r e v i s i ó n · adhesión celular, la fagocitosis de células apoptóticas, la autoinmunidad,...

R e v i s i ó n

Parasite calreticulin: possible roles in the parasite/host interface

V. FERREIRA1, Mª. C. MOLINA1, C. VALCK, A. ROJAS, A. FERREIRA

Programa de Inmunología, ICBM. Facultad de Medicina. Universidad de Chile. Santiago. Chile

VOL. 21 / NÚM. 3 / JULIO-SEPTIEMBRE 2002INMUNOLOGÍA, 2002; PP 156-168

156

INTRODUCTION

In the 12 years since rabbit and mouse calre t i-culin (CRT) cDNAs were isolated, numero u sother CRT cDNAs have been isolated in mam-

mals (1), insects (2), nematodes (3-6), pro t o z o a(7-9) and plants (10,11). There is a re m a r k a b l ec o n s e rvation of both the genomic org a n i z a t i o nand the amino acid sequence of CRT thro u g h o u tevolution (Fig. 1), in agreement with its role incellular functions.

Recent investigations on the functions of CRT,

RESUMENC a l reticulina es una proteína multifuncional altamente

c o n s e rvada que une calcio y que está presente en el re t í c u-lo endoplásmico de todas las células de organismos supe-r i o res, a excepción de eritrocitos. Entre las importantes ys o r p rendentes funciones de esta proteína se encuentranactuar como chaperona de tipo lectina y participar en pro-cesos tales como el almacenamiento de calcio y la señaliza-ción intracelular, la modulación de la expresión génica, laadhesión celular, la fagocitosis de células apoptóticas, laautoinmunidad, la angiogénesis, el crecimiento tumoral, laactividad lítica de perforinas en células T y NK, interaccio-nes potenciales con re c e p t o res del huésped y la inhibición,C1q-dependiente, de la actividad del complemento in vitro.Algunas de esas funciones podrían modular mecanismosinmunes efectores. También, calreticulina está presente envariados compartimientos sub-celulares. Una característi-ca fundamental del ciclo de vida de los parásitos, es su capa-cidad para adaptarse a cambios de temperatura, pH y estra-tegias de defensa del huésped. Como la calreticulina deparásitos está altamente conservada en sus dominios fun-cionales, sus contribuciones a las relaciones huésped / pará-sito deberían ser evaluadas, en particular la modulación dela infectividad del parásito y la evasión de la respuesta inmu-ne del huésped. Aquí se revisan estos aspectos, con espe-cial énfasis en calreticulina de Trypanosoma cru z i .

PALABRAS CLAVE: Calreticulina/ Parásito/ Complemen-to/ Trypanosoma cruzi.

ABSTRACTCalreticulin, a calcium-binding protein of the endoplasmic

reticulum, is a highly conserved multifunctional protein, pre -sent in every cell of higher organisms, except erythrocytes. Theamazing array of calreticulin-associated important functionsinclude lectin-like chaperoning, calcium storage and signa -ling, modulation of gene expression, cell adhesion, fagocyto -sis of apoptotic cells, autoimmunity, angiogenesis, tumoralg rowth, lytic activity of perforins from T and NK cells, poten -tial interactions with host receptors and inhibition of C1q-dependent complement activity in vitro. Some of these func -tions may modulate immune mechanisms. Also, calre t i c u l i nis present in a wide spectrum of subcellular compartments. Ahallmark of the parasite life cycle is its ability to adapt to chan -ges in temperature, pH and host defense strategies. Since para -site calreticulin is highly conserved in its functional domains,its contributions to the parasite / host relationship should beassessed, in particular modulation of parasite infectivity andevasion of the hosts’ immune system. These aspects are re v i e -wed herein, with special emphasis on Trypanosoma cruzi cal-reticulin.

KEY WORDS: C a l reticulin/ Parasite/ Complement/ Try p a-nosoma cruzi.

CALRETICULINA DE PARÁSITOS: POSIBLES ROLES ENLA INTERACCIÓN HUÉSPED/PARÁSITO

1Both authors contributed equally to this review.

INMUNOLOGÍA V. FERREIRA ET AL.

a calcium (Ca+ 2)-binding protein of the endoplas-mic reticulum (ER) (12,13), have revealed that itplays a variety of important roles in the re g u l a t i o nof key cellular functions (i.e., lectin-like chapero-ning, Ca+ 2 storage and signaling, gene expre s s i o n ,cell adhesion, autoimmunity, angiogenesis, tumo-ral growth, and the lytic activity of perforins fro mT and NK cells) (14,15), while being present inmany subcellular compartments (13).

CRT plays a crucial role in cell homeostasis. Forexample, CRT-deficient embrionic stem cells havei m p a i red integrin-mediated adhesion and inte-grin-mediated extracellular Ca+ 2 influx (16-19).Also, CRT-deficient mice die 14.5-16.5 days post-coitus, most likely from a lesion in cardiac deve-lopment, probably due to impaired ER Ca+ 2 t r a n s-p o rt. The CRT gene is activated during card i a cdevelopment, concomitant with an elevatedexpression of the protein, which decreases sharplyin the newborn heart (20).

