Conserved regions of protein disulfide isomerase are targeted by natural IgA antibodies in humans

Conserved regions of protein disul®deisomerase are targeted by natural IgAantibodies in humans

Bob Meek1, Jaap Willem Back2, Vincent N. A. Klaren1, Dave Speijer3 and RonPeek1

1Department of Molecular Immunology, The Netherlands Ophthalmic Research Institute, Meibergdreef 47,1105 BA Amsterdam, The Netherlands2Swammerdam Institute for Life Sciences, Mass Spectrometry Group, and 3Department of Biochemistry,University of Amsterdam, 1105A2 Amsterdam, The Netherlands

Keywords: antibody, antigen, cross-reactivity, epitope, human, lacrimal gland, parasitic protozoan,peptide, repertoire development

Abstract

Secretory IgA (sIgA) antibodies in human tears and milk were found to recognize protein disul®deisomerase (PDI) on a Toxoplasma gondii lysate immunoblot (IB). These antibodies were alreadydetectable in tears of infants. To determine the epitope containing-regions on PDI, we generatedtruncated versions of recombinant PDI that differ by 8±10 amino acids in length. By IB, it wasfound that the sIgA epitopes were con®ned to conserved regions of PDI, including the functionallyessential thioredoxin-like domain. This suggested the capacity of sIgA to react with PDI of otherspecies, which was con®rmed by recognition of human PDI by IgA in tears. In contrast, anti-T.gondii PDI antibodies generated by immunization were not able to cross-react. Binding to thethioredoxin-like domain on IB could be gradually abrogated by incubation with peptide constitutingthe same domain. By consecutive investigation of the function of the protein targeted by sIgA, thepresence of antibody in relation to age and analysis of the epitope constituting regions on PDI wedemonstrate that sIgA directed against PDI are self-reactive natural antibodies. Furthermore,analysis of antibody epitopes on an antigen is a useful method to distinguish conventional,af®nity-matured antibodies from natural antibodies. The presence at early age and continuity ofanti-PDI sIgA in relation to age suggests the existence of B cells secreting germline-encodedantibodies in human mucosa outside of the gut. Overall, the PDI-speci®c antibodies are clearly partof the natural antibody repertoire, suggesting an active role for these antibodies in the innatedefense against pathogens.

Introduction

The humoral component of the mucosal immune system (MIS)

mainly consists of secretory IgA (sIgA) and IgM antibodies.

The most important functions of these antibodies are ìmmune

exclusion' and ìmmune elimination', achieved by binding to

surface antigens on potential pathogens. Ìmmune exclusion'refers to prevention of adherence to epithelial cells and

subsequent infection, while ìmmune elimination' involves

pathogen clearance in the lamina propria and neutralization

of intracellular viruses (1±3). Both conventional (monospeci®c)

and natural (polyspeci®c) antibodies are important for optimal

protection against pathogens (2,4).Conventional antibodies are produced by B2 cells and are

af®nity matured upon exposure to speci®c antigens at induct-

ive sites of the MIS, such as the Peyer's patches in the ileum,

the mesenteric lymph nodes (MLN) and the tonsils. Once

induced, mature B cells migrate to effector sites that can be

Correspondence to: B. Meek; E-mail: [email protected]

Transmitting editor: T. Honjo Received 11 June 2002, accepted 1 August 2002

International Immunology, Vol. 14, No. 11, pp. 1291±1301 ã 2002 The Japanese Society for Immunology

by guest on June 7, 2015http://intim

m.oxfordjournals.org/

Dow

nloaded from

http://intimm.oxfordjournals.org/

located distant from the inductive site (5±7). Whether speci®cIgA can be detected at mucosal surfaces apart from thoseclose to an inductive site depends on immunogenicity andquantity of the antigen(s) presented at these site(s) (5,7).Natural antibodies can be found in any mucosal secretion inman and mice. Characteristically, natural antibodies targetphylogenetically conserved structures of commensals, patho-gens and autoantigens (8±10). Most murine B cells secretingnatural antibodies at mucosal sites express their antibodies inor near the germline con®guration (11). In humans, there doesnot seem to be a well-de®ned population of B cells secretingIgA antibodies in the germline con®guration (12±15), althoughnot all mucosal sites have been studied. Nevertheless, naturalpolyreactive IgA have been demonstrated in human mucosalsecretions (10,16). General features of both natural andconventional antibodies recognizing proteins are summarizedin Table 1.

