Interactions between anthrax toxin receptors and protective antigen

Interactions between anthrax toxin receptors andprotective antigenHeather M Scobie1,2 and John AT Young1

Since the anthrax mail attacks of 2001, much has been learned

about the interactions between anthrax toxin and its receptors.

Two distinct cellular receptors for anthrax toxin have been

identified and are designated capillary morphogenesis protein

2 (CMG2) and anthrax toxin receptor/tumor endothelial marker

8 (ATR/TEM8). The molecular details of the toxin–receptor

interactions have been revealed through crystallographic,

biochemical and genetic studies. In addition, a novel pathway

by which anthrax toxin enters cells is starting to be uncovered.

Addresses1 Infectious Disease Laboratory, The Salk Institute for Biological Studies,

10010 North Torrey Pines Road, La Jolla, CA 92037, USA2 Program in Cellular and Molecular Biology, University of Wisconsin-

Madison, 413 Bock Labs, 1525 Linden Drive, Madison, WI 53706-1596,

USA

Corresponding author: Young, John AT ([email protected])

Current Opinion in Microbiology 2005, 8:106–112

This review comes from a themed issue on

Host–microbe interactions: bacteria

Edited by Pascale Cossart and Jorge Galan

Available online 7th January 2005

1369-5274/$ – see front matter

# 2005 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.mib.2004.12.005

IntroductionAnthrax is caused by the spore-forming, Gram-positive

bacterium Bacillus anthracis. Anthrax toxin is secreted by

the bacteria and is thought to play a critical role in anthrax

pathogenesis and immune system impairment during

infection. Systemic disease is usually fatal if not treated

with antibiotics before symptoms develop, presumably as a

result of high levels of toxin. Therefore, it is thought that a

successful treatment strategy would include the use of

antitoxins [1,2]. In 2001, when the anthrax mail attacks

occurred in the USA, the cellular receptors used by the

anthrax toxin to enter cells had not been described. During

the past three years significant advances have been made in

understanding the interactions between the anthrax toxin

and its receptors. Now we have a three-dimensional,

molecular picture of a toxin-receptor interaction, increased

knowledge of toxin internalization pathways into cells, and

an idea of how toxin–receptor interactions and receptor

biology contribute to pathogenesis.

In this review, we focus upon recent developments that

have dramatically increased our understanding of anthrax


toxin entry into cells, and we point the way to future

approaches for anthrax treatment.

Anthrax toxinAnthrax toxin is a bipartite AB-type toxin with a single

receptor-binding B moiety, known as the protective anti-

gen (PA), and two alternative catalytic A moieties: edema

factor (EF) and lethal factor (LF). PA assembles with EF

or LF to generate edema toxin (ET) or lethal toxin (LT),

respectively.

LF is a zinc-dependent metalloprotease that cleaves the

N-terminus of various mitogen-activated protein kinase

kinases (MKKs) [3–7] resulting in their inactivation and

disruption of various cellular signal transduction path-

ways. LT causes the death of endothelial cells and

sensitized macrophages [8–10], impairs dendritic cell

function [11], disrupts glucocorticoid receptor signaling

[12] and kills mice in a hypoxia-mediated manner [13].

EF is a calcium- and calmodulin-dependent adenylate

cyclase [14] that raises intracellular cAMP levels [15],

which causes swelling [16] and blocks neutrophil-depen-

dent phagocytosis [17].

Toxin entryThe current model of toxin entry into host cells is as

follows. A full-length, 83 kDa form of PA (PA83) binds cell

surface receptors (Figure 1a,b) and is subjected to a

necessary cleavage by a cellular furin-like enzyme

[18,19]. The remaining activated PA63 protein then

self-assembles into a heptameric ring structure, or pre-

pore (Figure 1c,d). Binding sites for EF and LF are

generated at the dimer interface between two PA mono-

mers so that each heptamer can bind up to three mole-

cules of either EF and/or LF [20]. PA oligomerization is

accompanied by receptor clustering into lipid raft

domains of the plasma membrane. The receptor–toxin

complex is then taken up into cells by clathrin-dependent

endocytosis (Figure 1a) [21�].

