or CD22 (Siglec-2), a mammalian B cell-specic

lectin that modulates BCR activation and thereby alters

humoral immune responses. Like most Siglecs, CD22

contains both an extracellular lectin domain that binds

to specic sialic acid-bearing glycans and intracellular

protein sequence motis that can bind to intracellular

signal transduction proteins and regulate phosphory-

lation. The cytoplasmic domain o CD22 plays a nega-

tive regulatory role by recruiting the Shp-1 phosphatase

and thereby downmodulating immune signaling when

CD22 is associated with the BCR (Tedder et al., 1997).

Genetic deciency o the ST6Gal-I glycosyltranserase

in the mouse results in loss o CD22 Siglec ligands and

diminishes BCR activation and signaling. This correlated

with increased colocalization o BCRs with CD22, con-stitutive Shp-1 recruitment to CD22, decreased protein

phosphotyrosine levels, elevated BCR tracking to

clathrin microdomains, and enhanced endocytosis o

BCRs (Collins, et al., 2006; Grewal et al., 2006) . In mice

decient or both CD22 and ST6Gal-I, BCR signaling as

well as microdomain association and endocytotic rate

were restored to normal. Moreover, the reduced level

o humoral immunity in mice with ST6Gal-I deciency

urther prevented the development o autoimmune dis-

ease, unlike ndings in their normal counterparts that

bore lower levels o BCR-CD22 interactions. This lec-

tin-ligand system thereore modulates the threshold o

B cell immune activation in a mechanism linked to BCRtracking and endocytosis.

Diseases of Glycosylation

Endocytosis and tracking to lysosomes are typically

involved in degrading proteins and glycans. Deects in

these catabolic steps include glycosidase deciencies

that orm the bases or cellular storage disorders such

as Gauchers, Niemann-Pick type C, Sandhos, and

Tay-Sachs diseases. A subset o these maladies can

now be clinically treated using a small-molecule analog

o a plant-derived organic compound that reduces glu-

cosyltranserase I activity. This compound represents

the rst drug marketed or human disease therapy that

inhibits an endogenous mammalian glycosyltranserase

as a mechanism o action (Butters et al., 2005). Remark-

ably, unlike protein kinase inhibitors that compete with

the donor substrate ATP, glycosyltranserase inhibitors

generally do not compete with nucleotide sugar donor

substrates but are competitive with specic glycan

acceptor substrates.

Deects in the anabolic process o glycan ormation

are more typically considered as human diseases o gly-

cosylation. I cell disease was the rst to be identied and

was shown to result rom ailure to produce the mannose

6-phosphate modication on N-glycans in the Golgi. This

modication acts as a signal that is necessary or tra-

cking o hydrolases to the lysosome. Absence o this


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signal thereore also causes a storage disorder due to a

deciency in protein catabolism (Korneld and Sly, 1985).

The genetic basis o I cell disease encompasses muta-

tions in the gene encoding the GlcNAc-phosphotrans-

erase / subunit precursor (Kudo et al., 2006).

An increasing number o diseases o glycosylation

are being discovered, especially in the pediatric clinic

among children during the rst ew years o lie. The con-

genital disorders o glycosylation (CDGs) reer to a large

number o syndromes that include severe morphogenic

and metabolic deects associated with general ailure to

thrive, most o which have been linked to distinct steps

in glycan ormation (Jaeken and Carchon, 2004). Those

among the most prevalent grouping (CDG type 1a) are

due to hypomorphic mutations in thePMM2 gene. Muta-

tions in this gene diminish synthesis o the dolichol-oli-

gosaccharide precursor, which is essential or initiation

o N-glycosylation, and hence decrease the requencyo N-glycosylation. A small number o CDG subtypes

refect deects in the ormation o nucleotide sugar

donor substrates, and some can be treated by dietary

intake o precursor saccharides, as exemplied by man-

nose ingestion, which reverses signs o CDG type 1b

disease (Niehues et al., 1998). At present, at least 20

separate genes and more than 100 allelic variants have

been identied among the CDGs, many o which involve

hypomorphic mutations that impact on glycosyltrans-

erase and glycosidase enzyme activities, as well as

genetic deects in synthesis and transport o nucleotide

sugar donors (Aebi and Hennet, 2001; Freeze, 2006).