In a recent study we found that IgA produced by the humanlacrimal gland predominantly recognize 49-, 70- and 74-kDaantigens of Toxoplasma gondii (17). The 49-kDa antigen wasidenti®ed as protein disul®de isomerase (PDI) (18), an essen-tial enzyme that functions in the endoplasmic reticulum ofeukaryotic cells (19). T. gondii is prevalent in northwesternEurope (20), where the previous studies were conducted,resulting in a gradual increase in prevalence of anti-toxoplas-ma serum IgG with age (20,21). Toxoplasmosis is usually self-limiting and benign in immunocompetent individuals, but maycause serious complications in immunocompromised patientsand neonates (20). Penetration of the gut epithelial barrier byT. gondii is required to initiate systemic infection, and is usuallyaccompanied by a vigorous common mucosal sIgA responsein humans and mice (22,23). This suggests that the anti-toxoplasma IgA in tears are the result of occasional encoun-ters with T. gondii and implicates that they are conventionalantibodies. On the other hand, the anti-toxoplasma IgAstaining pattern on immunoblot remained remarkably stablein time (17) and PDI is a highly conserved protein (19),implying a natural origin of these antibodies. Since PDI hasboth species-speci®c as well as highly conserved regions, wehypothesize that conventional antibodies are preferentiallydirected against T. gondii-speci®c, non-`self' regions, whilenatural antibodies will primarily recognize conserved regionsof T. gondii PDI, presumably being molded by `self' antigens.

The relation between age and presence of these antibodiesin tears was determined to see whether they conformed to thecharacteristics of conventional or natural antibodies assummarized in Table 1. Next, we tested our hypothesis byidentifying the protein regions involved in antibody recognitionand determined whether there was cross-reactivity with PDIfrom other species. Our ®ndings strongly support an innate

origin for these antibodies. To our knowledge, this is the ®rstreport of a detailed analysis of a natural antibody response inman.

Methods

Volunteers and sample collection

Tear ¯uid was collected from eyes of healthy volunteers withglass capillaries (Assistent; Karl Hecht, Sondheim, Germany)(24). Spontaneously produced tears were collected frominfants by their parents. Tears were stored at ±20°C, untiluse. Paired tear ¯uid and milk samples were obtained fromthree women at different stages of lactation. Tears of adultsand infants were diluted 200 and 150 times respectively,except when stated otherwise. Human milk was diluted 25times. This study was conducted in accordance with theDeclaration of Helsinki.

Parasites

T. gondii tachyzoites of the RH strain were propagated in vitrounder normal cell culture conditions in RK13 cells in RPMImedium supplemented with 3% FCS. Plasmodium falciparumschizonts and gametocytes were provided by H. Eling(Academic Hospital Nijmegen, Nijmegen, The Netherlands).

Immuno¯uorescence (IF)

IF was performed as described earlier (25), except thatparasites were not ®xated. Brie¯y, 105 ®lter-puri®ed parasiteswere double-labeled by incubation with tears and a mousemAb against surface antigen (SAG) 1 (HyTest, Turku, Finland),diluted 500 times, in PBS containing 2% BSA, at 4 °C for 30min. Following washes, parasites were incubated with anti-human IgA conjugated to FITC, diluted 50 times, and anti-mouse Fab conjugated to indocarbocyanine (Cy3), diluted750 times, for 30 min. Following washing and drying, labeledparasites were embedded in Vectashield (Vector, Burlingame,CA) and visualized using a laser scanning microscope (ZeissLSM 410; Carl Zeiss Microscopie, GoÈttingen, Germany).

Preparation of antigens

T. gondii tachyzoites were ®lter puri®ed using ®lters with a poresize of 3 mm supplemented with a pre-®lter. P. falciparumparasites were provided as pellets and resuspended in PBSwith protease inhibitors before use. Human embryonic kidneycell extract derived from cell line 293T was provided by C.Neefjes (NORI, Amsterdam, The Netherlands). Parasites wereprocessed as described (17). The extracts of P. falciparumand the human cell line were concentrated 8±10 times to allowdetection of PDI.