Translocation of LF and EF into the cytosol requires a

low pH-dependent conformational change in the PA

prepore that results in pore formation in an intracellular

membrane (reviewed in [22]; Figure 1a). It was long

believed that the PA heptamer was inserted solely into

the membrane of an endosome, or an endosomal carrier

vesicle, and that the toxin A-moieties were translocated

directly from those compartments into the cytosol. How-

ever, a new model has instead invoked that the PA

heptamer inserts predominantly into the membranes of

intraluminal vesicles within early endosomes and that LF

www.sciencedirect.com

Interactions between anthrax toxin receptors and protective antigen Scobie and Young 107

Figure 1

CMG2

I

IIIII

IV

D683

Earlyendosome

Lipid raft

MVB

LFEF

(a) (b)

(c) (d)

Current Opinion in Microbiology

ATR/TEM8or CMG2

PA83 (PA63)7

Clathrincoated pit

Lateendosome LF cleaves

MKKs

EF raisescAMP levels

H+

H+

H+

H+H+

H+

H+H+

H+H+

H+

H+

H+

H+

H+ H+

H+

H+ H+

H+ H+

H+

H+

PA83

β3-β4Loop

PA63

PA20

Anthrax toxin–receptor binding and cellular intoxication. (a) Model of anthrax toxin entry and trafficking leading to intoxication of host cells. A full-length

form of PA (PA83) binds to ATR/TEM8 or CMG2 receptors and is cleaved by cell-surface furin. PA63 spontaneously forms a heptamer (PA63)7, or

prepore, which results in receptor clustering and migration of receptor–toxin complexes into lipid raft microdomains in the plasma membrane.

www.sciencedirect.com Current Opinion in Microbiology 2005, 8:106–112

108 Host–microbe interactions: bacteria

translocates into the lumen of these vesicles before trans-

port to late endosomes via multivesicular bodies. LF can

then be released into the cytosol when these vesicles fuse

with the limiting membrane of the late endosome

(Figure 1a) [23��]. However, many of the details of this

proposed toxin entry pathway, such as the trafficking

itineraries followed by the two types of anthrax toxin

receptor, remain to be characterized.

Anthrax toxin receptorsAnthrax toxin receptor/tumor endothelial marker 8 (ATR/

TEM8) was the first anthrax toxin receptor identified

using an approach that combined somatic cell mutagen-

esis in chinese hamster ovary cells with human cDNA

complementation [24]. The related capillary morphogen-

esis gene 2 (CMG2) protein was subsequently shown to

have anthrax toxin receptor function [25��].

ATR/TEM8 and CMG2 are type 1 transmembrane

proteins with single membrane spanning domains.

Their most distinctive feature is an extracellular region

approximately 200 amino acids long that binds directly

to PA and is related to the von Willebrand factor A

(vWFA) domains and most closely related to a-integrin-

like inserted (I) domains (Figures 1b and 2). Although

the amino acid sequences of both anthrax toxin recep-

tors are only 40% identical overall, their I domains are

approximately 60% identical. At present, three differ-

ent protein isoforms of each anthrax toxin receptor have

been described and are the result of alternative mRNA

splicing (Figure 2) [24,25��]. Although the precise dis-

tribution and levels of each of these proteins in differ-

ent tissues remain to be established, northern blot and

RT-PCR analysis has indicated that the CMG2 gene is

likely to be expressed in most human tissues, whereas

ATR/TEM8 expression may be restricted to tumor

endothelial and cancer cells [25��,26–30]. However,

ATR/TEM8 expression is modestly upregulated in

response to interleukin-1b treatment [31], raising the

possibility that the level and tissue distribution of this

receptor might fluctuate during the course of a bacterial

infection.