How many diseases o glycosylation exist in the humanpopulation, and what are their requencies o occur-

rence? The answers are not yet known. Their detection

is serendipitous due to inrequent clinical application o

serum transerrin isoelectric ocusing and other electro-

phoretic techniques to the analysis o specic glycopro-

teins. Although these are presently essential diagnostic

tools, they are nevertheless unable to detect most gly-

can linkage deects. Thus, human glycosylation disor-

ders identied so ar are primarily severe syndromes,

many o which refect the disruption o early steps in

the pathways o glycan biosynthesis. In contrast, mouse

models o deective glycosylation have been engineered

to ablate both early and late biosynthetic steps. Fromthese studies, it has become evident that the later the

deect in glycan synthesis, the less likely it is that a single

glycosyltranserase or glycosidase deciency will cause

a severe multisystemic disorder leading to dysmorphic

eatures, ailure to thrive, and lethality. The only interspe-

cies and biochemically comparable model documented

at present is loss o GlcNAcT-II glycosyltranserase activ-

ity, which is the basis or the human CDG-IIa syndrome.

Mice lacking GlcNAcT-II activity closely phenocopy

human CDG-IIa disease signs, and strain-associated

variations in disease severity were also observed (Wang

et al., 2001). These ndings suggest that animal models

may be useul in studying the molecular and pathogenic

bases or human diseases o glycosylation.

Connections between glycans and human disease

are now being made every year and have expanded to

include mild as well as severe syndromes, with timing o

onset that can span rom early neonatal to adult lie. The

deects and symptoms urther oten imply cell- and tis-

sue-specic dysunction. Human spondylocostal dysto-

sis has been recently linked to inactivation o the Lunatic

Fringe glycosyltranserase corresponding to a deect in

Notch signaling events essential in ontogenic patterning

o the axial skeleton during embryogenesis (Sparrow et

al., 2006). Humans lacking a unctional ST3Gal-V glyco-

syltranserase, also known as GM3 synthase, develop an

early neurological disorder termed inantile-onset symp-

tomatic epilepsy (Simpson et al., 2004). Mice lacking this

enzyme at rst appear unaected, but later, some exhibit

seizures as adults. Human hereditary multiple exostoses

is an autosomal-dominant bone disease characterized

by multiple cartilaginous tumors that occur throughoutchildhood and is caused by deects in the glycosyltrans-

erases required or synthesis o the heparan sulate gly-

cosaminoglycan (Duncan et al., 2001). Paroxysmal noc-

turnal hemoglobinuria usually occurs in adulthood and

results rom somatic mutation within the bone-marrow

stem cell population resulting in a deect in GPI anchor

synthesis (Bessler et al., 1994).

The participation o lectins and altered lectin binding

would also be expected to contribute to pathogenesis

among human diseases and refect the physiologic unc-

tions o glycosylation; however, ew examples currently

exist. This may imply that the roles o human lectins

overlap in vivo, a possibility consistent with the minimalphenotypic consequences o most lectin deciencies

thus ar induced and studied in the mouse. Alternatively,

some lectin mutations in humans may have a more

severe impact and disrupt embryogenesis, resulting in

early lethality. However, some human muscular dystro-

phies are due to mutations in laminin as well as in glyco-

syltranserases operating in the pathway o O-mannose

glycan ormation on -dystroglycan (McGowan and

Marinkovich, 2000; Yoshida et al., 2001; Michele et al.,

2002). These deects occur coincident with disruption o

-dystroglycan attachment to laminin in what resembles

a lectin binding mechanism.

Diseases that alter glycosylation have revealed newmechanisms regarding how glycan ormation is regu-

lated. Inherited deciencies in the conserved oligomeric

Golgi (COG) complex members COG1 and COG7 in

humans result in a severe childhood disease and peri-

natal death, respectively (Wu et al., 2004; Foulquier et

al., 2006). Cells rom these patients lack expression o

many dierent glycans, which may contribute to the

pathogenic basis o these syndromes. And the elusive

cause o human Tn syndrome, an adult hematologic dis-

order, was recently identied as a genetic mutation in the

COSMC gene encoding a protein with chaperone unc-

tion required or 1-3 galactosyltranserase activity in O-

glycan biosynthesis (Ju and Cummings, 2005). Human

diseases o glycosylation are not necessarily linked to


10/13


changes in glycosyltranserase and glycosidase activity.

Endogenous sporadic or germline modications o the

primary structure o single proteins may also contribute.