Table 1.

Protein function (10,16,29,44±46) species-speci®c function conventional antibodyconserved function natural antibody

Agea (20,29,30,47) relation with age conventional antibodyno relation with age natural antibody

aBased on the dynamics of natural IgM and conventional IgM titers.

1292 Human natural IgA antibodies target protein disul®de isomerase



Dow

nloaded from


SDS±PAGE, Western blotting and detection of peroxidase-conjugated antibodies

Procedures were performed as described earlier (17,26). b-

Mercaptoethanol was added to a concentration of 5% prior to

electrophoresis. Following this, proteins were transferred to

PVDF membranes. Membranes were blocked with Tris-buf-

fered saline (TBS: 50 mM Tris pH 10, 150 mM NaCl),

containing 0.5% Tween 20 and 2% non-fat milk powder. All

samples and conjugates were diluted in TBS containing 0.5%

Tween 20 and 0.03% non-fat milk powder (TBS-T). A

peroxidase-conjugated polyclonal antibody preparation spe-

ci®c for human secretory component was diluted 4000 times.

Bound antibodies were visualized by chemiluminescence,

according to the manufacturer's description (ECL; Amersham

Biosciences, Little Chalfont, UK) and exposed to X-ray ®lm.

Primers and peptides

For primers, see Table 2. Synthetic peptide FYAPWCGHCK-

COOH was manufactured commercially (Pepscan Systems,

Lelystad, The Netherlands). PCR products were ligated into

the pGEM-T Easy cloning vector as described by the manu-

facturer (Promega, Madison, WI) or digested and ligated into

expression vector pRP261, a derivative of vector pGEX-3X

(Amersham Biosciences).

Construction of the PDI expression vectors (Fig. 2)

Construction of vectors expressing full length and PDI D2 has

been described previously (18). The PDI D1 mutant was

obtained after EcoRI digestion of full-length PDI in pRP261

and subsequent ligation (see Fig. 2). To determine the location

of the antibody-binding regions in PDI D2, the fragment

encoding PDI D2 was split in two parts, named PDI D3 and PDI

D4. PDI D3 was generated by digestion of pRP261-D2 with SalI

and NcoI, followed by treatment with 1 U S1 nuclease in 100

mM NaCl for 30 min at 30°C and subsequent ligation. The

vector expressing PDI D4 was generated by digestion with

SalI, Klenow ®lling and further digestion with SmaI.For ®ne mapping of the D4 region, truncated versions of PDI

D4 were constructed by cloning NcoI- and EcoRI-digested

PCR products, obtained with PDI 12 as forward primer and

PDI 13±21 as reverse primers into pRP261 (see above).

Expression, puri®cation and detection of recombinantproteins coupled to GST

All proteins were expressed in Escherichia coli strain BL21

according to standard procedures (27). Expression was

induced by adding isopropyl-b-D-thiogalactopyranoside (1

mM) at 30°C. GST fusion proteins were puri®ed using

glutathione Sepharose 4B beads according to the manufac-

turer's instructions (Amersham Biosciences). For protein gels,

beads were directly solubilized in sample buffer (26). The

amount of recombinant protein used for immunoblots was

normalized based on the intensity of Coomassie gel staining

and staining with anti-GST antibody on blot. For the detection

of GST fusion proteins, antibodies were removed using erase

buffer [62.5 mM Tris, pH 6.8, 100 mM b-mercaptoethanol and

2% (w/v) SDS] for 1 h at 70°C. Subsequently, blots were

blocked for 1 h and incubated with anti-GST±peroxidase

(diluted 10,000 times). Visualization by chemiluminescence

was performed described above.