A hallmark of the parasite life cycle is its abilityto adapt swiftly to the unique physiology of bothits invertebrate and vertebrate hosts. This involv e sadapting to changes in temperature, pH and hostdefense strategies. This is particularly true forendo parasites and even more so for intracellular

parasites. In this context, the upregulation of CRTRNA from pro to amastigote forms has been obser-ved in Leishmania, indicating that CRT expressionresponds quickly to environmental changes in thes etrypanosomatids (7).

The identification of CRT protein homologuesin various parasites (3,4,7-9,13,21,22) suggeststhat this protein could have many conserved roles.The sharing of several functional domains byv e rtebrate and parasite CRT encourages furt h e rinvestigation on the contributions of this molecul eto the biology of parasites and to their interactionswith their hosts. The major conserved functionsof CRT in parasites and vertebrates are discussedh e re, focusing on their potential contribution toparasite biology and host / parasite interactions,with special emphasis on the Trypanosoma cru z i(T. cruzi) model (Chagas’ disease).

CRT: SEQUENCE AND FUNCTIONALDOMAIN SIMILARITIES

Human CRT (huCRT) is approximately 50%identical to CRT from O n c h o c e rca volvulus,Schistosoma mansoni, Leishmania donovani a n d

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Figure 1. Amino acid sequences of selected CRTs were compared using the Scanps program (NCBI protein matrixapplication). CRT amino acid sequences are presented as follows: (1) Trypanosoma cruzi (Acc. No. Q9U9N9); (2)Trypanosoma congolense (Acc. No. Q967S4); (3) Dirofilaria immitis (Acc. No. O97372); (4) Onchocerca volvulus(Acc. No. P11012); (5) Necantor americanus (Acc. No. O76961); (6) Amblyomma americanum (Acc. No. Q16893);(7) Mouse (Acc. No. P14211); (8) Human (Acc. No. P27797). The black segments represent homologies versus theTrypanosoma cruzi sequence (1). The white segments represent mismatches and the dashes represent gaps in the aminoacid sequences. The arrows indicate where the N, P and C domains start.

PARASITE CALRETICULIN: POSSIBLE ROLES IN THE PARASITE/HOST INTERFACE VOL. 21 NÚM. 3 / 2002

T. cruzi (Fig. 1). The consensus features of all CRTp roteins are a globular N-terminal, a pro l i n e - r i c hP and acidic C-terminal domains (13,23,24) (Fig.2). The amino acid sequences of both the N-ter-minal and P domains of CRT are well conserv e damong species, suggesting important roles in thebasic functions of this protein. The primarysequences of CRT initiate with a signal peptide andterminate with a KDEL ER–retention sequence, oran acceptable variant, which functions in theretrieval of ER resident proteins (14) (Fig. 2).

—N-domain (Fig. 1): It interacts with the DNA-binding domain of the glucocorticoid receptor i nv i t ro (25), with rubella virus RNA (14), with al-pha-integrins (17) and with autoantibodies (26).This domain also includes the N-terminal half ofthe complement component binding S domain(27-29) (Fig. 2).

—P-domain (Fig. 1): It comprises a proline-richsequence with three repeats of the amino acidsequence PXXIXDPDAXKPEDWDE (repeat A)followed by three repeats of the sequenceGXWXPPXIXNPXYX (repeat B). This region ofthe protein binds Ca+ 2 with high affinity (30,31).T. cru z i C RT (Tc C RT) has three consensus Ca+ 2

binding motifs, the same as the human counter-part (KPEDWDE or its conserved variations), andalso both Cys residues present in conserved posi-tions in other CRTs (9, and our unpublished data)(Fig. 2). Repeats A and B are critical for the lectin-like chaperone activity of CRT (32). The P-domainof CRT interacts with perforin (15,33), a compo-nent of the cytotoxic T-cell granules. This domainis one of the most interesting and unique re g i o n sof the protein because of its lectin-like activity andamino acid sequence similarities to other Ca+2 bin-ding chaperones, including calnexin (34). It alsoincludes the C-terminal half of the S domain.

—C - d o m a i n : This domain is poorly conserv e damong CRTs from various sources; however, it hasretained low aff i n i t y, high capacity Ca+ 2 – bindingactivity (13). It is highly acidic and terminates withthe KDEL-ER retrieval sequence (23,24) (Fig. 2).

It combines with over 25 mol of Ca+ 2 / mol of pro-tein (30) and binds to blood clotting factors (35).C a+ 2 binding to this domain of CRT plays a re g u l a-tory role in the control of CRT interaction with cer-tain chaperones (36).

Available evidence indicates that CRT has a con-sensus site(s) for N-linked glycosylation, which isutilized in a species- and/or tissue–specific manner.L e i s h m a n i a C RT is among the few CRTs that havebeen shown to be glycosylated (37). Tc C RT has twopotential O-linked glycosylation sites (9, and ourunpublished data) (Fig. 2). Although multiple sitesfor several protein kinases have also been identified(13), phosphorylation and glycosylation of CRT,appears to be species dependent (37). Little isknown about the glycosylation or phosphory l a t i o nstatus of O n c h o c e rc a a n d Schistosoma C RT.

