complex n-glycan number and degree

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Complex N-Glycan Number and Degree

of Branching Cooperate to RegulateCell Proliferation and DifferentiationKen S. Lau,1,2 EmilyA. Partridge,1,3 Ani Grigorian,4 Cristina I. Silvescu,6 Vernon N. Reinhold,6

Michael Demetriou,4,5 and James W. Dennis1,3,*1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue R988, Toronto, ON M5G 1X5, Canada2Department of Biochemistry, University of Toronto, ON M5S 1A8, Canada3Departments of Molecular & Medical Genetics, Laboratory Medicine and Pathology, University of Toronto, ON M5G 1L5, Canada4Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697-4025, USA5Department of Neurology, University of California, Irvine, CA 92862-4280, USA6Chemistry, University of New Hampshire, Durham, NH 03824, USA

*Correspondence:[email protected]

DOI 10.1016/j.cell.2007.01.049

SUMMARY

The number of N-glycans (n) is a distinct feature

of each glycoprotein sequence and cooperates

with the physical properties of the Golgi N-gly-

can-branching pathway to regulate surface

glycoprotein levels. The Golgi pathway is ultra-

sensitive to hexosamine flux for the production

of tri- and tetra-antennary N-glycans, which

bind to galectins and form a molecular lattice

that opposes glycoprotein endocytosis. Glyco-proteins with few N-glycans (e.g., TbR, CTLA-4,

and GLUT4) exhibit enhanced cell-surface ex-

pression with switch-like responses to increas-

ing hexosamine concentration, whereas glyco-

proteins with high numbers of N-glycans (e.g.,

EGFR, IGFR, FGFR, and PDGFR) exhibit hyper-

bolic responses. Computational and experi-

mental data reveal that these features allow

nutrient flux stimulated by growth-promoting

high-n receptors to drive arrest/differentiation

programs by increasing surface levels of low-n

glycoproteins. We have identified a mechanismfor metabolic regulation of cellular transition

between growth and arrest in mammals arising

from apparent coevolution of N-glycan number

and branching.

INTRODUCTION

Developmental sequences in metazoan embryogenesis,

tissue repair, and adaptive immunity frequently involve

cell proliferation, followed by differentiation and cell-cycle

arrest. Although intracellular nutrient-sensing pathways,

such as SCH9 andRAS-cAMP, arelargely conserved from

S. cerevisiaeto mammals (Sobko, 2006), additional path-

ways that are sensitive to extracellular factors have

evolved in metazoans to establish more complex body

plans. In metazoans, extracellular growth factors bind re-

ceptor tyrosine kinases (RTKs) and activate PI3K/ERK sig-

naling, stimulating glucose metabolism and proliferation

as well as membrane remodeling (Di Paolo and De Camilli,

2006). Equally important in metazoans are the opposing

pathways that antagonize growth and are sensitive to

extracellular morphogens, cell adhesion, and, indirectly,

nutrient levels. In this regard, TGF-b receptor II (TbRII), cy-totoxic T-lymphocyte-associated antigen-4 (CTLA-4), and

glucose transporter-4 (GLUT4) are examples of glycopro-

teins with tyrosine and dileucine adaptor-binding motifs

that promote rapid constitutive endocytosis (Al-Hasani

et al., 2002; Bradshaw et al., 1997; Ehrlich et al., 2001). Re-

markably, surface levels of these glycoproteins increase

to restrict proliferation despite increased endocytosis

downstream of growth signaling. Here, we explore the

possibility that cell-surface levels of glycoproteins are

regulated in a conditional manner by metabolic flux to

N-glycan biosynthesis.

For most cell-surface receptors and transporters in

eukaryotes,70% of the Asn-X-Ser/Thr (X s Pro) motifs

exposed to the lumen of the endoplasmic reticulum are

cotranslationally N-glycosylated (Glc3Man9GlcNAc2 /

Asn; Apweiler et al., 1999). The N-glycans are trimmed

and remodeled upon transit through the Golgi, but, due

to inherent inefficiencies of the pathway enzymes, the

resulting structures on mature glycoproteins are hetero-

geneous. The activities of medial Golgi-branching N-

acetylglucosaminyltransferases I, II, IV, and V (encoded

by Mgat1, Mgat2, Mgat4a/b, and Mgat5 in mammals)

are dependent on the availability of the common donor

substrate UDP-N-acetylglucosamine (UDP-GlcNAc; Fig-

ure S1; Schachter, 1986). UDP-GlcNAc biosynthesis

through the hexosamine pathway utilizes substrates (fruc-

tose-6P, glutamine, acetyl-CoA, and UTP) that are key

Cell 129, 123134, April 6, 2007 2007 Elsevier Inc. 123
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metabolites in carbon, nitrogen, and energy homeostasis

(Broschat et al., 2002). Hence, the extent of GlcNAc

branching is dependent on branching enzyme kinetics,

and metabolic flux through the hexosamine pathway to

generate UDP-GlcNAc (A.G., unpublished data; Sasai

et al., 2002). Branched N-glycans are further modified in

the trans Golgi by b1,4galactosyltransferases, variably

elongated with poly N-acetyllactosamine (linear repeats

of Galb1,4GlcNAcb1,3) by b1,3N-acetylglucosaminyl-

transferases (iGNTs), and capped with sialic acid and

fucose. The Mgat5 intermediate (tetra-antennary b1,

6GlcNAc-branched) is preferentially elongated with poly

N-acetyllactosamine, producing N-glycans with higher af-

finities for galectins than less-branched structures (Hira-

bayashi et al., 2002). Thus, Mgat5/ cells display reduced

galectin-3 binding to complex N-glycans on the T cell re-

ceptor (TCR; Demetriou et al., 2001) as well as EGF recep-

tor (EGFR) and TbRII in epithelial cells (Partridge et al.,2004). Disruption of galectin-3 binding in Mgat5/ cells

or by lactose competition in Mgat5+/+ cells increases

TCR and EGFR mobility in the plane of the membrane

and, thus, movement into both caveoli and coated-pits

(Demetriou et al., 2001; Partridge et al., 2004; unpublished

data). As such, the lattice of interactions resulting from

galectin/glycoprotein crosslinking limits receptor internal-

ization and maintains downstream signaling sensitivity.