Results

Anti-T. gondii IgA in tears and milk samples

Different mucosal samples were analyzed to determine

whether the secretion of PDI-speci®c IgA antibodies was a

phenomenon restricted to the lacrimal gland or a more general

feature of the common MIS. The latter seems to be the case as

PDI-speci®c IgA was detectable in both milk and tear samples

of lactating women (Fig. 1, M2 and M3), although human milk

had to be used at low dilutions (25 times) compared with tears

(200 times).In an earlier analysis of tears, we only included adults. In

order to shed light on the natural or conventional origin of anti-

T. gondii IgA, we analyzed tears of children for whom it was

highly unlikely that they already were exposed to T. gondii

[prevalence of anti-toxoplasma IgG is very low in these age

groups (21)]. Anti-T. gondii IgA staining patterns from tears

obtained from children and infants were compared with those

of adults (Fig. 1). We readily detected T. gondii PDI-speci®c

IgA antibodies in tears of young children (D1, D2 and D3,

aged 6, 9 and 11 years) as well as in tears of infants fed

exclusively on human milk (B1 and B2, 3 and 5 months of age

respectively). The presence of sIgA in infant tears is not

surprising as infants already produce (natural) sIgA anti-

bodies within 3 days post partum (28).Unexpectedly, three sisters of 6, 9 and 11 years of age

displayed an almost identical IgA staining pattern of total T.

gondii extract blots (Fig. 1, D1±3), with both clear differences

and similarities in comparison to parental IgA staining patterns

(Fig. 1, M1 and F). In addition, the tear IgA staining pattern of

the infant brother (Fig. 1, B1) not only differed markedly from

all his siblings, it also retained its simplicity even after long

exposures; only PDI and the other common constituents of

tear IgA staining patterns comprising a T. gondii 70/74-kDa

antigen doublet (17) being recognized by IgA. The paucity of

antigens recognized and the predominance of anti-PDI anti-

bodies in tears was con®rmed with a tear sample from an

unrelated infant (Fig. 1, B2).

Table 2. Primers used for the production of truncated PDI

PDI 12 5¢-GAGACCATGGTACCCCGTGCCA-3¢ sPDI 13 5¢-GAGAGAATTCGCCCAGGGCGTTCTCAGC-3¢ asPDI 14 5¢-GAGAGAATTCAGCGTCCTTCAGGGAGGT-3¢ asPDI 15 5¢-GAGAGAATTCGGCTTCGTCTTGCTTCTC-3¢ asPDI 16 5¢-GAGAGAATTCGACTCCTTGTACTTCTC-3¢ asPDI 17 5¢-GAGAGAATTCCTCCTTGTAGATGGGCTC-3¢ asPDI 18 5¢-GAGAGAATTCCTTGCAGTAGCCGCACCA-3¢ asPDI 19 5¢-GAGAGAATTCGATCTCCAGCATCACATC-5¢ asPDI 20 5¢-GAGAGAATTCGATGACCATCTCTTCGAA-3¢ asPDI 21 5¢-GAGAGAATTCGTTCTTTCCGACGACAAA-3¢ as

s: sense; as: anti-sense.

Human natural IgA antibodies target protein disul®de isomerase 1293



Dow

nloaded from


Finally, we con®rmed that anti-PDI antibodies are of the sIgA

class using a polyclonal antibody speci®c for secretory

component (SC; Fig. 1, V7).

Expression and analysis of regions involved in epitopeformation

In view of the high percentage (~80%) of volunteers with anti-

T. gondii PDI IgA in their tears (17), it became important to

determine whether different PDI regions were recognized by

IgA in tears of different individuals. Assuming natural anti-

bodies are not speci®c for T. gondii PDI, their ability to cross-

react with PDI of other species would imply that natural

antibodies should be mainly directed against conserved

regions of PDI. To identify T. gondii PDI regions involved in

IgA epitope recognition, mature PDI lacking the N-terminal

signal sequence was expressed as a recombinant GST fusion

protein (Fig. 2). In addition, series of PDI mutants truncated at

either the N- and/or C-terminal end were constructed. Strips of

Western blots with recombinant PDI were incubated with tears

from individuals at dilutions resulting in recognition of PDI on a

T. gondii lysate Western blot. Tear IgA from all individuals

tested recognized the full-length PDI and various large

fragments of PDI (PDI D1, PDI D2 and PDI D4) with the

exception of the region between amino acids 402 and 471

(PDI D3, see Figs 2 and 3).To further delineate the epitope(s) constituting regions

within PDI D4 (amino acids 266±400) several truncated

proteins were produced having their C-terminal end within

this region (PDI D4, D10±12). As shown in Fig. 3 (left panels) all

but one sample (F) failed to bind to PDI D10, indicating that the

regions involved in IgA epitope formation were located at the

C-terminal end of PDI D4, and not in the common sequences of

PDI D1 and PDI D4. Importantly, the region present in PDI D4

and absent in PDI D10 (amino acids 351±400) contains the

thioredoxin-like domain, a motif highly conserved in eukaryotic

PDI sequences (19) (see Fig. 4).