The possible functional implications of thesesequence and functional domain similarities be-tween mammalian and parasite CRT will be dis-cussed throughout this review.

CRT CELLULAR LOCALIZATIONAND FUNCTION

ER retrieval signals have been predicted fro mthe deduced amino acid sequences of the CRTfrom Leishmania (KDEL) (7), Schistosoma (HDEL)(38) and Trypanosoma (KEDL) (8,9). Interestingly,CRT has non-ER locations, which include: cytoto-xic granules in T cells (15,33,39), cell surf a c e(21,40-46), tick saliva (47), blood serum (48),nucleus (49), cytoplasm (17,37,49), sperm acro-somes (50), and the extracellular space of severalcell types stimulated in vitro (51,52).

The important issue of how CRT escapes the ERretention and is translocated to the cell surf a c eremains to be answered. CRT is strongly bound tothe cell surface, but it does not possess a trans-membrane domain. There, it orchestrates a num-ber of cellular events, including cellular adhesionand migration. Localization of CRT to the cell sur-

158

SH

C-domainP-domainN-domain

KEDL COO -

399281 307A193 266

133103

21

NH3+

LeaderSequence

SH

G

Ca+2Ca+2

36 185

1Amino acid

279 365 373

S-domain

151G

Figure 2. Schematic diagram of Trypanosoma cruzi CRT depicting the four domains and the putative recognitionsites for various functions. Repeat sequence, which includes the consensus Ca

+2binding motif (KPEDWDE); IgG CH2-

like domains (ExKxK); Putative O-glycosylation sites.


face can be induced by subjecting cells to physio-logical stresses such as viral infection and ultra-violet light exposure (37). Pre s e n t l y, we are inves-tigating whether CRT is expressed on the parasitecell surface and its participation in its interactionswith its mammalian host cells (Fig. 3).

C RT can also be released from the cell by eitheractive secre t o ry processes or cell death, mediatingvarious functions. There is now good evidencelocalizing CRT to the secre t o ry pathway from stu-dies on plant cells (53), B16 mouse melanoma cells(54), rat hepatocytes (55) and Ve ro cells (56).I n t e re s t i n g l y, the tick Amblyomma americanum,while feeding on its host, secretes CRT (57), pre-sumably as a mechanism to divert the host’s re s-ponse. The protein becomes a target for both cell-mediated and innate immune responses (possiblygenerating antibodies cro s s - reactive with hostC RT), and parasites might exploit the anti-thro m-botic and complement-inhibiting characteristicsof CRT to supress host defense actions (Fig. 3).S u rface bound CRT on endothelial cells can pro-voke inflammatory events, for example stimu-lation of nitric oxide production. An N-term i n a lfragment of CRT called vasostatin plays an activerole in preventing angiogenesis and tumor growth(14). The extracellular presence of CRT may havea variety of origins. For example, interaction be-tween CTL and its target cells stimulates re l e a s eof granule contents, including CRT, into the extra-cellular space. Also, stimulated human neutro -phils actively secrete CRT and HuCRT has beenfound in the sera of normal individuals (58).

The functional consequences of the presence ofextracellular CRT is an area of extremely activere s e a rch. The presence of CRT in penetrationgland cells of schistosome cercariae suggests are g u l a t o ry influence on Ca+ 2-dependent pro t e a s e sin skin penetration and parasite migration (4).Also, we have shown that seropositive humansp roduce easily detectable antibodies againstTc C RT (8,59,60), strongly suggesting that themolecule should also be accessible to C1q andmannose-binding lectin (MBL), with possibleimplications in the classical and lectin comple-ment pathways, respectively (Fig. 3). One studyindicates that epimastigote Tc C RT is found in them i c rosomal subcellular fraction of the parasite(9), compatible with the presence of an ER re t r i e-val sequence. Localization of Tc C RT using othermethods, such as confocal microscopy in the infec-tive forms of the parasite, are under way in ourl a b o r a t o ry, in order to determine possible surf a c eexpression.

CRT AND CHAPERONE FUNCTION

Like calnexin, CRT has been shown to have lec-tin-like pro p e rties and act as a molecular chape-rone for the correct folding of glycopro t e i n s(61,62). These lectin-like chaperones interact withg l y c o p roteins possessing monoglucosylated N-lin-ked oligosaccharides (Glc1M a n9 - 7G l c N A c2) that aregenerated both by the trimming of outer glucoseresidues by glucosidases and reglycosylation of

159

Chaperone function1

2 Regulation of Ca+2 levels ?3 Modulation of gene expression ?4 Membrane expression ?

CRT

5 CRT secretion ?

8

6

Membrane CRT-C1q or MBL interaction ?

6

7

7 8

9

Parasite cell

Mammalian host cell Hu-CRT

9 Host cell invasion ?

Antigenic properties Autoimmunity ?Protection ?