Although mapping binary interactions between galec-

tins and N-glycans is one approach to study the structural

complexity of the lattice, the dependency of multiple

glycoprotein receptors and transporters on the lattice

suggests that a systems approach may lead to a morethorough understanding of lattice-dependent regulation.

Moreover, the number of N-glycans (n, also defined as

multiplicity in this report) is highly variable for different gly-

coproteins and may interact with N-glycan branching to

regulate surface residency in a glycoprotein-specific man-

ner. To explore this possibility and its impact on respon-

siveness to extracellular cues, we considered a relatively

basic set of N-glycan-mediated interactions between

galectins and cell-surface glycoproteins downstream of

metabolic flux to the Golgi. We show that the N-glycan-

branching pathway cooperates with the number of N-gly-

cans on glycoproteins to differentially regulate their sur-

face levels in response to hexosamine flux. Our results

suggest that coevolution of N-glycan multiplicity of glyco-

proteins and hexosamine/N-glycan-branching kinetics

integrates nutrient metabolism with cellular transition be-

tween cell growth and arrest/differentiation in mammals.

RESULTS

N-Glycan Branching Is Ultrasensitive to Hexosamine

Galectins bind to complex N-glycans with affinities pro-

portional to N-acetyllactosamine content (Hirabayashi

et al., 2002). We reasoned that increasing the molar frac-

tions of triantennary N-glycans on glycoproteins produced

inMgat5/ tumor cells might be sufficient to rescue ga-

lectin binding and surface levels of EGFR and TbR. There-

fore, cells were cultured with GlcNAc, which is taken up by

bulk endocytosis, phosphorylated at the six position, and

converted to UDP-GlcNAc. UDP-GlcNAc (i.e., UDP-Hex-

NAc to UDP-Hex ratio) increased in a Michaelis-Menten

manner with GlcNAc supplementation to both Mgat5/

and Mgat5+/+ tumor cells (Figure 1A). The increase was

greater inMgat5/ cells, possibly due to reduced feed-

back inhibition by downstream high-affinity products.

GlcNAc enhanced surface TbR complexes in Mgat5/

and Mgat5+/+ cells and restored surface levels of EGFR

bound to galectin-3 in Mgat5/ cells (Figure 1B). Cell-

surface galectin-3 was reduced in Mgat5/ cells but

rescued by GlcNAc (Figure 1C).

To characterize the role of hexosamine and Golgi-

dependent regulation in growth and arrest signaling, we

considered the intermediary steps downstream of UDP-

GlcNAc, notably N-glycan branching, receptor retention

at the cell surface, and intracellular signaling (Figure 1D).The hexosamine-dependent changes in branching were

determined by mass spectrometry in Mgat5/ and

Mgat5+/+ cells (Figures 2A, 2B, and S2). Triantennary N-

glycan levelsdoubledin Mgat5/cellswhen supplemented

with GlcNAc, suggesting that less-branched N-glycans in

larger quantities are sufficient to restore galectin binding

and surface receptor levels (Figure 2A). In contrast,

GlcNAc supplementation ofMgat5+/+ tumor cells only pro-

duced small increases in tri- plus tetra-antennary N-gly-

cans (Figure 2B). Oncogenic transformation increases

gene expression ofMgat4,Mgat5, and iGNT by 5- to 10-

fold (Buckhaults et al., 1997; Ishidaet al., 2005; Takamatsu

et al., 1999), which appears to reduce the requirement forhexosamine supplementation by supporting higher levels

of tri- and tetra-antennary N-glycan biosynthesis in

Mgat5+/+ tumor cells. Interestingly, we observed that the

triantennary N-glycan fraction, the Golgi-pathway end

product in Mgat5/ cells, increased with an ultrasensitive

response to GlcNAc (Hill coefficient nH5.5;Figure 2A).

Ultrasensitivity describesa stimulus/response relationship

where the response rises sharply over a narrow range

of stimulus, producing a sigmoid-shaped curve with a

delay preceding the rise in response and a Hill coefficient

(nH) [ 1 (Koshland et al., 1982). The more common

Michaelis-Menten relationship describesa responsecurve

resembling a hyperbolic function (nH1), which is typical

of a simple one-step substrate-product relationship.

Ultrasensitive responses are switch-like (all-or-none) re-

sponses that have been observed in many biological

systems (Ferrell and Machleder, 1998). For example, non-

ordered phosphorylation of yeast SIC1 increases progres-

sively with advancing cell cycle, and, at more than five of

nine sites occupied, SIC1 affinity for CDC4 increases in

a switch-like manner (Klein et al., 2003).

Computational Model of Golgi Ultrasensitivity

To better understand branching ultrasensitivity to UDP-

GlcNAc, we constructed a computational model of ele-

mentary processes using ordinary differential equations

(ODEs) with enzyme concentrations and kinetic

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parameters fromliterature values (TableS1; Krambeckand

Betenbaugh, 2005; Umana and Bailey, 1997). N-glycan

structures aregenerated up to a maximum size of tetra-an-tennary with N-acetyllactosamine and (Galb1-4GlcNAcb1-

3)1-2 extensions fora total of 14 structures (TableS2; struc-

tures). Substitutions of the antennae with sialic acid,

fucose, and sulfate can alter the affinity for galectins, but

we did not include these modifications in order to empha-

size the effects of N-glycan branching. The GlcNAc-

branching enzymes have well-characterized specificities

foracceptors (Schachter, 1986), and, therefore, the medial

Golgi pathway is modeled as a distributive series of reac-

tionsand not physicallyorderedin the Golgicompartments

(Figure S3A). In Mgat5/ cells, simulations of N-glycan

profiles as functions of increasing UDP-GlcNAc con-

centrations revealed that triantennary N-glycans indeed

increased in a switch-like fashion (nH5.6;Figure 2C).We next investigated the source of branching ultrasen-

sitivity by computationally perturbing the Mgat5/model.