Additional truncated PDI proteins (PDI D5±D10), differing byonly a few amino acids in length (Fig. 2), allowed much moreprecise localization of regions essential for IgA epitopeformation. Two major epitopes were delimited within thisregion, represented in the 9-amino-acid difference betweenPDI D6 and D7 (amino acids 375±384) and the 8-amino-aciddifference between PDI D9 and D10 (amino acids 351±359,Fig. 3). The sequence between PDI D6 and D7 contains thethioredoxin-like domain with the H ® Y amino acid substitution(18), while the sequence between PDI D9 and D10 is a morehydrophobic region with a VKVVV motif. This region is alsoparticularly well conserved between T. gondii and various

Fig. 1. Tear and milk antibody staining patterns on T. gondii lysate blots. To determine whether secretion of anti-PDI antibodies was correlatedwith age or mucosal site, tear samples of siblings D1±3 (aged 6, 9 and 11 years), infants B1 and 2 (aged 3 and 5 months), mothers M1±3,father F, and volunteers V1±7, human milk samples (Milk) and serum sample (Ser) were incubated on T. gondii lysate blots. The serum sampleused was a pool of four sera obtained from patients recently infected by T. gondii. All samples were stained for anti-toxoplasma IgA, exceptfor the `Ser' and `SC' lanes where samples were stained for anti-toxoplasma IgG and anti-secretory (SC) IgA antibodies respectively. Theantibody speci®cally generated against recombinant PDI stained a 49-kDa antigen, as expected (a-PDI). Samples M1 and V7 were diluted500 times; Ser, 1000 times.

Fig. 2. Schematic representation of native (top) and recombinantstructures encoding PDI. PDI was expressed without the predictedsignal sequence. Amino acid positions indicated at each N- and C-terminus refer to their position in the translated sequence of PDI, thepredicted signal sequence included. GEEL is the presumedendoplasmic reticulum retention signal.




Dow

nloaded from


known human PDI sequences (cf. entries XM 053104.1, BC

000425.1, XM 016522.2, AL356378.17 and U75886.1, and

Fig. 4). The sibling analysis depicted in Fig. 3 (D1 and D2)

demonstrated that the thioredoxin-like domain is the main IgA

epitope within PDI D2 for the two sisters. The newborn brother

(B1) did not display IgA reactivity against this region but,

similar to the mother (M1), relied on the VKVVV motif present

between PDI D9 and D10 (amino acids 351±359).These results show that healthy human subjects produce

sIgA recognizing at least three different highly conserved

regions on T. gondii PDI, including the thioredoxin-likedomains.

IgA in tears recognizes thioredoxin-like domains, and cross-react with human and P. falciparum proteins

To con®rm recognition of thioredoxin-like domains by IgA intears, tear samples of V1 and V7 were incubated with blots oftruncated PDI with (PDI D6) and without (PDI D7) a thioredoxin-like domain. Subject V1 was already shown to recognize onlyPDI D6 in this region and not the slightly smaller truncated

Fig. 3. Recognition of truncated versions of recombinant T. gondii PDI by IgA in tears. Each sample contained IgA that recognized PDI D2.Subsequently, samples were incubated with strips containing stepwise truncated (~90 amino acids) versions of PDI D2: PDI D4 and D10±D13(see Fig. 1). All samples that recognized PDI D4 only were subjected to additional ®ne mapping with stepwise truncated (6 8 amino acids)versions of PDI D4: PDI D5±D9 (see Fig. 1). Strips were stained for GST after the PD-speci®c antibodies were removed (upper panels markeda-GST). Fine mapping demonstrated that PDI D6 and D9 contain regions essential for binding of IgA antibodies. In cases of PDI D9recognition, PDI D8 signals were always considerably weaker than PDI D7 and D9 signals. Subject ID and dilutions of tears used correspondwith Fig. 1.