CRT-C1q and

CRT-MBL

binding

Inhibition of clasical andlectin complement pathwaysImpai red immune complexprocessing and apoptotic cellclearance ?

RE

Golgi

CRT

nucleus

3

4

51 2

10 Increase in CRT levels during cell stressresponse ?

10

C1

or MBL

C1qor MBL

C1qor MBL

Figure 3. Schematic representation of a Trypanosoma cruzi trypomastigote interacting with the host. The numbersrepresent proposed CRT functions in the parasite, based on analogies with the functions in mammalian cells. Questionmarks represent possible functions.


non-glycosylated unfolded proteins by UDP-Glc:g l y c o p rotein glucosyltransferase (63). This ro l ehas been shown for the variant surface glycopro-tein of African trypanosomes, gp63, related pro-teins of L e i s h m a n i a and other glycophosphatidylinositol (GPI) anchored proteins, which comprisethe major antigenic determinants of most unice-llular parasites (64). Thus, Tc C RT specificallyrecognizes free monoglucosylated high-mannose-type oligosaccharides. Mature monoglucosylatedc ruzipain, the principal, highly immunogenicT. c ru z i cysteine proteinase involved in infectivity(65-69), was found to interact with re c o m b i n a n tC RT (9). Thus, the quality control of glycopro t e i nfolding appeared early in evolution. Tc C RT bindsmonoglucosylated oligosaccharides but not thep rotein moiety of cruzipain (9).

CRT AND IMMUNOGENICITY

Tc C RT was first isolated in our laboratory in1991 and named Tc45. It is a 45 kDa immunodo-minant (22), dimorphic antigen, with variablec h romosomal gene localization (8,59). We havecloned, sequenced, and expressed the TcCRT gene(8). Tc C RT from another T. cru z i strain has alsobeen characterized (9).

I n t e re s t i n g l y, native Tc C RT is highly immuno-genic in humans (59,60) and mice (22). Thus,Tc C RT could either be shed by live and / or leakedf rom dead parasites, since B cells can respond tothis antigen (Fig. 3). Tc C RT is immunogenic inA.SW ( H 2

s) mice, both infected or immunized

with total parasite extracts. These animals, uponchallenge, develop a chronic infection. On theother hand, A.CA (H2f) mice, which fail to respondto Tc C RT, develop acute infection. Sepharo s e -P rotein A-purified IgG from chronically infectedA.SW mice passively protects the A.CA congenicc o u n t e r p a rt (22,70). These observations might beindicative of immune protection due to a Tc C RT-specific response in these animals. Antibodiesagainst released or shed Tc C RT may modulate itsputative extracellular functions in the host, withconsequences for parasite infectivity (Fig. 3). Also,S. mansoni C RT is a good T- and B-cell antigen,representing a potential vaccine candidate (71).

An immunometric assay was developed todetect human antibodies against re c o m b i n a n tT. c ruzi or S c h i s t o s o m a C RT. This assay, adequa-tely validated, could complement available diag-nostic methods in terms of specificity and sensiti-vity (59). Additionally, in sera from infectedindividuals suffering from cardiac problems, thepossibility that anti TcCRT antibodies might cross-react with huCRT is investigated in our laboratory.If such antibodies exist, implications with autoim-mune phenomena could be envisaged. For exam-ple, CRT has been identified as a new rh e u m a t i c

disease autoantigen that is associated intimatelywith the Ro/SS-A soluble ribonucleoprotein com-plex, consisting of at least four cytoplasmic RNAcomponents (72). Immune responses againsthuman and parasitic CRT have been detected inautoimmune patients (73).

Autoantibodies to CRT are found in a signifi-cant number of patients with autoimmune di-seases such as systemic lupus ery t h e m a t o s u s(SLE), Sjögren´s syndrome (26), mixed connecti-ve tissue diseases (37), rheumatoid arthritis (74),celiac disease (75) and halothane hepatitis (76).Autoantibodies have also been identified inpatients and transgenic mice overe x p ressing CRT,both suffering from complete congenital heartblock (CCHB) (73,77). Since CRT is involved inC a+ 2 storage, anti CRT antibodies might influencethe development of CCHB in children upon fetaltransfer of IgM autoantibodies leading to passivelya c q u i red autoimmune disease. In patients withactive coeliac disease, there are higher levels ofserum IgA that react with CRT than in healthy con-trols (75).

Similarly, RAL-1, a homologue of CRT found inthe filarial parasite Onchocerca, the causative agentof river blindness, is an immunodominant antigenin onchocercasis (5). These patients have antibo-dies to huCRT, indicating a putative autoimmuneresponse. Indeed, some of the clinical abnorm a l i-ties in human Chagas’ disease as well as onchocer-casis are similar to autoimmune responses (5,78-80), although this is still a rather contro v e r s i a lissue (81,82). Because the RAL-1 protein lacks theER retention signal it could be expeditely secre t e dby the parasite and recognized by the immunes y stem.