(Figure 1 legend continued) Up to three molecules of LF or EF can bind t

clathrin-mediated endocytosis. The toxin–receptor complex is trafficked in a

PA domain II and domain IV is thought to prevent membrane pore formatio

translocated into the vesicle lumen and gain access to the cytoplasm prima

endosome where they back-fuse with the limiting membrane, or alternativel

mitogen activated kinase kinases (MKKs) and EF raises cAMP in a calmodu

determined for the full-length and catalytic portions of these proteins, respe

monomer (Protein Database 1T6B [33��]). The CMG2 I domain (purple) adopt

a site of PA binding. PA domain I (orange and yellow) contains two Ca2+ ions

PA63 and PA20 fragment (orange). PA domain II (red) lines the PA pore, is in

that results in membrane pore formation. b3–b4 loop contacts with CMG2 a

involved in oligomerization and domain IV (green) is a discrete receptor-bind

(pink stick representation) makes contact with the metal cation at the recep

(Protein Database 1TZN [40��]). CMG2 I domains (purple) punctuate the low

domains IV (green) and II (red). The CMG2 MIDAS Mg2+ and PA Ca2+ ions a

in panel (c) modeled on a schematic of the plasma membrane. The structur

solved and it is depicted as a purple stalk. LF and/or EF (not shown) bind t


In the case of ATR/TEM8, mRNA isoforms are known as

splice variants (sv) 1–3. The ATR/TEM8 proteins

encoded by sv1 and sv2 are transmembrane proteins with

differing cytoplasmic regions and both function as anthrax

toxin receptors (Figure 2) [24,32�]. Interestingly, the

cytoplasmic domains of ATR/TEM8 proteins are not

essential for toxin binding and entry since they can be

deleted or replaced with a glycosylphosphatidylinositol

(GPI) anchor sequence and still support intoxication

[32�]. The sv3 mRNA encodes a secreted ATR/TEM8

protein that does not serve as an anthrax toxin receptor

(Figure 2) [32�].

The three CMG2 protein isoforms are transmembrane

proteins consisting of either 386, 488 or 489 amino acids

that we have designated as CMG2386, CMG2488 and

CMG2489, respectively. CMG2489 and CMG2488 are iden-

tical except for the 12–13 amino acids located at their

C-termini (Figure 2), and both proteins function as

anthrax toxin receptors ([25��]; HM Scobie et al., unpub-

lished). The cytoplasmic domain of CMG2386 is the same

as CMG2489 but is lacking two C-terminal residues of the

I domain as well as a 100 amino acid membrane proximal

domain present in the two longer protein isoforms

(Figure 2) [25��]. Although the CMG2386 I domain can

bind PA [33��], the full-length version of this protein

cannot support intoxication (HM Scobie et al., unpub-

lished). The precise defect associated with the CMG2386

protein remains to be determined.

ATR/TEM8 and CMG2 proteins are conserved

between diverse species, including mouse, rat and zeb-

rafish, suggesting that they fulfill an important physio-

logical function. Their precise function is unknown, but

evidence suggests they are cell adhesion molecules

involved in angiogenesis. ATR/TEM8 proteins bind

to collagen a3(VI) [29]. ATR/TEM8 is preferentially

expressed in the vasculature of different types of human

tumors [26,29,30,34], as well as developing mouse

embryos [30]. CMG2 binds to collagen IV and laminin,

and its gene is specifically upregulated in human umbi-

lical vein endothelial cells induced to undergo capillary

he PA heptamer, and after assembly, the complex is taken up via

n intraluminal vesicle to the early endosome. Receptor contact with

n until exposure to this low pH environment. Toxin A moieties are

rily after vesicle trafficking via multivesicular bodies (MVB) to the late

y after fusion with the early endosomal membrane. LF cleaves cellular

lin- and calcium-dependent manner. Crystal structures have been

ctively [3,14]. (b) Ribbon diagram of a CMG2 I domain bound to PA83

s a Rossmann fold with a metal cation (Mn2+; cyan) bound at its MIDAS,

(cyan) and is cleaved by furin to generate EF and LF binding sites and a

volved in oligomerization, and undergoes a conformational change

re hypothesized to inhibit this change. PA domain III (blue) is primarily

ing domain. The carboxylate side-chain from PA residue Asp683

tor MIDAS. (c) Overhead view of CMG2 I domain/PA heptamer complex

er, outer surface of the PA prepore (grey) making contact with PA

re colored cyan. (d) Side-view of the CMG2–PA heptamer complex

e of the CMG2 receptor membrane proximal domain (MPD) was not

o the top surface of the PA heptamer.



Figure 2

5641 27 44 216 320 343

3191 27 44 216 333

sv1

sv2

sv3

1 27 44 216 320 343 368364

1 33 44 212 215 233 386

1 33 44 214 318 336 488477

1 33 44 214 318 336 489

489

488

386

MIDAS

SP TMMPD

Current Opinion in Microbiology

ATR/ TEM8:

CMG2:

vWFA/ I domain

Schematic representation of known CMG2 and ATR/TEM8 protein isoforms. These products are the result of alternative splicing events from the