Dow

nloaded from


versions lacking this domain (Fig. 3). Subject V7 had an anti-

PDI staining pattern similar to M1 and B1 (Fig. 3), recognizing

PDI D9 containing the VKVVV motif. This motif is present in

both PDI D6 and D7.Before incubation, peptide FYAPWCGHCK representing the

thioredoxin-like domain was added in various concentrations.

In both cases, increasing amounts of peptide resulted in a

marked decrease in recognition of PDI D6, while recognition of

the other PDI versions was hardly affected (Fig. 5A). This

con®rmed that IgA in tears from subject V1 recognized the

thioredoxin-like domain and indicated that these particular

antibodies form part of the repertoire of subject V7 as well. The

PDI D7 recognition of subject V7 and the `full' recognition of

subject V1 remained intense despite the absence of a




Dow

nloaded from


contribution by the thioredoxin-like domain. It seemed that a

reduction in binding to the thioredoxin domain is compensated

by an increase in binding to the VKVVV motif. Possibly,

binding to these different nearby IgA epitopes in PDI is

mutually exclusive.More importantly, recognition of the highly conserved

thioredoxin-like domains by subjects V1 and V7 implies

species cross-reactive capabilities. To study cross-reactivity,

we compared the reactivity of tear IgA with the reactivity of two

polyclonal antibody preparations directed against T. gondii

PDI and bovine PDI, the latter known to cross-react with

human PDI. The anti-T. gondii PDI polyclonal generated in

rabbit revealed bands at expected heights in our preparations

of tachyzoites and recombinant T. gondii PDI, but not in the

human cell line extract or the P. falciparum extract (Fig. 5B).

The polyclonal against bovine PDI reacted with a 55-kDa

antigen in the human cell extract, but not with antigen in the

other preparations. In marked contrast, the IgA stainingpatterns of infant B1 and volunteer V1 on blots containinghuman or Plasmodium extracts were remarkably similar in theircomplexity (Fig. 5B). The patterns had bands at the expectedheights for human, T. gondii and P. falciparum PDI (Fig. 5B). Athird sample, V7, did not show such a complex stainingpattern on strips with human or plasmodium extract, but alsostained bands at expected heights for the various PDI (resultsnot shown).

T. gondii PDI is a surface antigen

To con®rm our previous ®nding that anti-toxoplasma IgA arecapable of binding to the parasite surface and thus befunctional in defense against T. gondii (Fig. 6A), intactparasites were incubated with anti-SAG1 antibodies, speci®cfor the major membrane protein of T. gondii (25), and dilutedtears of ®ve individuals. While the anti-SAG1 antibody gave a

Fig. 4. Clustal W (1.81) multiple sequence alignment of T. gondii, P. falciparum, Cryptosporidium parvum and Homo sapiens PDI. Overall, theprotein has the highest similarity to PDI from other species of the phylum Apicomplexa: P. falciparum (AJ250363) and C. parvum (U48261),with 210/444 and 171/448 identities respectively, followed by PDI homologues of Ostertagia ostertagi (AJ419174), Humicola insolens (P55059)and Onchocerca volvulus (U12440). There is considerable homology with human PDI (P55, P07237) as well: 163/453 identities. Of the ®vedomains of PDI denoted A±B±B¢±A¢±C (102±60±68±104±16 amino acids), the A domains characteristically display the highest similarity withother PDI, mainly due to conservation of the thioredoxin-like domains (32,33). The alignment pattern of the A¢ domain is similar to the overallalignment, with T. gondii PDI (AJ306291) having most homologues with A¢ domains of the above-mentioned protozoan family members (56/104 and 59/104 identities respectively) and human PDI (53/105 identities). Remarkably, the A domain of T. gondii displays more homologywith PDI of O. ostertagi (62/102 identities), Caenorhabditis briggsae (AJ005807, 61/102 identities), Schistosoma mansoni (S34275, 61/100identities) and Caenorhabditis elegans (S71862, 57/102 identities) than with P. falciparum (54/101 identities). In contrast to the A regions, bothB regions of T. gondii have only limited homology with B regions of PDI of P. falciparum (17/58 and 29/61 identities respectively). A and A¢regions, B and B¢ regions, and C region of human PDI according to (48).