A recent analysis o glycoprotein mutations in the human

disease database revealed a larger-than-random occur-

rence o amino acid changes in proteins that predict a

gain-o-N-glycosylation, and those that were tested

indeed acquired an N-glycan concurrent with glycopro-

tein dysunction (Vogt et al., 2005).

Summary and Future Directions

Research on the unction o mammalian glycosylation

demonstrates that glycans are involved in multiple dis-

ciplines spanning ontogeny, immunology, neurobiol-

ogy, hematology, and metabolism and are linked to a

number o genetic diseases. It is increasingly appar-

ent that biological networks exploit both glycosylation

and phosphorylation by using cell-surace glycans toorganize plasma membrane receptors and control the

recruitment o intracellular signal transduction media-

tors. Hence, discoveries encompassing glycobiology

will contribute to the knowledge needed to decipher the

biological systems that comprise living organisms.

In some cases, glycans are the only source o variation

between otherwise identical glycoproteins produced

within the same cell, resulting in microheterogeneity.

Such heterogeneity and its regulation may play a role

in setting thresholds or molecular interactions that gov-

ern cellular responses. For example, cer tain glycoorms

representing IgG microheterogeneity are increased in

arthritis (Parekh et al., 1985). In addition, the anti-infam-matory property o IgG binding to Fc receptors is ele-

vated among the subset o total circulating IgGs that are

sialylated, compared to the raction o IgGs that have

ewer or no sialic acid linkages (Kaneko et al., 2006).

When glycosylation does occur, it may not always alter

the olding, interactions, unction, or ate o all proteins

similarly modied. The unction o an enzyme in modi-

ying many substrates does not dictate that all such

modications will maniest a biological purpose. This

possibility is conspicuous in recent analyses o mouse

models o glycan deciencies in which the phenotype

can be attributed to the modulation o single glycopro-

teins including CD22, glucose transporter 2, TGF-1receptor, and von Willebrand actor (Ellies et al., 2002;

Ohtsubo et al., 2005; Wang et al., 2005; Collins et al.,

2006; Grewal et al., 2006). Glycans attached to some

proteins and lipids may be physiologically inert. Such

nondeleterious glycosylation might impart subtle char-

acteristics to glycoproteins and glycolipids that become

advantageous in response to new selective pressures

applied by exogenous and pathogenic stimuli. In con-

trast, those endogenous glycans with essential physi-

ologic purpose would urther maintain the sequence

conservation evident among glycosyltranserase and

glycosidase gene orthologs.

Although it is not possible to predict a cells glycan

repertoire rom analyses o genomic DNA sequences,

it is clear that the highly ordered, regulated, and con-

served pathways o glycan diversication have evolved

to play specic biological roles, many o which are

essential and have persisted throughout mammalian

speciation. As biomedical connections to glycobiol-

ogy expand, therapeutic rationales or modulating and

detecting glycan production and variation continue to

emerge. Glycomics-based initiatives can assist by acili-

tating the development o techniques to produce com-

plex glycan structures, detect glycan interactions with

other molecules, and enable glycan-based methodolo-

gies to modulate cell processes (Schweizer and Hinds-

gaul, 1999; Seeberger and Werz, 2005; Paulson et al.,

2006). Research encompassing glycobiology is highly

interdisciplinary and increasingly successul in explain-

ing how extracellular signals originate and how cell-

cell communication is established among multicellular

organisms. The amount o the genome invested in gly-cosylation compared to what has been understood thus

ar implies that only a small raction o glycan unction

has been decoded. The integration o glycobiology into

mainstream education and research will urther prepare

the next generations o scientists to oversee the merg-

ing o now disparate biological disciplines into a cohe-

sive rendering o the molecular mechanisms that govern

physiology and disease.

ACknowLEDGMEnts

The authors apologize to colleagues and researchers who have made

important contributions that could not be incorporated in this review.

The authors thank S. Dowdy, P. Drckes, T. Hennet, R. Schnaar, and

P. Stanley or helpul comments. The authors are supported by the

Howard Hughes Medical Institute and research grants rom the NIH

(DK4247, HL57345, and GM62116). J.D.M. is a ounder o Abaron

Biosciences, Inc., a company that is developing drugs related to the

research described in this review. The University o Caliornia, San

Diego, is also an equity holder. The terms o this arrangement have

been reviewed and approved by the University o Caliornia, San Diego,

in accordance with its confict o interest policies.

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