Uncovered: the family relationship of a T-cell-membrane protein and bacterial toxins

IMMUNOLOGY

TODAY

Friedrich Mono-ADP-ribosylation originally

was

discovered as the

mechanism by which diphtheria ono-ADP-ribosylatlon a post-translational tein modification was originally

is prothat

discov-

ered as the mechanism by which bacterial toxins interfere with protein synthesis and signal transduction in human host cells. It involves the transfer of the ADP-ribose moiety from NAD+ to a specific ammo acid in a target protein while the nicotinamide moiety is released (Fig. 1). Mono-ADP-ribosylation of diphthamide in elongation factor 2 by diphtheria and Pseudomonas toxins inactivates protein synthesis*. A number of other potent bacterial toxins, including cholera, pertussis and Escherichia colt heatlabile enterotoxins, interfere with signal transduction in human host cells by ADPribosylating regulatory G proteins on arginine or cysteine residues. Since cellular protein functions are profoundly affected by ADP-ribosylation, these toxins have found wide application in cell and molecular biol-

toxin inactivates

protein synthesis.

It is now apparent that higher organisms use analogous mechanisms to regulate endogenous metabolism.

Here,

the relationship

between bacterial foxins and

vertebrate mono-ADP ribosyltransferaseserases is explored. ample evidence has been put forth to support this hypothesis. However. the responsible enzymes eluded molecular cloning until the groups of Moss3 and Shiioyama4 succeeded in purifying proteins with enzyme activities akin to those of bacterial monoADP-ribosyltransferases from rabbit skeletal muscle (designated RABNAART) and chicken bone marrow cells (designated CHAT-l and CHAT-21, respectively (Fig. 1). Homology searches revealed significant sequence similarity of RABNAART and the

ogy, for example, as recombinant toxins for the directed killing of specific cells (diph-

CHATS to only one other previously known eukaryotic protein of unknown function:

theria and Pseudomonas toxins) and to identify proteins involved in signal transduction

the T-cell alloantigen RT6 (Refs 5,6). Furthermore, RT6 was quickly shown to possess the

(cholera and pertussis toxins). The suspicion that mono-ADP-ribosyl-

enzyme activities predicted by the strmtural homology’-lo.

ation might also be used as a mechanism to regulate endogenous metabolism was first corroborated in photosynthetic bacteria. In Rhodos@itlizrm ru5rum and Azospirillium bruziIiense, fixation of nitrogen is regulated by reversible ADP-ribosylation on arginine residues of the key enzyme dinitrogenase reductase2.

Endogenous relatives of ADPribosylating toxins in vertebrates Theidea that endogenous relatives of bacterial toxins regulate protein functions in vertebrates has long aroused the curiosity of biochemists and cellular biologists, and

proteins of approximately 25-30 kDa (Refs 5, 6). Expression of RT6 is restricted to peripheral T cells: thymic lymphocytes and recent thymic migrants do not show any cellsurface expression

of RT6 (Ref. 12). RT6 is

not expressed by B cells or macrophages, nor by cells of any other tissues examined, including liver, muscle, brain and kidney. The restricted expression pattern of RT6 suggests that it has an immunological function. As in the case of several other GPI-anchored T-cell-surface proteins, T cells are activated by treatment with RT6-specific antisera13. Molecular cloning revealed that the rat RT6 antigens are encoded by a single copy gene with two known, highly divergent allele@. The mouse has been shown to carry two functional copies of the gene (designated Rt6.1 and Rt6.2Y4. As in the rat, Rt6 alleles of inbred mouse strains are strikingly polymorphici5. The haplotypes of the closely linked mouse Rt6 genes correlate with the typing of mouse strains for the Hl minor histocompatibility

locus, suggesting

that

Rt6 and HZ may be identicali5. To date, only a single Rt6 homologue has been cloned from the human, and, remarkably, this gene is inactivated by premature stop codor&‘. We have recently identified and cloned a novel human gene family member from the

Discovery and molecular characterization of RT6 RT6 was independently discovered in the mid-1970s by several groups who observed

EST database, which is specifically expressed in spleen and encodes a GPI-anchored protein (E Koch-Nolte et al., unpublished). Possibly, this gene product compensates for that of the inactivated human Rt6 pseudogene.