Ultrasensitivity appears to be distributed over multiple re-

actions and therefore is a robust property of the pathway

(Table S3). However, in simulations where GlcNAc-

branching enzymes are removed sequentially, ultrasensi-

tivity is stepwise diminished, demonstrating the necessity

of multistep usage of UDP-GlcNAc (Table S2). Multistep

ultrasensitivity in a biochemical pathway, unlike allosteric

sensitivity, occurs with repetitive use of either enzymes or

substrates in sequential reactions that decline in efficiency

(Ferrell and Machleder, 1998). In this regard, affinities for

UDP-GlcNAc and enzyme concentrations decrease step-

wise fromMgat1 to Mgat5(Table S1). Furthermore, simu-

lation of branching as a linear pathway with no removal

of intermediate products (i.e., transit out of the Golgi) re-sults in a hyperbolic increase of end product in response

to UDP-GlcNAc (Figure S3B). These results identified the

two conditions required for branching ultrasensitivity: (1)

sequential decreasing efficiency of Golgi enzyme reac-

tions and (2) removal of intermediate products. In keeping

with (1), simulation of a 15-fold increase of late Golgi en-

zymes Mgat4, Mgat5, and iGNT) characteristic of trans-

formed cells (Buckhaults et al., 1997; Ishida et al., 2005;

Takamatsu et al., 1999), displayed response profiles with

unchanging tri- and tetra-antennary N-glycans due to

a pathway D50 below the physiological concentration of

1.5 mM UDP-GlcNAc, similar to that observed experimen-

tally forMgat5+/+ tumor cells (Figure 2D).

N-Glycan Multiplicity Cooperates with Branching

to Regulate Glycoproteins

Next, we confirmed that hexosamine enhancement of

surface receptors and signaling is dependent on complex

N-glycans. Wild-type CHO cells and the Mgat1 mutant

Lec1 cells were compared for changes in b1,6GlcNAc-

branched N-glycans (which bind L-PHA, P. vulgaris

leukoagglutinating lectin) and responsiveness to TGF-b

following GlcNAc supplementation. Lec1 CHO cells are

deficient inMgat1 activity, blocking all GlcNAc-branching

reactions (Kumar and Stanley, 1989), and GlcNAc supple-

mentation to these cells did not increase L-PHA reactivity

nor enhance responsiveness to TGF-b (Figures 3A and

Figure 1. Hexosamine Rescues Surface

Galectin-3 and EGF/TGF-b Surface-

Receptor Levels in Mgat5/ Cells

(A) shows UDP-HexNAc to UDP-Hex ratio

[UDP-GlcNAc + UDP-GalNAc] / [UDP-Gal +

UDP-Glc] in Mgat5+/+ and Mgat5/ tumorcells

after48 hr ofculture with GlcNAcsupplementa-

tion. Normalized mean SE (standard error of

the mean) of two independent experiments is

shown.

(B) Galectin-3 association is shown with EGFR

determined by 125I-EGF crosslinkingto cell sur-

face EGFR and immunoprecipitated with anti-

bodies to galectin-3 with and without 50 mM

GlcNAc. 125I-TGF-b1 bound to surface TbR in

cells.

(C) FACS analysis of surface galectin-3 in

Mgat5+/+ and Mgat5/ cells supplemented

with indicated amounts of GlcNAc is shown.

Mean SE.

(D) is a schematic of subsystems consideredin the rest of this report.



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S4A). In contrast, GlcNAc enhanced both end points with

switch-like responses in wild-type CHO cells (nH = 4.6

6.0). Without supplementation, L-PHA binding was 2-

fold higher in Mgat5+/+ tumor cells than CHO cells, consis-

tent with PyMT oncogene-dependent increases in the

late-branchingMgat4/5 enzymes.

With the multistep structure of the branching pathway,

a gain inMgat1 activity is predicted to limit the availability

of UDP-GlcNAc to late enzymes, thus replacing high-affin-

ity tri- and tetra-antennary N-glycans with low-affinity

monoantennary structures (Figure S3B;Table S2). Over-

expression (253) ofMgat1 inMgat5/ cells suppressed

GlcNAc-induced EGFR (Figure 3B). Simulations sup-

ported the above result (Figure S5A) and also confirmed

that EGFR suppression by Mgat1 overexpression is de-

pendent on both declining efficiency of the pathway and

on intermediate product removal with Golgi trafficking;

these are the key properties required for ultrasensitivity

(Figures S5C and S3B). Indeed, overexpression of

Mgat1 inMgat5+/+ tumor cells did not inhibit GlcNAc-de-

pendent increases in EGFR signaling, as condition (1) for

ultrasensitivity was not met. (Figure S5D).

Next, we explored the effect of hexosamine-dependent

N-glycan changes on the surface levels of EGFR and TbR.

Surface TbR increased with the expected switch-like re-

sponse (nH4.4) to GlcNAc, reflecting the Golgi-pathway

response, but, surprisingly, EGFR increased in a linear

manner (Figures 3C, 3D, andS4B). GlcNAc also restored

responsiveness to EGF and TGF-b in signaling assays

with hyperbolic (nH 1.2) and switch-like (nH 5.4) re-

sponse curves, respectively (Figures 3E, 3F, S4C, and

S4D). Downstream intracellular signaling does not appear

to be a confounding factor as both ERK and SMAD activa-

tion increase hyperbolically with increasing surface recep-

tor when stimulated by excess ligand, based on computa-

tional models (Figures S4E and S4F; Schoeberl et al.,

2002; Vilar et al., 2006) and experimental data (Wiley

et al., 2003). Therefore, we conclude that branching ultra-

sensitivity to UDP-GlcNAc is a regulatory feature of the

pathway that interacts with glycoprotein-specific informa-

tion to differentially regulate its surface levels. In this

regard, the number of N-glycosylation sites (n) is a distinct

feature defined by the protein sequence that determines

avidity for the galectin lattice. Murine TbRI and TbRII

have one and two N-glycans, respectively (Koli and Ar-

teaga, 1997), while EGFR has eight occupied N-X-S/T

sites (Zhen et al., 2003).

Computational Model of Glycoprotein Regulation

by N-Glycan Multiplicity and Branching

To explore the potential for N-glycan multiplicity and hex-

osamine/N-glycan branching to differentially regulate

Figure 2. Hexosamine Flux Stimulates

N-Glycan Branching

Distribution of branched N-glycans in (A)

Mgat5/ and (B) Mgat5+/+ cells is shown.

Branched N-glycan structures from mass

spectrometry analysis are pooled as in

Figure S2, and branching is indicated as 0 to

4+ with variable extensions as in the cartoon.