CMG2 and ATR/TEM8 genes. Each protein contains a signal peptide (SP) and a PA-binding region related to von Willebrand factor A (vWFA) or

integrin-like inserted (I) domains. Within the I domains of these proteins is a metal ion dependent adhesion site (MIDAS), or series of five non-

consecutive amino acid residues (represented by stars) that chelate a divalent cation. The C-termini of ATR/TEM8 proteins diverge so that sv1 and sv2

contain single-pass transmembrane (TM) domains with differing cytoplasmic tails, whereas sv3 is a soluble protein. Similarly, CMG2489 and CMG2488

are identical except for their C-terminal 12–13 amino acids. Although the cytoplasmic tail of CMG2386 matches that of CMG2489, CMG2386 is generated

from a splicing event resulting in exclusion of the membrane proximal domain (MPD) and the C-terminal two amino acids of the I domain.

formation within three-dimensional collagen matrices

[35]. Mutations in the CMG2 gene, some of which are

predicted to destabilize the protein’s I domain, result in

two related human autosomal recessive syndromes,

juvenile hyaline fibromatosis and infantile systemic

hyalinosis [28,36]. Fibroblasts from patients with these

diseases have been shown to be deficient in adhesion to

laminin [36]. Understanding how the loss of CMG2

expression contributes to these diseases should point

the way toward better understanding the natural func-

tions of these proteins.

Interactions between receptor I domainand PABiochemical, genetic and structural studies have revealed

that receptor–PA interactions resemble a-integrin/ligand

binding. As with the I domains of a integrins ATR/TEM8

and CMG2 contain a metal ion dependent adhesion site

(MIDAS) motif (DxSxS. . .T. . .D) that binds a metal

cation directly involved in ligand binding (Figures 1b


and 2). Binding of PA to both types of anthrax toxin

receptor I domains has been shown to occur in the

presence of the metal cations Mg2+ and Mn2+, which is

similar to a-integrin/ligand binding, but binding between

CMG2 I domain and PA also occurs in the presence of

Ca2+ [25��,37�]. Involvement of the MIDAS motif has

been further demonstrated by the fact that binding of

ATR/TEM8 to PA was abolished by mutating MIDAS

residue Asp50, a change predicted to result in loss of

cation binding [37�].

Despite their similarities to a-integrin I domains, it is

currently unknown if ATR/TEM8 and CMG2 I domains

can exist in both open (high affinity ligand binding) and

closed (low affinity ligand binding) states associated with

integrin I domains [33��,38,39,40��]. Specific changes in

MIDAS metal ion coordinating residues are associated

with the different integrin I domain conformational

states, including the MIDAS Thr residue that switches

from indirect metal ion coordination in the closed state to



direct coordination in the open state [41]. Mutation of the

corresponding residue (T118) in ATR/TEM8 reduced

receptor activity by 100–1000-fold, a result that was

consistent with the idea that this receptor might have

to adopt an open-like binding state to bind PA [37�]. This

view is supported by X-ray crystallographic studies that

have demonstrated that residue T118 of CMG2 makes

direct contact with the metal ion in PA–CMG2 complexes

[33��,40��].

Ligands bound to the open conformation of a-integrin I

domains contribute a carboxylate-containing amino acid

side-chain that completes the MIDAS metal ion coordi-

nation sphere [41]. On the basis of mutagenesis studies,

the D683 residue of PA was postulated to perform a

similar role in the ATR/TEM8 interaction [37�,42].

Again, crystallographic studies of PA–CMG2 complexes

have revealed that D683 makes direct contact with the

metal ion bound at the receptor MIDAS (Figure 1b).

Other conformational changes that accompany the

a-integrin I domain conversion between closed and open

conformations include a dramatic 10A movement of the

C-terminal a-helix and insertion of different hydrophobic

residues into the domain’s hydrophobic pockets to stabi-

lize the two conformations [39,41]. Because the CMG2 I

domain appears to lack the hydrophobic residue for

stabilizing a putative closed conformation, it has been

argued that this receptor might never adopt a closed-like

state [39]. However, a crystal structure of the CMG2386 I

domain lacking the residue that stabilizes the open

CMG2489 I domain is able to bind to PA in an open

conformation manner using a different stabilizing hydro-

phobic residue [33��]. This raises the possibility that other

local hydrophobic residues might help stabilize alterna-

tive conformations of this domain.