that, in certain rat strain combinations, alloimmuniiations with lymph node cells induced the production of high-titre T-cellspecific alloantisera. A committee at the 4th

Association of defects in RT5

International

susceptibility for awtoimmune

Workshop

on Alloantigenic

structure and/or expression with

Systems of the Rat assigned the name ‘RT6’ to the cognate antigens**. Subsequent bio-

disease Greiner, Rossini and co-workersi

chemical and molecular analyses showed that the RT6 alloantigens are glycosylphos-

an intriguing coincidence of defective Rt6 expression and increased susceptibility for autoimmune disease in the diabetes-prone

phatidylinositol

(GPIl-anchored

membrane

described

IMMUNOLOGY

I I I I I I I

Bacterial ectoenzymes

L-r CT PT \

Vertebrate ectoenzyrnes

I\]

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CHAT

Mono-ADP ribosyltransferases

FIT6 c3 EDIN ‘\

N

RABNAART ESTs

N

SST-1

Arginine-protein-ADP ibosylglycohydrolases

C Aplysia

cyclase

Fig. I. Mono-ADP-ribosylafion

involves the transfer of the ADP-ribose

moiety from NAD’

(shown as red sfnrs), to a specific anzino acid (skown in

brackets) in a target protein, wkile the nicofinamide moiety is released. At lenst four subfamilies of bncterial mono-ADP-ribosylfransferases Tjze catalytic Sl subunits of diphtheria and Pseudomonas factor 2 (EFZJ by ADP-ribosylafiotz.

kolofoxins

are klzown.

(DTand ETA) penetrate into the cytoplasm of kost cells and inactivate elongation

Similarily, the catalytic A subunits of perfussis, cholera and Escherichia coli toxins (PT, CT nnd LT) penetrate

into the cytopla.=:n nnd ADP-ribosylafe

the 1yszrbunif of regr;lutoy

Staphylococci K’3, EDIN) ADP-ribosylate

small GTP-binding

keferofrimeric

G proteins. Other bacterial exoenzymes [e.g. from Clostridia and

proteins of the ras superfamily

the membrane bartie): Cytop’nsmic dinitroge,2ase-redzIctase-ADP-ribosyltransferases

in vitro. However, it is not clear how they penetrate

(DRAT) in pkotosy>itketic bacteria ADP-ribosylate

inactivate diniiro$enrrse r.edur;ase. Removal of the ADP-ribose moiety by dinifrogenase-reductase-ADP-ribosylglycokydrol~ses key enzyme of nitrogen fixufion. Altkougk

and thereby

(DRAG) reactivates this

members of distinct subfamilies show almost no discernible amino acid sequence identity, the core folds nre

remarkably similar in the fozlr toxin structures known to date: 44 amino acid residues lining a jaw-like nctive-site crevice are superimposabl$O. Knozun vertebrate

mono-ADP-ribosylfransferases

include RT6 and related ectoenyzmes

(CHAT). We have identified addifional genes encoding glycosylpkosphafidylinositol

expressed by muscle cells (RABNAART) (GPiJ-anchored

and bone marrow cells

relatives of RT6 in the EST d&base. The pkysio-

logical target proteins of these enzymes are unknown but several candidate proteirzs are being investigated. A distinct family of membrnne-bound NAD +metabolizing

ecfoenzymes

including CD38 and BST-l/BP-3’ catalyzes the synthesis of cyclic-ADP-ribose

BB rat. Transfusion of RT6+T cells can prevent susceptible rats from developing diseaseIs, and, conversely, treatment of rats with RT6specific antibodies can induce diabetes under certain conditions17. These observations led to the hypothesis

that RT6 marks

a regulatory T-cell subset that mediates protection from autoimmune disease. Consistent with this hypothesis, we have found defects in RT6 structure and/or expression in the (NZBxNZWIFl and BXSB mouse models for autoimmune lupus nephritislg.