(C) Simulation of steady-state N-glycans in

Mgat5/ and (D) Mgat5+/+ cells is shown as

a function of GolgiUDP-GlcNAcconcentration.

The gray box represents UDP-GlcNAc below

the estimated physiological D0 of 1.5 mM,

from where the Hill coefficient is calculated.

The larger 4+ structures are underrepresented

relative to smaller structures in the MALDI pro-

files, but the slopes of the response curves to

UDP-GlcNAc are consistent with computa-

tional simulations.



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surface glycoprotein levels, we extended our mathemati-

cal model to calculate the fractional changes in surface

glycoproteins under varying degrees of N-glycan multi-

plicity and Golgi enzyme activities. A glycoform is defined

here as a protein isoform with a distinct combination of N-

glycan structures distributed randomly over the possible

N-X-S/T sites of the glycoproteins extracellular domain.

The protein environment of N-X-S/T sites can limit the

accessibility to Golgi enzymes (Do et al., 1994), but we

assume that site-specific bias is not a significant determi-

nant of glycoform generation. The number of glycoforms

follows a combinatorial relationship with respect to the

number of N-glycans on a glycoprotein and the number of

N-glycan types. Although this number is actually much

larger, the 14 N-glycan types used in the model are suffi-

cient to illustrate the concepts.

The steady-state distribution of glycoforms at the cell

surface is calculated based on the binding avidities of gly-

coforms for galectin-3 (Ahmad et al., 2004), a prototypic

galectin that is not limiting in this model. Avidity for the lat-

ticeis modeled to beproportional tothe sum of binding af-

finities of the N-glycans on each glycoform (SKa). The af-

finity threshold for stable association of a glycoform with

the galectin lattice corresponds to a single tri-, two bi- or

6 monoantennary N-glycans (SKa> 1.28mM1) and cor-

responds to in vitro galectin-binding data (Hirabayashi

et al., 2002). Glycoforms are retained on the cell surface

with half-lives proportional to their avidities for the lattice

and are countered by constitutive endocytosis, which is

generally held constant in our simulations to illustrate the

effects of N-glycan-dependent surface interactions.

Surface retention of glycoproteins by the lattice with a

single N-glycan follows the switch-like response of tri-

and tetra-antennary N-glycan production. However, with

larger values of n, the number of glycoforms and their

mean avidity for the lattice increase, which essentially

suppresses the lag phase of the sigmoidal response

(Figure 4A). Moreover, the larger glycoform distributions

traverse the affinity threshold in response to increasing

UDP-GlcNAc in a graded manner, and, importantly, at

lowerUDP-GlcNAc concentrations (Figures S6A and S6B).

Thus, the slopes for high-n glycoprotein responses are

steeper at low hexosamine flux (1.5 mM UDP-GlcNAc)

than at high flux (>3 mM), while the trend is reversed for

Figure 3. Downstream Signaling Re-

flects GlcNAc Dose Responses of Sur-

face EGFR and TbR Receptors

(A) L-PHA staining (binds b1,6GlcNAc-

branched N-glycans) of Mgat5+/+, Mgat5/,

CHO, and Lec1 (Mgat1/CHO) cells supple-

mented 48 hr with GlcNAc is shown. Mean

SE.

(B) shows overexpression of Mgat1 in

Mgat5/ cells supplemented with GlcNAc

and acutely stimulated with EGF (5 min). Infec-

tion with the Mgat1 vector and transient ex-

pression for 48 hr increased Mgat1 activity

by25-fold, while pools of stably transfected

cells displayed an 2.5-fold increase in

Mgat1 activity. Normalized mean SE is of

minimum of six replicates from two to seven

independent experiments.

Surface (C) EGFR and (D) TbR in Mgat5+/+ and

Mgat5/ tumor cells quantified by FITC-EGF

binding and 1 25I-TGF-b1 binding, respectively.(E) ERK-p nuclear translocation measured 5

minafterstimulation with EGF(acute) or vehicle

(autocrine).

(F) SMAD2/3 nuclear translocation measured

45 min after stimulation with TGF-b1 or vehicle.

Normalized mean SE of three independent

experiments with at least 200 cells each is

shown.



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low-n glycoproteins (Figure 4A).Mgat1 overexpression at

high levels (253) suppresses hexosamine-dependent up-

regulation of EGFR signaling, suggesting that replace-

ment of all high-affinity N-glycans with monoantennaryN-glycans puts glycoforms near the threshold for lattice

association, which is functionally similar to removing N-

glycans. Thus, moderate Mgat1 overexpression (2.53)

changes the GlcNAc response curve from hyperbolic to

sigmoidal, characteristic of a low-n glycoprotein (Fig-

ure 3B).

Examples of Other Glycoproteins with Low and High

N-Glycan Multiplicities

The glucose transporter GLUT4 has one N-glycan, and

a dileucine motif mediates its constitutive endocytosis

(Al-Hasani et al., 2002). Intracellular stores of GLUT4 in

muscle and adipocytes arerecruited into the recycling en-

dosomes by insulin receptor activation and to the cell sur-

face in a poorly defined manner. If GLUT4 retention at the

surface is dependent on hexosamine/N-glycan branching

and galectin binding, its upregulation by GlcNAc is ex-

pected to display a high degreeof ultrasensitivity. Accord-

ingly, GlcNAc supplementation to HEK293T cells express-

ing myc-GLUT4-GFP enhanced cell-surface GLUT4 levels

with an expected switch-like response (nH 10.0; Figures

4B andS7). Moreover, mutation of the single N-X-S/T site

(N 57 Q) blocked GlcNAc-dependent enhancement of sur-

face GLUT4 (Figure 4B). In contrast, receptors of FGF,

IGF, and PDGF possess high numbers of N-glycans (8

16), and GlcNAc increased responsiveness to their cog-

nate ligands hyperbolically (Figure 4C).

N-Glycan Multiplicity and Receptor Kinase Function

Our results suggest that hexosamine/N-glycan branching

may differentially regulate EGFR/PI3K/ERK and TGF-b/

SMAD signaling, which are antagonistic for G1/S transi-tion (Matsuura et al., 2004; Siegel and Massague, 2003).