A striking difference between CMG2–PA and a integrin/

ligand interactions is found at the level of binding affi-

nities. The binding affinities that are associated with

a-integrin interactions with physiological ligands are

typically in the mM to mM range [38]. By contrast,

PA binds the CMG2 I domain with an extremely high

affinity (KD = 170 pM or 780 pM in the presence of Mg2+

or Ca2+ ions, respectively) [43�]. Indeed, this receptor can

still bind PA in the absence of metal ions at a level

(KD = 960 nM) [43�] on par with a-integrin/ligand inter-

actions in the presence of metal ions. These differences

in binding affinity are likely explained by the fact that

the PA–CMG2 interaction buries approximately 2000A2

of protein surface, whereas a-integrin/ligand interactions

typically bury only 1300A2 of protein surface [33��,40��].In contrast to CMG2, the ATR/TEM8 I domain binds

much less tightly to PA (HM Scobie et al., unpublished); a

property that may be explained by several amino acid

differences between the PA-binding interfaces of both

receptors [33��,40��]. The high binding affinity between


CMG2 and PA is currently being exploited for the devel-

opment of soluble receptor-based antitoxins [2,25��].

A role for receptor in low pH-dependentPA pore formationPA consists of four discrete domains (I–IV) with different

functions (Figure 1b). Prior to the crystal structures of

monomeric PA83 with the CMG2386 I domain (2.5A

resolution; Figure 1b) and heptameric PA63 with the

CMG2489 I domain (4.3A resolution; Figure 1c,d), it

was believed that domain IV was the only receptor-

binding region of PA [33��,40��]. Indeed, domain IV

contains the D683 residue that binds the MIDAS metal

and also contains other residues that make contact with

the CMG2 I domain (Figure 1b) [33��,40��]. However,

the co-crystal structures unexpectedly revealed that the

b3–b4 loop of PA domain II, a domain that is primarily

involved in forming the pore thought to translocate EF

and LF, also contributes significantly to the receptor

interaction (Figure 1b,c) [33��,40��].

The binding of the CMG2 I domain to regions of PA

domain II and IV have helped to explain one of the

puzzling features of PA pore formation, namely that it

occurs at a higher pH value in artificial membranes than in

cells that have receptors [44]. The bound CMG2 I domain

serves as a clamp that, at neutral pH, blocks subsequent

rearrangements of PA domain II necessary for pore for-

mation. This action would ensure that the PA pore would

form only after proper trafficking of the toxin–receptor

complex to an endosomal compartment with low pH

(Figure 1a) [33��,40��]. Consistent with this idea, PA63

prepore to pore conversion in solution has been demon-

strated to occur at neutral pH in the absence of the

receptor but only at acidic pH in its presence [40��]. It

has been postulated that this triggering mechanism

involves the protonation of histidine residues located at

the PA–receptor binding interface and at regions of PA

that are thought to undergo structural rearrangements

upon pore conversion [33��].

ConclusionsThe first step in anthrax intoxication involves PA binding

to the I domain of either of two cell surface receptors,

ATR/TEM8 or CMG2. These binding interactions

resemble those between a-integrins and their ligands

in that the MIDAS of the receptor, as well as a carbox-

ylate-containing residue of PA, serve important roles. The

recently obtained X-ray crystal structures of monomeric,

and heptameric PA with the CMG2 I domain have

revealed the molecular details of the toxin–receptor inter-

action. These structures have led to the identification of

shared residues that are presumably important for PA-

specific binding, and have uncovered an unexpected role

for the receptor in acting as a molecular clamp to ensure

that the PA heptameric pore forms only within the acidic

lumen of an endosomal compartment. Cell biological



studies have led to the proposal that pore formation and

LF translocation occur in intraluminal vesicles within

endosomes and that LF is not delivered into the cytosol

directly, as was previously thought, but instead when

these vesicles fuse with limiting endosomal membranes.

The continued study of anthrax toxins and their receptors

promises to yield more exciting discoveries in the fields of

microbiology and cell biology, and should aid in the

development of antitoxins aimed at preventing toxin

entry into the cell.

AcknowledgementsThe authors acknowledge support for this work from NIH grantsAI48489 and AI56013, and thank G Jonah Rainey and Kenneth Bradleyfor their critical feedback on the manuscript.

References and recommended readingPapers of particular interest, published within the annual period ofreview, have been highlighted as:

� of special interest�� of outstanding interest

1. Inglesby TV, O’Toole T, Henderson DA, Bartlett JG, Ascher MS,Eitzen E, Friedlander AM, Gerberding J, Hauer J, Hughes J et al.:Anthrax as a biological weapon, 2002: updatedrecommendations for management. JAMA 2002,287:2236-2252.

2. Rainey GJA, Young JAT: Antitoxins: novel strategies to targetagents of bioterrorism. Nat Rev Microbiol 2004, 2:721-726.