Immune responses to simple foreign moleculesthat associate with or mimic host molecules couldpotentially initiate complex autoimmune re s p o n-ses. Ignorance of self-antigens, like CRT, is re a s o-nable because of the low levels of extracellularC RT present under normal physiological condi-tions. Non-tolerogenic self-epitopes might be trig-g e red by molecular mimicry or formation of larg ecomplexes of self antigens, which are no longerrecognized as such. The recognition of RAL-1 andTc C RT, for example, by the host immune systemsuggests that it may be secreted by the parasite.Our unpublished work shows that rabbit anti-huCRT antibodies cross-react with TcCRT, sugges-ting that the opposite (i.e. human anti-TcCRT anti-bodies, generated by the infection, reacting withhuCRT) could also occur (Fig. 3).

The cellular infiltrates in chagasic heart mus-cle lesions involve macrophages and lymphoidcells, together with intense necrosis, which hasbeen shown to appear at the same time immuno-globulin and complement deposition is observ e d(83-85), generating a dangerous source of autoan-tigens, such as host CRT.

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CRT AND COMPLEMENT

Binding to collectins and implications in thecomplement pathways

H u C RT binds to the collagenous region of C1qand members of the collectin family, such as MBL,lung surfactant protein A, bovine conglutinin, andcollectin 43 (86). A cell membrane associated formof CRT may serve as a receptor for C1q and collec-tins (27-29,46,86). The C1q (globular heads andcollagenous portions) and collectin binding siteof HuCRT was defined on its 12 kDa S subdomain/ N-terminal portion (27,29,57,58). An import a n td e g ree of similarity between HuCRT and parasiteCRT, in relevant functional domains, suggests thatparasite CRT may also bind to host C1q, MBL andother collectins, thus interfering with the immun eresponse to certain parasites by inhibiting the clas-sical or lectin pathways of complement activation(Fig. 3). HuCRT has sequences similar to the C1q-binding CH2 domains of IgG (ExKxK, and otherrelated ones) (57,87), a property shared by TcCRT(our unpublished data) and Necantor americanusCRT (4).

In the T. cru z i model, very important infectiveparasite molecules that participate directly in thestage-specific inhibition of the alternate pathway ofcomplement activation, such as CRP, and DAF-likep rotein (88-91), have been described. Thus, F(ab’)2

and Fab fragments directed against these pro t e i n smake the parasite susceptible to the action of thea l t e rnate pathway of complement (92,93).A p p a re n t l y, the classical pathway of complementactivation would be playing an amplifying role inthe T. cru z i model, but it would not be able to elicitan efficient lytic response on its own.

As above mentioned, C1q binds to the CH2domain of IgG via the motif ExKxK, with possiblereplacement of E by T or N and of K by R. TheH u C RT protein sequence contains six short ami-no acid sequences with similar motifs to the C1qglobular head-binding site on IgG (57,87).I n t e re s t i n g l y, the amino acid sequence of Tc C RTcomprises various binding motifs homologous tothe human counterpart, 1 8 5E S K A K1 8 9; 3 6T S K H R40;279T R R T R283; 365E K R K K369; and 373E E R E K377. Onthe other hand, the collagenous tails of C1q andMBL bind to the S sub-domain of huCRT (inclu-ded in the N and P domain) (27,29,57,58) (Fig.2), with functional consequences in the corre s-ponding complement pathways. Import a n t l y, cer-tain regions within the S sub-domain are up to 80%identical between Tc C RT and huCRT. We haved e t e rmined that C1q and MBL bind to re c o m b i-nant Tc C RT S domain, in a dose-dependent, spe-cific and saturable manner. Moreover, this bindinginhibits complement-mediated hemolysis ofimmunoglobulin sensitized ery t h rocytes in vitro(our unpublished data). Recombinant hookworm

CRT also binds to and inhibits the biological func-tion of human C1q and binds specifically to thecytoplasmic signaling domains of a number ofintegrins, adhesion molecules considered impor-tant to leukocyte and platelet function (4).

C1q-mediated immune complex processing

A major contributing factor to autoimmunedisease such as SLE is the failure to clear immu-ne complexes, a process largely mediated by thefirst component of the classical pathway of com-plement, C1q. This mechanism is highlighted bythe fact that patients who lack C1q fre q u e n t l ydevelop active SLE (94). Significantly, it has beendemonstrated that CRT can bind to C1q (57)and, furt h e rm o re, can compete with antibody forbinding to C1q and inhibition of C1q-mediatedhemolysis. There f o re, extracellular CRT mayhave consequences in the etiology of diseasessuch as SLE (94) and Chagas’ (83,95,96), amongothers, where immune complex formation anddeposition participate directly in their pathoge-nesis.

The role of complement in promoting tissuei n j u ry when bound to immune complexes is veryi m p o rtant in the development of an autoimmuneresponse. In the absence of complement, immunecomplexes may escape clearance by the mononu-clear phagocytic system and end up in tissues whe-re they trigger an inflammatory response, with therelease of autoantigens, leading to development ofan autoimmune response (94). Most interestingly,functional hypocomplementaemia could bemediated by parasite CRT by virtue of its capacityto capture C1q (Fig. 3).