If more broadly applicable, this observation might be re-

flected in the N-glycan multiplicity of different functional

classes of receptors. To explore this possibility in a rela-

tively well-studied set of human receptors, we ranked

the receptor kinases by the incidence of canonical N-gly-

cosylation motifs (N-X-S/T not followed by P and X not P)

in their extracellular domains. The number of N-X-S/T in

mammalian receptor kinases varies considerably, but re-

ceptors that stimulate cell proliferation, growth, and onco-

genesis have more N-X-S/T sites, longer extracellular do-

mains, and an increased number of sites per 100 amino

acids (30 receptors designated RK1; 11.33 5.05 sites/re-

ceptor; 1.89 0.58 sites /100 amino acids; Figures 4D and

S10A; Table S4). In contrast, receptors known for their

roles in vasculature formation and organogenesis, such

as TIE1, MUSK, LTK, ROR1/2, DDR1, the EPH receptors,

and TGF-b/BMP receptors, have lower N-glycan multi-

plicities (27 receptors designated RK2; 2.48 1.28 sites/

receptor; 0.86 0.54 sites/100 amino acids; Figure 4D).

The occurrence of N-X-S/T sites per 100 amino acids in

a random selection of human cell-surface glycoproteins

is 1.21 0.89, which is greater than 0.60, a value calcu-

lated based solely on the amino acid composition of the

extracellular domains in this set (Figure S10A). The actual

frequency may in part reflect the conservation of some

N-X-S/T sites, where their substitution in the ER is critical

Figure 4. N-Glycan Branching Cooper-

ates with N-Glycan Multiplicity

(A) Simulation of surface glycoprotein fractions

in Mgat5/ cells as a function of Golgi UDP-

GlcNAc concentration and N-glycan multiplic-

ity (n) is shown.

(B) Change in surface expression of myc-

GLUT4-GFP (wild-type, n = 1; mutant, n = 0)

in HEK293T cells supplemented with GlcNAc

for 48 hr and imaged with confocal microscopy

is shown. Normalized mean SE surface/inte-

rior GLUT4 from three independent experi-

ments of transfected cells from 10 random

fields is shown for each GlcNAc concentration.

(C) shows dose responses of ERK activation in

Mgat5/ tumor cells to IGF, PDGF, FGF, or

EGF as a function of GlcNAc supplementation.

Mean SE.

(D)Scatterplotof N-X-S/T multiplicityas a func-

tion of extracellular domain length for mamma-

lian receptor kinases is shown. RK1 are recep-tor kinases with growth promoting activities

(closed, 11.3 5.1 sites/receptor), and RK2

are those with arrest/morphogenic activities

(open, 2.5 1.3 sites/receptor).



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for protein folding. However, where it has been examined

(such as the insulin receptor), most sites are not required

for protein folding or activity (Elleman et al., 2000). These

observations suggest that differences in N-glycan multi-

plicity of receptorkinases are conservedalong with growth

and differentiation functions in mammals and are determi-

nants of regulation via hexosamine/N-glycan branching.

Hexosamine/N-Glycan Branching Balances

Responsiveness to Growth and Arrest Cues

Epithelial-to-mesenchymal transition (EMT) requires ERK

and PI3K activation downstream of RTKs as well as auto-

crine TGF-bsignaling at intermediate levels that are con-

duciveto theinduction of a mesenchymal program of gene

expression but below the threshold for SMAD2/3-depen-

dent cell-cycle arrest (Oft et al., 2002). Nontransformed

NMuMG cells respond to TGF-b and undergo EMT, which

is also observed during embryogenesis and carcinoma

progression. Therefore, provided that the N-glycan-

branching pathway is ultrasensitive to UDP-GlcNAc in

NMuMG cells, we anticipated that hexosamine flux

through the Golgi might enhance responsiveness to growth

cues first, then TGF-b leading to EMT and growth arrest.

GlcNAc supplementation to NMuMG cells increased UDP-

GlcNAc concentration (nH 1.3), b1,6GlcNAc-branched

N-glycans (nH 6.0), and responsiveness to acute TGF-

b/SMAD (nH 6.0) and to FCS/ERK (nH 1.3), all with

the expected response behaviors (Figures 5A and 5B).

Mgat1 overexpression in NMuMG cells also suppressed

GlcNAc-dependent signaling, confirming the branching

pathway to be multistep ultrasensitive (Figures S8AS8D).

In addition to acute signaling, hexosamine also

alters cellular responsiveness to autocrine stimulation in

NMuMG cells. In normal growth conditions, the ratio of

activated ERK/SMAD increased and then decreased in

response to hexosamine flux, mirroring changes in cell-

proliferation rates (Figure 5C). Accompanying growth

arrest, GlcNAcinduced loss of E-cadherin and themesen-

chymal morphology (Figure 5D). TGF-b/SMAD signaling in

Mgat5/ tumor cells was ultrasensitive to GlcNAc, which

also induced EMT with the expected response as auto-

crine TGF-b stimulation (Figures S8E and S8F). EMT was

Figure 5. N-Glycan Branching and

Multiplicity Impose Growth-to-Arrest

Sequence

(A) L-PHAstaining,UDP-GlcNAc, and UDP-Gal

in NMuMG cell lysates following GlcNAc sup-

plementation are shown.

(B) SMAD2/3 and ERK-p nuclear translocation

in NMuMG cells preconditioned in GlcNAc for

120 hr and stimulated with either vehicle (auto-

crine) or 3 ng/ml of TGF-bor 5% FBS, respec-

tively, are shown. Mean SE.

(C) ERK-p-to-SMAD2/3 activation and cell

density of NMuMG cells in GlcNAc-supple-

mented medium (cell viability >95% through-

out). Normalized mean SE of three indepen-

dent experiments.

(D) E-cadherin and F-actin staining of NMuMG

cells with or without GlcNAc supplementation

is shown.

(E) shows simulation of the relative surface

levels of glycoprotein receptor pairs (X and Y)as a function of N-glycan multiplicity and Golgi

UDP-GlcNAc concentration.

(F) Simulation of the dynamic range of change

in surface glycoproteins as a function of endo-

cytosis rate with an increase of Golgi UDP-

GlcNAc from 1.5 to 6 mM is shown.



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reversible upon GlcNAc withdrawal in NMuMG cells and

Mgat5/ tumor cells. In contrast, pathologic overexpres-

sion of late-branching enzymes in PyMT Mgat5+/+ tumor

cells supplies high-affinity galectin ligands at lower UDP-

GlcNAc concentrations, rendering proliferation and EMT

independent of hexosamine (Figure S8E).