3. Pannifer AD, Wong TY, Schwarzenbacher R, Renatus M, Petosa C,Bienkowska J, Lacy DB, Collier RJ, Park S, Leppla SH et al.:Crystal structure of the anthrax lethal factor. Nature 2001,414:229-233.

4. Vitale G, Bernardi L, Napolitani G, Mock M, Montecucco C:Susceptibility of mitogen-activated protein kinase kinasefamily members to proteolysis by anthrax lethal factor.Biochem J 2000, 352:739-745.

5. Vitale G, Pellizzari R, Recchi C, Napolitani G, Mock M, MontecuccoC: Anthrax lethal factor cleaves the N-terminus of MAPKKsand induces tyrosine/threonine phosphorylation of MAPKs incultured macrophages. Biochem Biophys Res Commun 1998,248:706-711.

6. Duesbery NS, Webb CP, Leppla SH, Gordon VM, Klimpel KR,Copeland TD, Ahn NG, Oskarsson MK, Fukasawa K, Paull KD etal.: Proteolytic inactivation of MAP-kinase-kinase by anthraxlethal factor. Science 1998, 280:734-737.

7. Chopra AP, Boone SA, Liang X, Duesbery NS: Anthrax lethalfactor proteolysis and inactivation of MAP-kinase-kinase.J Biol Chem 2003, 278:9402-9406.

8. Park JM, Greten FR, Li ZW, Karin M: Macrophage apoptosis byanthrax lethal factor through p38 MAP kinase inhibition.Science 2002, 297:2048-2051.

9. Kirby JE: Anthrax lethal toxin induces human endothelial cellapoptosis. Infect Immun 2004, 72:430-439.

10. Kim SO, Jing Q, Hoebe K, Beutler B, Duesbery NS, Han J:Sensitizing anthrax lethal toxin-resistant macrophages tolethal toxin-induced killing by tumor necrosis factor-alpha.J Biol Chem 2003, 278:7413-7421.

11. Agrawal A, Lingappa J, Leppla SH, Agrawal S, Jabbar A, Quinn C,Pulendran B: Impairment of dendritic cells and adaptiveimmunity by anthrax lethal toxin. Nature 2003, 424:329-334.

12. Webster JI, Tonelli LH, Moayeri M, Simons SS Jr, Leppla SH,Sternberg EM: Anthrax lethal factor represses glucocorticoidand progesterone receptor activity. Proc Natl Acad Sci USA2003, 100:5706-5711.


13. Moayeri M, Haines D, Young HA, Leppla SH: Bacillus anthracislethal toxin induces TNF-alpha-independent hypoxia-mediated toxicity in mice. J Clin Invest 2003, 112:670-682.

14. Drum CL, Yan SZ, Bard J, Shen YQ, Lu D, Soelaiman S,Grabarek Z, Bohm A, Tang WJ: Structural basis for theactivation of anthrax adenylyl cyclase exotoxin by calmodulin.Nature 2002, 415:396-402.

15. Leppla SH: Anthrax toxin edema factor: a bacterial adenylatecyclase that increases cyclic AMP concentrations ofeukaryotic cells. Proc Natl Acad Sci USA 1982, 79:3162-3166.

16. Pezard C, Berche P, Mock M: Contribution of individual toxincomponents to virulence of Bacillus anthracis. Infect Immun1991, 59:3472-3477.

17. O’Brien J, Friedlander A, Dreier T, Ezzell J, Leppla S: Effectsof anthrax toxin components on human neutrophils.Infect Immun 1985, 47:306-310.

18. Beauregard KE, Collier RJ, Swanson JA: Proteolytic activation ofreceptor-bound anthrax protective antigen on macrophagespromotes its internalization. Cell Microbiol 2000, 2:251-258.

19. Klimpel KR, Molloy SS, Thomas G, Leppla SH: Anthrax toxinprotective antigen is activated by a cell surface protease withthe sequence specificity and catalytic properties of furin.Proc Natl Acad Sci USA 1992, 89:10277-10281.

20. Mogridge J, Cunningham K, Collier RJ: Stoichiometry of anthraxtoxin complexes. Biochemistry 2002, 41:1079-1082.

21.�

Abrami L, Liu S, Cosson P, Leppla SH, van der Goot FG: Anthraxtoxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process. J Cell Biol 2003,160:321-328.

This study shows that PA oligomerization leads to receptor redistributioninto lipid rafts of the plasma membrane followed by clathrin-dependentendocytosis of toxin–receptor complexes.