The regulatory

role played by

RT6 itself in the pathogenesis of autoimmune disease is still unclear, but the recent finding that treatment of activated cytotoxic T lymphocytes (CTLs) with exogenous NAD+ profoundly downregulates their cytotoxic activities20 may point to an immunosuppressive

function of RT6.

xtracellular target proteins In order to clarify the function of RT6, it will be of great interest to identify its physiological target protein(s). Considering that

(shown as red circles).

the entire polypeptide chain of RT6 is extracellular, an extracellular target is an obvious possibility. The target could, in principle, be in ‘cis’ on the same cell, in ‘tmrzs on the surface of another cell, or soluble. It is also conceivable that RT6, like its bacteriai toxin equivalents, can translocate across membranes to target intracellular ligands.

The problem of NAD+ access If the physiological targets of ecto-ADP-

ribosyltransferases

SEPTEMBER

indeed

turn out to be

I996

extracellular, the question arises: how is access to the required substrate NAD+ assured, considering that NAD+ is an intracellular metabolite for which celI membranes are impermeable? Considering that CTLs have the job of lysing other cells, it has been suggested that they may thus create a local supply of extracellular NAD+, at least for the CTL-surface transferasezo. However, it is difficult to envision an analogous mechanism for the surface enzymes of muscle and bone marrow cells. Perhaps, the possibility of regulated secretion of NAD+, analogous to that of other classic intracellular metaholites (e.g. ATP), deserves investigation. The problem of NAD+ access is posed also for another recently discovered family of NAD+metabolizing ectoenyzmes, the ADPribosylcyclases, which include lymphocytcmembrane proteins CD38 and BST-l/BP-3 (Ref. 21; Fig. 1). Here, too, it is still unclear whether the cyclase itself, its substrate NAD+ or its product cyclic-ADP-ribose crosses the membrane barrier.

Adhesion protein targets? An ecto-ADP-ribosyltransferase on mouse muscle cells catalyzes mono-ADP-ribosylation of the c+ integrin chain upon incubation of intact cells with ecto-NAD+ (Ref. 22). Related integrins influence the binding of activated T cells to other cells as well as the ‘homing’ behaviour of T cells to lymphatic and inflammatory tissues. An attractive hypothesis is that RT6 modifies adhesion proteins. The inhibition of binding of activated CTLs to target cells by an ectc+mono-ADPrlbosyltransferase activity on these cells20 is consistent with this hypothesis. It is likely, but not yet established, that this enzyme is RT6. If 50, it follows that Interference with RT6-mediated downregulation of CTL function5 could lead to enhanced T-cell (autokeactivity, possibly explaining why defective RT6 expression is associated with enhanced susceptibility for autoimmune disease. Of interest in this context is the finding that treatment of nonobese diabetic (NOD) mice with @ntegrin-specific antibodies markedly suppresses progression to autoimmune diseas@. It ls tempting to speculate that the function of ~14integrin is affected similarly by these antibodies as by hypothetical RT6-catalyzed ADP-rlbosylation.

Microbial targets? The ‘star-wars’ (secreted-T-cell ADP-ribosy!transferase) hypothesis of RT5 function proposes that RT6, like its bacterial toxin equivalents, is part of the arsenal of host-parasite interactions. Considering that bacteria and lymphocytes both release mono-ADPribosyltransferases, it could be hypothesized that these enzymes are ancestors of a primitive defence mechanism. We have observed an unusually high degree of allelic polymorphism in the Rt6 genes of laboratory rats and mice as well as of their wild relatives. This could reflect evolutionary pressures to adapt to a polymorphic ligand, for example, a component of the microbial world.

,Cytoplasmic target proteins Bacterial toxins are exoenzymes that have evolved mechanisms for penetrating the cell membrane to reach cytosolic target proteins. Given the structural and functional relatlonship of the vertebrate ecto-ADP-ribosyltransferases to bacterial toxins, it is also conceivable that there exists an analogous mechanism for vertebrate transferases to translocate from the extra- to an intracellular environment. The GPI-linkage of RT6 provides an inherent potential cleavage mechanism, for example, by the action of bacterial or endogenous GPIspecific phospholipases. Indeed, Dennert and co-worker9 have recently shown that the CTL mono-ADP-ribosyltiansferase is released

detergent-insoluble

microdomains,

which

appear to play an important role in signal transduction27, may be of relevance for RT6. RT6 is resistant to solubilization by nonionic detergents5 and treatment of T cells xith RTb-specific antibodies provides a mitogenie stimuIus’3. It can thus be hypothesized that RTb-mediated ADP-ribosylation regulates a component of signal transduction. Indeed, it was recently shown that the GPI-anchored mono-ADP-ribosyltransferase of activated CTLs can use ecto NAD+ to ADP-ribosylate a membrane protein of 40 kDa that exists as a complex with the src-family protein tyrosine kinase Lck (Ref. 28). This ADP-ribosylation was shown to downregulate the tyrosine kinase activity of 1 thereby to suppress CDS-mediated Lc! tra.,snrembrane signalling. Also of interest in this context is the finding that the (Y subunit of the stimulatory heterotrimeric G protein in muscle cells is a target not only for cholera toxin but also for an as yet unidentified transfera5e29.