Computational simulations show that ratios of surface

receptors taken in pairs change in proportion to the

difference in n. Glycoproteins with high multiplicities (nu-

merator) increase at the surface early in the UDP-GlcNAc

titration, while branching ultrasensitivity creates a delay

before low-n glycoproteins (denominator) accumulate

(Figure 5E). Simulations where endocytosis rates are var-

ied indicate that low-n receptors are markedly more sen-

sitive to removal from the surface due to inherently lower

avidities for the lattice (Figure 5F). Therefore, conditional

regulation of branching by nutrient flux through the hexos-

amine pathway stimulates growth first and at higher fluxarrest and differentiation.

Regulation of T Cell Growth Arrest

by Hexosamine/N-Glycan Branching and CTLA-4

Next, we investigated whether hexosamine/Golgi ultra-

sensitivity regulates growth arrest mechanisms in other

cellular systems. Naive T cells from Mgat5/ mice have

reduced thresholds for activation due to the lower affinity

of TCR for the galectin lattice, which enhances antigen-

driven TCR clustering and signaling at the immune syn-

apse(Demetriou et al., 2001). Once activated, naive T cells

undergo multiple rounds of division followed by growth ar-

rest and differentiation into effector T cells. Growth arrestof blasting T cells is dependent on induction of cell-sur-

face CTLA-4, a glycoprotein with low N-glycan multiplicity

and high constitutive endocytosis (Alegre et al., 2001).

CD28 and CTLA-4 are sequence-related receptors that

compete for CD80/86 ligand on antigen-presenting cells

to positively and negatively regulate T cell proliferation, re-

spectively (Alegre et al., 2001). CD28 is constitutively ex-

pressed at the cell surface and has five N-glycan sites

(3.8/100 amino acids), while surface CTLA-4 is induced

35 days after TCR signaling and has only two sites (1.6/

100 amino acids).

Surface CTLA-4 was lower onMgat5/ andMgat5+/

than Mgat5+/+ T cells following anti-CD3 stimulation but

reached comparable levels with strong costimulation by

anti-CD3 and CD28 antibodies (Figure S9A). Tracking T

cells by the number of divisions revealed that the propor-

tion of cells expressing surface CTLA-4, as well as their

associated surface levels (i.e., MFI), is always lower in

Mgat5/ T cells following low, but not high, levels of TCR

stimulation (Figure 6A). In contrast, surface CD28 levels

are similar in the two genotypes (Figure S9B). CTLA-4

gene expression increases in proportion to TCR signaling

strength (Chikuma and Bluestone, 2002), and as ex-

pected, TCR hypersensitivity of Mgat5/ T cells is

associated with increased intracellular CTLA-4 following

activation (Figure 6B), consistent with a specific defect

in surface retention. CTLA-4 surface expression was

reduced by lactose treatment, a competitive inhibitor of

galectin binding, but not by control disaccharide sucrose

(Figure S9C). Increased membrane remodeling with T

cell activation presumably increases the requirement for

lattice-dependent retention of CTLA-4. Consistent with

this notion, constitutive CTLA-4 expression in resting

CD4+FOXP3+ regulatory T cells is not different between

Mgat5+/+ andMgat5/ cells (Figure S9E).

GlcNAc enhanced surface CTLA-4 in Mgat5+/+ more

than in Mgat5/ T cells (Figure 6C), and, as predicted

by our model, induced CTLA-4 surface expression with a

switch-like response (nH 6.4), while the UDP-HexNAc/

UDP-Hex ratio increased in a linear manner to 3-fold

(Figure 6D). Swainsonine, an inhibitor of Golgi a-mannosi-

dase II, suppressed high-affinity galectin ligands and

blocked GlcNAc-induced surface CTLA-4 (Figure S9D).

TCR signaling induces large increases in glucose uptake

(Frauwirth et al., 2002) and Mgat5 gene expression(Morgan et al., 2004) as well as UDP-GlcNAc, surface

b1,6GlcNAc-branched N-glycans, and, finally, CTLA-4

(Figure 6E). We conclude that TCR signaling increases

metabolite flux through the hexosamine and N-glycan-

branching pathways, upregulating surface CTLA-4 with a

delay that is dependent on Golgi multistep ultrasensitivity.

DISCUSSION

Branching-Pathway Ultrasensitivity and N-Glycan

Multiplicity

Our results demonstrate multistep ultrasensitivity of

N-glycan branching downstream of hexosamine flux tobe a structural and regulatory feature of the pathway. Re-

markably, ultrasensitivity of N-glycan branching appears

to be transformed inversely by N-glycan multiplicity (n),

an encoded feature of glycoprotein sequences. Receptors

engineered to have different numbers of N-glycan sites

should conform to this model provided that the alterations

do not disrupt other aspects of receptor function. Without

exception, the hexosamine response profiles for the

glycoproteins tested herein with high (EGFR, PDGF,

IGFR, and FGF) and low (TbR, CTLA-4, and GLUT4) multi-

plicities are consistent with a model where high-n glyco-

proteins are associated with growth and low-n with arrest/

differentiation. Although not a signaling receptor, GLUT4

accelerates the systemic disposal of glucose (Tsao

et al., 1996), thus reducing hexosamine flux and maintain-

ing signaling homeostasis. Indeed, failure to reduce

glucose associated with type II diabetes induces matrix

deposition in glomerular mesangial cells in a TGF-b-

dependent manner (Kolm-Litty et al., 1998). Taken together,

our results indicate that differences in N-glycan multi-

plicities cooperate with Golgi multistep ultrasensitivity

to enhance differential regulation of surface glycopro-

teins downstream of metabolic flux into the hexosamine

pathway.

In addition to N-glycans, UDP-GlcNAc is utilized in

mucin, glycolipid, and hyaluronate biosynthesis and in

O-GlcNAc modifications to cytosolic proteins on Ser/Thr



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(Slawson et al., 2006). However, suppression of GlcNAc-

dependent surface-glycoprotein regulation by mutation

of the N-X-S/T site in GLUT4 and by knockout (Lec1) or

overexpression ofMgat1 confirms the involvement of N-

glycan branching. Many details of N-glycan-lectin interac-

tions remain to be defined, such as metabolic and genetic

factors regulating Golgi sugar-nucleotide pools, further

substitutions to the antennae of N-glycans, and binding

at the cell surface by other lectins. For example, cell-sur-

face retention of B cell receptors is positively regulated by

sialoadhesin CD22, which binds a2,6sialic acid found on

the termini of lactosamine antennae (Collins et al., 2006).