22. Collier RJ, Young JA: Anthrax toxin. Annu Rev Cell Dev Biol 2003,19:45-70.

23.��

Abrami L, Lindsay M, Parton RG, Leppla SH, Van Der Goot FG:Membrane insertion of anthrax protective antigen andcytoplasmic delivery of lethal factor occur at different stagesof the endocytic pathway. J Cell Biol 2004, 166:645-651.

This recent study, which employed inhibitors of endosomal trafficking andfunction, has led to a new model for anthrax toxin trafficking andtranslocation. The PA pore inserts predominantly into the membraneof intraluminal vesicles within early endosomes. Lethal factor is thentranslocated into the lumen of these vesicles and trafficked via multi-vesicular bodies to late endosomes, where toxin is finally released intothe cytoplasm after vesicle back-fusion with the limiting endosomalmembrane.

24. Bradley KA, Mogridge J, Mourez M, Collier RJ, Young JA:Identification of the cellular receptor for anthrax toxin.Nature 2001, 414:225-229.

25.��

Scobie HM, Rainey GJ, Bradley KA, Young JA: Human capillarymorphogenesis protein 2 functions as an anthrax toxinreceptor. Proc Natl Acad Sci USA 2003, 100:5170-5174.

CMG2 is identified as a second anthrax toxin receptor based upon itsability to confer PA binding and susceptibility to intoxication whenexpressed in PA receptor-deficient cells. Northern blot analysis showsbroad expression of the receptor in human tissues that may be relevantfor intoxication. Also, a soluble version of the CMG2 I domain is shown tobe a very efficient receptor decoy, blocking cell intoxication when mixedwith PA at a 3:1 ratio.

26. Davies G, Mason MD, Martin TA, Parr C, Watkins G, Lane J,Matsumoto K, Nakamura T, Jiang WG: The HGF/SF antagonistNK4 reverses fibroblast- and HGF-induced prostate tumorgrowth and angiogenesis in vivo. Int J Cancer 2003,106:348-354.

27. De Preter K, Pattyn F, Berx G, Strumane K, Menten B, Van Roy F,De Paepe A, Speleman F, Vandesompele J: Combinedsubtractive cDNA cloning and array CGH: an efficientapproach for identification of overexpressed genes in DNAamplicons. BMC Genomics 2004, 5:11.

28. Hanks S, Adams S, Douglas J, Arbour L, Atherton DJ, Balci S,Bode H, Campbell ME, Feingold M, Keser G et al.: Mutations in



the gene encoding capillary morphogenesis protein 2 causejuvenile hyaline fibromatosis and infantile systemic hyalinosis.Am J Hum Genet 2003, 73:791-800.

29. Nanda A, Carson-Walter EB, Seaman S, Barber TD, Stampfl J,Singh S, Vogelstein B, Kinzler KW, St Croix B: TEM8 interactswith the cleaved C5 domain of collagen alpha 3(VI).Cancer Res 2004, 64:817-820.

30. Carson-Walter EB, Watkins DN, Nanda A, Vogelstein B,Kinzler KW, St Croix B: Cell surface tumor endothelial markersare conserved in mice and humans. Cancer Res 2001,61:6649-6655.

31. Rmali KA, Al-Rawi MA, Parr C, Puntis MC, Jiang WG:Upregulation of tumour endothelial marker-8 by interleukin-1beta and its impact in IL-1beta induced angiogenesis.Int J Mol Med 2004, 14:75-80.

32.�

Liu S, Leppla SH: Cell surface tumor endothelium marker 8cytoplasmic tail-independent anthrax toxin binding,proteolytic processing, oligomer formation, andinternalization. J Biol Chem 2003, 278:5227-5234.

The authors demonstrate that, as with ATR/TEM8 sv2, ATR/TEM8 sv1also functions as an anthrax toxin receptor. In addition, the cytoplasmictail of the receptor is shown to be dispensable, because replacement ofthis region with a GPI anchor does not impair the ability of the receptor tosupport intoxication.

33.��

Santelli E, Bankston LA, Leppla SH, Liddington RC: Crystalstructure of a complex between anthrax toxin and its host cellreceptor. Nature 2004, 430:905-908.