endogenous

ADP-ribosyl-

.’

ectives These observations have opened new fields of experimental investigation at the interfaces of enzymology, immunology, signal transduction and molecular biology. Considering the extracellular localization of RT6, it is both a potential agent of and target for experimental interventions. It will be of

from the cell surface upon triggering of the T-cell receptor. Mon~ver, a soluble form of RT6 has been demonstrated in sen.mP. It Is

great interest to probe whether recombinant RT6 and/or RTG-specific antibodies modu-

possible that release of RT6 from its membrane anchor is a preparatory step for subsequent

late immune functions in vitro and/or in vivo. Moreover, RtG-knockout mice are eagerly

tmnslocation of the soluble protein across the membrane of a target cell. A ubiquitously expressed cytoplasmic arginine-protein-AD& ribosylglycohydmlas (ARH; see Fig. 1) has been described26 that catalyzes removal of ADP-ribose from linkage to arginine, that is, the reversal of RT6-catalyzed modifications.

awaited as they may provide clues to the physiological function of this intriguing Rlative of ADP-ribosylating toxins on the T-cell

Conceivably, this enzyme counteract5 the effects of intracellular mono-ADP-rlbosylation.

Targets in signal-tra.nsduction cascade(s)? Finally, the fact that many GPI-anchored proteins of lymphocytes associate with

surface.

This article is dedicated to Heinz-Giinter Thiele on the occasion of his retirement. Friedrich Koch-Nolte and Friedrich Haag are at the Dept ofZmmunolo~~, UniversihJ Hospital, D-20246 Hamburg, Germany; Robert Kastelein and Fernando Bazan are at the DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304, USA.

IMMUNOLOGY

Qf@kWbCQS

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with the host’s immune system has recently been achieved. fhe development has been stimulated by the long-acknowledged involvement of immune mechanisms in the pathogenesis and clinical course of melanoma, and by the steady increase in its incidence and mortality rate worldwide (R.M. Ma&e, Glasgow). As a result, more-specific immunotherapy trials are under way.

elanoma progression In horizontal- and Vera,-a!-~o~vth-ph:!+p primary melanomas, hemangiogenesis isul not lymphangiogenesis occurs. However, preexisting lymphatics show signs of acti-

vation and i&alumina1

neoplastic cells are

present, which may contribute to local tumor spread (D.J. Ruiter, Nijmegen). Tumor-associated hemangiogenesis might favor hematogenous spread via the melanoma-associated adhesion molecules sialyl-Lewis X (sLeX), sialyl-Lewis A (sLeA) (T. Kageshita, Kumamoto) and MUCl8 (J.l’. Johnson, Munich), whose expression increases with tumor thickness. In fact, sLeX and sLeA bind to endothelial E- and Pselectin, and MIJCZS interacis with a MLJClB ligand that is expressed on both melanoma and endothelial cells, inducing homotypic adhesion of melanoma cells

and their heterotypic interaction with endothelia. In contrast to melanocytes, which respond only to a limited number of growth factors, melanoma cells constitutively secrete a plethora of cytokines with tumorgrowth-stimulating or -inhibitory activity, or cytokines such as interleukin 10 (IL-lo), which have immunosuppressive properties (M. BGhm, Miinster). Therefore, immunotherapy based

with cellularvaccines

kines released

might

or synthetic-peptidebe affected

at vaccination

by cyto-

or tumor

(see below). The uncoordinated

sites

release of cytokines also influences angiogenesis, adhesion or invasion and the transition of melanocytic cells from common-acquired to dysplastic nevus (IL-8 and melanoma protein l), or from radial- to vertical-growthphase melanoma (platelet-derived growth