Regulation of Cell Growth and Differentiation

by Fine-Tuning Ultrasensitivity

Signalingdownstream of RTKs hasoutputs that differin ul-

trasensitivity as required to control cell polarity versus the

more switch-like cell-cycle transitions. The RAF-MEK-

ERK cascade is ultrasensitive as previously demonstrated

by computational modeling and in experiments using

Xenopus oocyte, where direct activation with progester-

one triggers maturation (Ferrell and Machleder, 1998).

EGF-EGFR binding stimulates tyrosine-phosphorylation

of AP-2, EPS15, CBL-2, clathrin, and other components

of coated-pit endocytosis and promotes rapid receptor in-

activation upstream of the RAF-MEK-ERK cascade (van

Delft et al., 1997). Consequently, the membrane-proximal

portion of the EGF-signaling pathway from EGFR to RAS/

PI3K is subsensitive (slope0.1; see Supplemental Data),

presumablymeeting temporal-spatial requirements for the

exploratory nature of membrane remodeling (Wang et al.,

2002). This effectively suppresses RAF-MEK-ERK ultra-

sensitivity (nH 10), resulting in a near hyperbolic re-

sponse for EGFR to ERK. With similar considerations, high

N-glycan multiplicities of receptors produce much larger

glycoform distributions and, thus, a gradation of avidities for

the lattice that effectively suppresses Golgi ultrasensitivity

at the level of glycoprotein recruitment to the cell surface.

Importantly, higher N-glycan multiplicities increase glyco-

protein avidities for the lattice at lower hexosamine levels,

Figure 6. Hexosamine and N-Glycan

Branching Promote CTLA-4 Cell-Surface

Expression

(A) Surface CTLA-4+ (%) and MFI in Mgat5+/+

and Mgat5/ T cells stimulated for 5 days

are shown, with cell division measured by

CFSE staining.

(B) Total CTLA-4 in Mgat5+/+ and Mgat5/ T

cells is shown after 4 days of stimulation with

250 ng/ml anti-CD3 antibody.

(C)SurfaceCTLA-4 MFI in T cells from Mgat5+/+

and Mgat5/ mice stimulated for 5 days in

the presence or absence of GlcNAc (40 mM).

Mean SE.

(D) shows UDP-HexNAc/UDP-Hex ratio at 24

hr andCTLA-4+ (%) at 4 days following GlcNAc

supplementation and stimulation of T cells with

250 ng/ml anti-CD3 antibody. Mean SE.

(E) Intracellular UDP-HexNAc/UDP-Hex ratio,

L-PHA staining,and surface CTLA-4are shown

following stimulation with 500 ng/ml anti-CD3antibody. Mean SE.



10/12

which eliminates the delay in the hexosamine-dependent

titration observed for low-n receptors. This delay, as well

as the switch-like response, is a defining feature of

ultrasensitivity. Therefore, N-glycan multiplicity and the

Golgi-branching pathway impose an order of glycoprotein

titration to the cell surface with respect to increasing hex-

osamine flux: first growth factor receptors, then TbR and

other low-n glycoproteins. Simulations suggest that acti-

vation of cells from a quiescent state may promote endo-

cytosis of low-n receptors at an early stage before lattice

avidity is strengthened by positive feedback to the hexos-

amine pathway (Figures5F and7).

Regulation of CTLA-4 and Growth Arrest in T Cells

After undergoing serial rounds of cell division, activated T

cells induce CTLA-4 to the cell surface and arrest in G1 of

the cell cycle (Alegre et al., 2001).b1,6GlcNAc-branching

deficiency in naive T cells reduces activation thresholds byweakening the galectin lattice, enhancing TCR clustering,

and signaling at the immune synapse (Demetriou et al.,

2001). Metabolic supplementation of the hexosamine

pathway with glutamine, acetoacetate, uridine, or GlcNAc

enhances surface b1,6GlcNAc-branched N-glycans in na-

ive T cells and reduces TCR sensitivity (A.G., unpublished

data). However, early changes in gene expression follow-

ing antigen-driven immunesynapse formation increase IL-

2 andMgat5 activities (Morgan et al., 2004), which drives

positive feedback to glucose metabolism (Frauwirth et al.,

2002), hexosamine flux, N-glycan branching, lattice

strength, and, finally, CTLA-4 surface retention. Thus,

hexosamine/Golgi/lattice negatively regulates T cell re-sponses early by inhibiting TCR/CD28 signaling in naive

T cells and later by promoting surface retention of CTLA-

4 in T cell blasts.

The elimination of pathogens requires rapid expansion

of reactive T cells followed by abrupt suppression to limit

inflammation and avoid autoimmune responses. Mgat5-

deficient mice, as well as inbred mouse strains with

deficiencies in GlcNAc-branching, are hypersensitive to

autoimmune disease (Demetriou et al., 2001; A.G., unpub-

lished data). Hexosamine flux enhances N-glycan branch-

ing in T cells, and GlcNAc supplementation of cultured T

cells during restimulation suppresses Experimental Auto-

immune Encephalomyelitis (EAE) upon transplant of the

cells into naive mice. Moreover, recent analysis of N-gly-

can branching in Multiple Sclerosis patients has revealed

novel alleles ofMgat1 that enhance activity and reduce

GlcNAc branching in a manner sensitive to hexosamine

flux, consistent with the data and model presented herein

(M.D., unpublished data).

Metabolic and Developmental Phenotypes

The hexosamine pathway and minimal branching by

Mgat1 and Mgat2 are required for mammalian embryo-

genesis (Boehmelt et al., 2000; Ioffe and Stanley, 1994).