C1q and apoptosis

Mammalian cell surf a c e - e x p ressed CRT inte-racts with complement C1q and MBL (46,97) andthe beta-chain of fibrinogen (40). C1q and MBLbind to apoptotic cells and stimulate their inges-tion by ligation of the multifunctional pro t e i n ,CRT (also known as cC1qR) on the phagocyte sur-face. cC1qR is bound to the endocytic re c e p t o rprotein CD91. Ingestion of apoptotic cells throughC RT/CD91 stimulation involves the process ofm a c ropinocytosis, implicated as a primitive andrelatively nonselective uptake mechanism forC1q- and MBL-enhanced engulfment of whole,intact apoptotic cells, as well as cell debris andf o reign organisms to which these molecules maybind (46,98-100). Thus, both non-infective T. cru -z i epimastigotes and vertebrate-stage tissue cultu-re trypomastigotes (TCT) bind C1q in a saturablefashion, at 4 °C. Internalization by mononuclearphagocytes and fibroblasts of TCT, but not epi-

161


mastigotes, bearing C1q is enhanced as compare dto untreated parasites. Purified C1q alone poten-tiates internalization of TCT without an additio-nal re q u i rement for C3 fragments or IgG deposi-tion on the target particle (101).

Parasite surface carbohydrates and mammalianlectins have been implicated in the invasion ofmammalian cells. It has been shown that humanMBL binds to T. cru z i, facilitating its uptake intophagocytic cells. Pre f e rential opsonization ofamastigotes (102) with MBL may account for theirclearance from the circulation and may contributeto the parasites' ability to invade diff e rent cell types.Since we have shown that Tc C RT interacts specifi-cally with C1q and MBL in a dose-dependent andsaturable manner (our unpublished data), the pos-sibility could be entertained that Tc C RT, if locatedon the surface of the parasite, could be acting as aligand for C1q and MBL (Fig. 3).

On the other hand, if C1q is sequestered byparasite CRT (soluble or on the parasite surf a c e ) ,this may affect the clearance of apoptotic cells,generating an increased pool of circulating dange-rous autoantigens (Fig. 3).

Concluding remarks on complement andTcCRT

C1q or MBL binding motifs in mammalian CRTmay represent an evolutionary remnant, devoid offunctional purpose, given the mainly intracellularlocalization of the protein. However, in parasites,they may re p resent pathogen associated molecu-lar patterns (PAMPs), recognizable directly bymammal C1q or MBL. However, our recent resultsindicate that this may not re p resent an innatedefensive strategy. Rather, Tc C RT may be used bythe parasite to modulate host defense mechanisms(i.e. diverting C1q and MBL innate defensive func-tions), by creating a privileged micro e n v i ro n m e n tat the parasite / host interface, mainly in its extra-cellular stage. Alternatively or concomitantly, the-se interactions may participate in invasion, mainlyt h rough CRT binding to C1q (46,101). There f o re ,it could be speculated that this activity of CRT mayhave been conserved by various parasites as amechanism for evading the immune system.

CRT AND CALCIUM BINDING FUNCTION

The contribution of CRT in regulation of Ca+ 2

was demonstrated by altering CRT levels in the cell(103,104). Ca+ 2 release from the ER is impaired inC RT-deficient mouse embryonic fibroblasts, sug-gesting that a role for CRT during cardiac develop-ment likely relates to its effects on ER Ca+ 2 t r a n s-p o rt (20). Ca+ 2 plays important roles in theregulation of metabolic pathways, hormone recep-

tor signal transduction, cell cycle control and acti-vation of nuclear processes such as gene transcrip-tion and the activation of nuclear DNA cleavageby nucleases during programmed cell death orapoptosis (105,106).

Leishmania, Schistosoma and T. cru z i C RTs pos-sess conserved Ca+ 2 binding domains and the firsttwo have been demonstrated to bind Ca+ 2 in vitro(6,7,9, and our unpublished data). Tc C RT hast h ree consensus Ca+ 2 binding motifs, the same asthe human counterpart (KPEDWDE or its conser-ved variations) (9, and our unpublished data). Thei m p o rtance of this function may go beyond themaintenance of Ca+2 homeostasis and impact uponthe release of secondary messengers in re s p o n s eto re c e p t o r-binding or interactions with solublehost proteins. Indeed, the interaction of T. cru z iwith mammalian host cells involves the release ofC a+ 2 into the cytosol. Thus, Ca+ 2 chelators, whichbuffer Ca+2 release, inhibit parasite invasion (107).Recent observations showed that T. cru z i t ry p o-mastigotes or their isolated membranes inducerepetitive cytosolic-free Ca+ 2 transients in indivi-dual normal rat kidney fibroblasts, in a pert u s s i stoxin-sensitive manner (108) and it has been pos-tulated that a trypomastigote membrane factortriggers cytosolic-free Ca+ 2 transients in host cells.C y t o s o l i c - f ree Ca+ 2 transients may be re q u i red forfocal re a rrangement of the cortical actin cytoske-leton allowing lysosome access to the plasmamembrane and lysosome fusion at the site of try-panosome entry (108). A role of Ca+ 2 in the pro-cess of cell invasion by diff e rent parasites such asT. cru z i (109-111), Plasmodium falciparu m ( 1 1 2 ) ,and Leishmania donovani (113) has been postula-ted on the basis of an increase in cytosolic [Ca+ 2]in the host cells after prolonged intracellular pre-sence of parasites. Later, other re p o rts (108,114)have described an early Ca+ 2 signal triggered byparasites. Furthermore, investigation into the con-tributions of CRT to the regulation of Ca+ 2 e ff l u xwithin the host cell may provide new insights intothe cellular mechanisms of parasite invasion andinduced gene regulation.