The crystal structure of monomeric PA with the CMG2 I domain isdescribed. The structure reveals a large buried protein surface at thePA-CMG2 interface and, along with the paper by Lacy et al. [40��], showsthat domain II of PA makes unexpected contact with the receptor. Theauthors suggest that the bound CMG2 moiety might serve as a molecularbrace to prevent domain II rearrangements necessary for pore formationuntil the complex is exposed to a low pH environment, at which time theprotonation of specific histidine residues located at the PA–CMG2 inter-face and within mobile segments of PA might drive PA prepore to poreconversion.

34. St Croix B, Rago C, Velculescu V, Traverso G, Romans KE,Montgomery E, Lal A, Riggins GJ, Lengauer C, Vogelstein B et al.:Genes expressed in human tumor endothelium. Science 2000,289:1197-1202.

35. Bell SE, Mavila A, Salazar R, Bayless KJ, Kanagala S, Maxwell SA,Davis GE: Differential gene expression during capillarymorphogenesis in 3D collagen matrices: regulated expressionof genes involved in basement membrane matrix assembly,cell cycle progression, cellular differentiation and G-proteinsignaling. J Cell Sci 2001, 114:2755-2773.

36. Dowling O, Difeo A, Ramirez MC, Tukel T, Narla G, Bonafe L,Kayserili H, Yuksel-Apak M, Paller AS, Norton K et al.: Mutationsin capillary morphogenesis gene-2 result in the allelic


disorders juvenile hyaline fibromatosis and infantile systemichyalinosis. Am J Hum Genet 2003, 73:957-966.

37.�

Bradley KA, Mogridge J, Jonah G, Rainey A, Batty S,Young JA: Binding of anthrax toxin to its receptor is similarto alpha integrin-ligand interactions. J Biol Chem 2003,278:49342-49347.

Anthrax toxin–receptor interactions are shown to resemble thosebetween a-integrin I domains and their physiological ligands in thatATR/TEM8–PA binding requires an open-like conformation of the recep-tor with a divalent cation bound to the MIDAS as well as a carboxylateside-chain from PA.

38. Shimaoka M, Xiao T, Liu JH, Yang Y, Dong Y, Jun CD, McCormackA, Zhang R, Joachimiak A, Takagi J et al.: Structures of the alphaL I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation. Cell 2003, 112:99-111.

39. Lacy DB, Wigelsworth DJ, Scobie HM, Young JA, Collier RJ:Crystal structure of the von Willebrand factor A domain ofhuman capillary morphogenesis protein 2: an anthrax toxinreceptor. Proc Natl Acad Sci USA 2004, 101:6367-6372.

40.��

Lacy DB, Wigelsworth DJ, Melnyk RA, Harrison SC, Collier RJ:Structure of heptameric protective antigen bound to ananthrax toxin receptor: A role for receptor in pH-dependentpore formation. Proc Natl Acad Sci USA 2004, 101:13147-13151.

The co-crystal structure of seven CMG2 I domains bound to the PAheptamer is presented and, along with the paper by Santelli et al. [33��],shows that there is a large CMG2–PA contact surface and that PA domainII makes unexpected contact with receptor. This structure also reveals theplacement of the domain II membrane insertion loop in the PA prepore.The authors demonstrate that CMG2 binding imposes a low pH depen-dence on pore formation, presumably preventing premature translocationof the toxin A subunits.

41. Emsley J, Knight CG, Farndale RW, Barnes MJ, Liddington RC:Structural basis of collagen recognition by integrinalpha2beta1. Cell 2000, 101:47-56.

42. Rosovitz MJ, Schuck P, Varughese M, Chopra AP, Mehra V,Singh Y, McGinnis LM, Leppla SH: Alanine-scanning mutationsin domain 4 of anthrax toxin protective antigen reveal residuesimportant for binding to the cellular receptor and to aneutralizing monoclonal antibody. J Biol Chem 2003,278:30936-30944.

43.�

Wigelsworth DJ, Krantz BA, Christensen KA, Lacy DB, Juris SJ,Collier RJ: Binding stoichiometry and kinetics of the interactionof a human anthrax toxin receptor, CMG2, with protectiveantigen. J Biol Chem 2004, 279:23349-23356.

The PA heptamer is shown to bind seven copies of the CMG2 I domain andthe binding affinity of the monomeric PA–CMG2 I domain interaction isfound to be very high in the presence of either magnesium or calcium ions.

44. Miller CJ, Elliott JL, Collier RJ: Anthrax protective antigen:prepore-to-pore conversion. Biochemistry 1999,38:10432-10441.


Documents

Interactions between anthrax toxin receptors and protective antigen