Mgat2 deficiency is postpartum lethal with morphogenic

defects in multiple tissues, similar to the homologous

deficiency in human type II congenital disorder of

glycosylation (Wang et al., 2001). Our model suggests

that Mgat2 expression provides minimal branching re-

quired for inclusion of most receptors in the galectin/gly-

coprotein lattice (Figure S6E). Mgat5/

mice develop nor-mally but display postnatal deficiencies including reduced

numbers of muscle satellite and bone marrow osteopro-

genitor cells with reduced nuclear ERK/SMAD ratios

(J.D., unpublished data). Mgat5/ mice also display re-

duced blood glucose and resistance to weight gain on

a high-fat diet and are similar in this regard to GLUT4-de-

ficient mice (Katz et al., 1995). Mgat4a/ mice display

a predisposition to type II diabetes, and their pancreatic

b cells are deficient in glucose-stimulated insulin secretion

due to a reduction in galectin binding to surface GLUT2

(Ohtsubo et al., 2005). Interestingly, our results demon-

strate that the hexosamine pathway enhances surface ex-

pression of GLUT4 and predicts a similar function for

GLUT2 in b cells during peak glucose flux. More generally,

fluctuations in metabolic pathways on the time scale of al-

losteric regulation (sec) appear to interact with N-glycan

branching to continually adapt cellular responsiveness to

growth and arrest cues and, consequently, homeostasis

in multiple systems.

Coevolution of Branching and Multiplicity

Finally, complex N-glycans are expressed in very low

amounts inC. elegansandD. melanogaster(Cipollo et al.,

2005), and most mature N-glycans are mannose-termi-

nated (i.e., paucimannose) rather than complex N-glycan

structures (Zhu et al., 2004). C. elegans expresses func-

tional Mgat1, Mgat2, and Mgat5 enzymes (but not

Figure 7. Model of Hexosamine Regulation of Surface Glyco-

proteins and Responsiveness to Growth and Arrest Cues

Growth factor receptors have high numbers of N-glycans and, there-

fore, high avidities for the galectin lattice, while TGF-b receptors I

and II have low multiplicities (n = 1 and n = 2). Glycoforms generated

in the Golgi are above (R*) or below (R) the affinity threshold for stable

association with the galectin lattice (Figure S6A). In Mgat5/ cells, in-

sufficient positive feedback to growth signaling (black) results in a pre-

dominance of arrest signaling (red). In wild-type cells, (1) stimulation of

RTKs in quiescent cells (2) increases PI3K signaling and promotes F-

actin remodeling and preferential internalization of low-n receptors

(e.g., TbR,Figure 5F). (3) This enhances positive feedback to hexos-

amine/N-glycan processing, which leads ultimately to (4) increasing

TbR association with galectins and autocrine arrest signaling.



11/12

Mgat4; Warren et al., 2002), while D. melanogaster has

Mgat1, Mgat2, anda sequenceortholog of the Mgat4 gene

(but not Mgat5;Figures S10BS10F). Our computational

model suggests that a minimum of 13 monoantennary

N-glycans is required to significantly increase cell-surface

glycoproteins downstream of hexosamine flux (Figures

S6CS6E). Although a survey of receptor kinases in mam-

mals revealed that N-glycan sites are reduced in number

and density on receptor kinases that mediate arrest/differ-

entiation, receptors in C. elegans and D. melanogasterhave

relatively high N-X-S/T multiplicity and do not segregate in

this manner (Figure S10A). This suggests that selection

pressures driving the coevolution of N-glycan branching

and N-glycan multiplicity in a subset of glycoproteins may

be associated with greater complexity of the immune sys-

tem and metabolic homeostasis in mammals.

EXPERIMENTALPROCEDURES

Computational Model

Each process of the ordered sequential bi-bi reaction mechanism of

Golgi-branching enzymes was modeled as an ODE:

dCi

dt =SVproduction SVconsumption

where [Ci] is the concentration of a component. Since the Golgi con-

centration of UDP-GlcNAc is 15 times that of cytosol (100 mM),

the Golgi UDP-GlcNAc is estimated to be in the millimolar range.

Theprobabilityof obtaininga specific glycoformis theproduct of the

probabilities of specific N-glycan occurrences at each N-X-S/T site:

Pglycoform =Yn

i1

PNglycanxi

was calculated using the Golgi-pathway output of P(N-glycan(x)), then

partitioned into glycoform fractions according to avidity.

Glycoform fractions are filtered on the cell surface by their avidities for

galectin-3 and modeled using another set of ODEs where membrane

recycling and protein degradation are uncoupled. The kinetic parame-

ters of membrane recycling are fitted to the avidity-enhancement scal-

ing factor for lattice multivalency (seeSupplemental Datafor details).

Cell Lines and Growth Conditions

Mgat5+/+(2.6) andMgat5/(22.9) mammary tumor cell lines were es-

tablished from spontaneous mammary carcinomas in polyoma middle

T (PyMT) transgenic mice as previously described (Granovsky et al.,

2000). Tumor cells, NMuMG (NMARU mammary gland), and

HEK293T cells were propagated in DMEM (Gibco) supplemented

with 4.5 g/L glucose and 10% fetal bovine serum plus 10 mg/ml insulin

(Sigma) for NMuMG. Cell cultures were supplemented with various

concentrations of GlcNAc (Sigma) for 48120 hr. For Mgat1 overex-

pression, cells were infected with pMX-PIE-carrying human Mgat1

and GFP separated by IRES. myc-GLUT4-GFP/mutant constructs

were transfected into HEK293T cells using Effectene (Qiagen), fixed,

and stained with anti-myc antibody (Santa Cruz) overnight. To reveal

cell-adhesion junctions, cells were fixed and stained with anti-E-cad-

herin antibodies as previously described (Partridge et al., 2004).

Supplemental Data

Supplemental Data include ten figures, four tables, Experimental Pro-

cedures, and References and can be found with this article online at

http://www.cell.com/cgi/content/full/129/1/123/DC1/.

ACKNOWLEDGMENTS

This research was supported by grants from CIHR and Genome Can-

ada through the OGI to J.W.D. and funding from NIH and NMSS USA

to M.D. K.S.L. was funded by a NSERC PGSD scholarship. We thank

P. Cheungand G. Ruan fortechnical assistance andDrs. H. Schachter,

R. Linding, J. Parkinson, Y. Haffani, A. Datti, G. Bader, I.R. Nabi, and

M. Rozakis-Adcock for advice.

Received: September 15, 2006

Revised: November 15, 2006

Accepted: January 24, 2007

Published: April 5, 2007

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