PARASITE CRT AND IMPLICATIONS OFOTHER POSSIBLE FUNCTIONS

MHC class I presentation of antigens

MHC class I molecules expressed in a CRT-defi-cient cell line (K42) assembled with beta2micro-globulin norm a l l y, but their subsequent loadingwith optimal peptides was defective. The peptide-loading function was specific to CRT since thedefect in K42 could be rectified by transfectionwith CRT, but not with a soluble form of calnexinwhich shares its lectin-like activity (116). CRTreleased by an intracellular parasite (i.e. T. cru z i ),

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capable of entering the cytoplasm, could be pro-cessed via MHC class I (80). The possibility couldbe entertained that certain Tc C RT-derived pepti-des could compete with HuCRT for the binding ofthe MHC molecule, and thus interf e re with pepti-de loading and presentation.

Association with perforins

Ingestion and destruction of T. cru z i by "pro-fessional" phagocytes is a major parasite clea-r a nce mechanism. However, T. cru z i can alsoaccess the cytoplasm of macrophages by penetra-ting the cell membrane and by exiting phagocyticvacuoles, a process facilitated by perf o r i n - l i k emolecules, showing immunological cro s s - re a c t i-vity to complement C9 (117,118). Intere s t i n g l y,in humans, perforin lytic activity of cytotoxic Tcells has recently been re p o rted to be contro l l e dby CRT, by preventing perforin from forming poresin the granule membrane, either by Ca+2–chelation(39) or direct interactions with perforin (33).H o w e v e r, more recent work has suggested thatC RT has a more active role in preventing autolysisof the lymphocyte by binding directly to the cells u rface (15). Experiments perf o rmed on ery t hro c y-tes showed that CRT bound to their membrane,w h e re it prevented the insertion of perforin andhence prevented cell lysis.

Many micro o rganisms have evolved successfulescape strategies to avoid immune-cell-mediatedattack. Epimastigote, amastigote, and try p o m a s t i-gote forms of T. cruzi a re resistant to purified per-forin at doses up to 100-fold larger than that suff i-cient to kill susceptible tumor cells. T. cru z i re s i s tp e rforin attack by avoiding transmembrane poref o rmation. Resistance to perforin is not trans-f e rred to host cells since infected macro p h a g e scould be easily destroyed by perforin while intra-cellular amastigotes remain intact (119). Perh a p sTcCRT plays a similar role in the parasite, possiblystabilizing the parasite membrane.

CRT and heat shock

The nucleotide sequences of the mouse and thehuman CRT gene show greater than 70% identity,indicating a strong evolutionary conserv a t i o n(120,121). The CRT promoter is activated by Zn+(122) and heat shock (123). Expression of CRT isalso induced by viral infection (124), by aminoacid deprivation (125) and in stimulated cytoto-xic T cells (126,127), further indicating that theC RT gene is activated by a variety of chemical andbiological stresses. Although, in general, the levelof protein expression correlates with transcriptionand translation re g u l a t o ry elements, positivecorrelations with the number of coding genes have

also been described. The glycosylation pattern ofthe protein seems to be heterogeneous and doesnot appear to be a conserved pro p e rty of the pro-tein. Heat shock may trigger glycosylation of CRT(128,129); however, the functional consequenceof this stress-induced glycosylation of the pro t e i nis presently not clear. Our unpublished observ a-tions indicate that Tc C RT has a moderate heatshock protein behavior, as shown by increases inboth mRNA and CRT, after in vitro transfer of epi-mastigotes from 28 °C (the vectors’ temperature )to 37 °C (the hosts’ temperature).

C RT overe x p ression and induction of apopto-sis

O v e re x p ression of CRT promotes the diff e re n-tiation-dependent apoptosis in H9c2 cells by sup-p ressing the Akt signaling pathway. This indicatesa novel mechanism by which cytoplasmic Akt sig-naling is modulated to cause apoptosis by CRT(130). It remains to be determined whether secre-ted parasite CRT is capable of inducing similare ffects, with pathological consequences. Accor-d i n g l y, apoptosis could play a role in the clea-r a nce of lymphomononuclear cells in the inflam-m a t o ry infiltrate in chronic chagasic myocard i t i s(84).

CRT overexpression in heart disease

Several studies have shed light on gene expre s-sion changes in several forms of heart disease,including heart failure, familial hypert rophic car-d i o m y o p a t h y, and primary dilated card i o m y o-p a t h y. Changes in the expression of Ca+ 2 - trans-p o rting proteins and their regulators have beeno b s e rved in many forms of acquired and genetich e a rt diseases, most notably in cardiac hyper-t rophy and heart failure. These changes seem tobe secondary to the primary cardiac dysfunction,but recent publications have implicated thesechanges as major contributors to systolic and dias-tolic dysfunction. Ion channels, as well as Ca+ 2-binding proteins such as calsequestrin and CRTa re important in the regulation of global cytosolicas well as localized Ca+ 2 concentrations in the dif-f e rent cell compartments. The normal adult myo-c a rdium expresses only low levels of CRT, raisingthe possibility that CRT is induced with the fetalgene program that is reactivated during card i a chypertrophy and failure. However, other fetal geneproducts normally induced in hypertrophy are notfound in mice overe x p ressing CRT (20,73,131).I n t e re s t i n g l y, approximately 20-30% of peopleinfected with T. cru z i p ro g ress to the cardiac formof the chronic phase, with high mortality due tocongestive heart failure and arrhythmias. There is

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a well-documented inflammatory character ofChagas’ heart disease, diff e rentiating itself fro mother clinically less severe non-inflammatory dila-ted cardiomyopathies (80,132). Although T. cru z ihas marked tropism for cardiac muscle cells (81),it remains to be determined whether parasite CRTlevels within the invaded cell (especially in theintracytoplasmatic stage) could affect the heart s ’physiological function.

CRT and cell adhesion

As previously mentioned, CRT modulates celladhesion. This modulation could be perf o rm e df rom inside the cell through an interaction withintegrin tails or through the regulation of focal-adhesion-associated proteins, as well as thro u g hthe modulation of cytosolic phosphotyro s i n elevels. Another possibility is that CRT can modu-late cell adhesion from the cell surface. CRT hasbeen demonstrated to bind to the extracellularmatrix proteins Bb fibrinogen (40) and laminin(41), and it has been re p o rted that cell-surface CRTcan complex with integrins (54,133). The integrinfamily of receptors possesses a CRT-binding motif(KxGFFKR) (18). Association of CRT with thecytoplasmic tail of integrins in vitro and in vivo i sC a+ 2 regulated and alteration of the expression ofintegrin receptors on cell surfaces was found toa ffect the ability of these cells to attach and spre a don substrates (18). There f o re, it is possible thatC RT bound to integrins could modulate the aff i-nity state or signaling activity of such re c e p t o r s .The apparent auto-kinase activity associated withmammalian (37) and leishmanial CRT may havephysiological implications in this situation. Thesestudies suggest that CRT may mediate parasite inte-ractions with host cell receptors and, thus, mayp rovide insight into the mechanisms used by para-sites for host cell invasion. In this context, it is inte-resting to note that S c h i s t o s o m a C RT was localizedin penetration gland cells of cercariae (115).

It has been shown that thro m b o s p o n d i n - i n d u-ced disassembly of focal adhesins is mediated byc e l l - s u rface CRT (44). Thro m b o s p o n d i n - re l a t e danonymous protein (TRAP), a candidate malariavaccine antigen, is re q u i red for Plasmodium s p o ro-zoite gliding motility and cell invasion. TRAP con-tains an A-domain, a well-characterized adhesivemotif found in integrins (134). If parasite TRAPinteracts with either parasite CRT or with host CRT,p resent on the cell surface, consequences on theparasites’ ability to invade cells are possible.

CONCLUDING REMARKS

Several precedents obtained with huCRT arefundamental for the study of parasite CRTs. HuCRT

has several functional C1q binding domains withfunctional consequences, has chaperoning activi-ties, modulates perforin activity, is present in nor-mal human plasma and on cell surfaces, and iss e c reted by neutrophils. On the other hand, cer-tain parasite CRTs have several putative C1q bin-ding domains, are associated, in a chapero n e - l i k efashion with molecules important for infectivity,a re secreted, are immunogenic in both mice andhumans and have diagnostic potential. Based onthese facts our laboratory undertakes re s e a rc horiented at understanding the contributions ofTc C RT to the biology of T. cru z i / host interactions.

ACKNOWLEDGEMENTS

S u p p o rted by grants 1010930 (A.F.) and2010069 (V. F.) from the Chilean National Fundfor the Development of Science and Te c h n o l o g y(FONDECYT). We are grateful to Mrs. JuanaO rellana for her expert technical assistance in theT. cruzi calreticulin-related work.

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CORRESPONDENCE:Arturo FerreiraPrograma Disciplinario de Inmunología, ICBMFacultad de MedicinaIndependencia 1027, casilla 13898, correo 21Independencia. Santiago. ChilePhone and fax: +56 2 7353346e-mail: [email protected]


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r e v i s i ó n · adhesión celular, la fagocitosis de células apoptóticas, la autoinmunidad,...

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