Fig. 5. Recognition of a thioredoxin-like domain and analysis of cross-reactivity of tear IgA antibodies. (A) Tears of subjects V1 and V7 wereincubated with Western blots containing full-length PDI, and two truncated versions with (PDI D6) and without (PDI D7) the thioredoxin-likedomain. Before incubation peptide FYAPWCGHCK was added in the amounts indicated. Strips were stained for GST after the PDI-speci®cantibodies were removed (lower panel marked a-GST). Note that the positions of the bands do not re¯ect the actual mol. wt. of the proteins,as small blot-strips were used in an attempt to minimize volumes required. Subject ID corresponds with Fig. 1. (B) Strips with Plasmodiumschizont extract (pl), human embryonic kidney cell extract (hu), PDI D2 (D2) and T. gondii lysate (tg) were incubated with diluted tears of B1and V1, and polyclonals speci®c for bovine and T. gondii PDI. The `tg' strips were taken from another blot. Strips incubated with tears fromsubject B1 were incubated with the polyclonal speci®c for bovine PDI after tear antibodies were removed. Even upon long exposure, anti-bovine and T. gondii PDI polyclonals showed no cross-reactivity. Cross-reaction by IgA in tears occurred despite considerably lower anti-PDIconcentrations: tears were diluted 100 times compared with 4000 and 20,000 times for bovine and T. gondii PDI respectively.




Dow

nloaded from


homogeneous ¯uorescent pattern contouring all parasites,IgA antibodies in tears gave a coarse ¯uorescence of mostparasites and a homogeneous ¯uorescence of a minority(Fig. 6B, using sample V4). There were differences in¯uorescence patterns between tear samples tested; sampleV1 only displayed a coarse pattern, weaker and more apicallylocalized (not shown). Two other incubations did not givedetectable ¯uorescence patterns. In general, ¯uorescencewas only observed with tear samples showing brisk recogni-tion of PDI.

Although suggesting a possible function, surface staining ofIgA does not help to distinguish between conventional andnatural antibodies as conventional IgM (and most likely alsosIgA) responses are directed against surface-exposed epi-topes of antigens (29), while natural sIgA are capable ofbinding to the surface of bacteria (11) and, as shown here,protozoan parasites.

Discussion

In a recent study we identi®ed T. gondii PDI as the majorantigen recognized by tear IgA (18). Now we demonstrate thatanti-PDI sIgA are present in different mucosal secretions(tears and milk) from adult healthy human subjects, and tearsfrom young children and infants. It is highly unlikely that theseinfants were exposed to T. gondii when nursed and there wereno symptoms of an acquired infection of the mothers duringgestation. Previous ®ndings already pointed out that there isno relation between the presence of these IgA antibodies intears and chronic infection by T. gondii, i.e. no correlation withpresence or absence of T. gondii antibodies in serum. The factthat human milk also contains anti-PDI antibodies con®rmsthat human milk is also a rich source of natural sIgA antibodies(10,16). Clearly, antibodies with speci®city for PDI form part ofthe sIgA repertoire responsible for the primary protection ofnursed infants and probably remain present in tears through-out life (17), stressing their importance.

PDI was one of the few antigens recognized by lacrimal IgAin infants and adults. The paucity of antigens recognized bynatural antibodies in infants as compared to older children andadults has been observed previously in natural IgM repertoires(30). Apparently IgA anti-PDI antibodies are secreted by aselect group of B cells naturally activated early in MISdevelopment. As anti-PDI antibodies are continuously se-creted irrespective of age (17), these B cells apparentlyremain present throughout lifeÐa property shared with naturalIgM-secreting B cells in mice (30). Remarkably, three siblingshad similar anti-toxoplasma IgA staining patterns, includinganti-PDI. Although autoimmune repertoires of natural IgMantibodies in sera of mice within a strain are almost identical,the autoimmune repertoires of congenic mice already differ(31). More research is required to determine the immunologi-cal framework responsible for identical patterns in siblings.

Detailed analysis of amino acid sequences recognized byanti-PDI antibodies showed at least two conserved regionsinvolved, including a thioredoxin-like domain of PDI. Thisdomain is highly conserved among PDI (19,32,33) (Fig. 4),suggesting that anti-T. gondii PDI IgA antibodies could reactwith PDI from other species. This was demonstrated withextracts of the closely related P. falciparum and cultured

Fig. 6. Tear antibody staining patterns of intact parasites. (A) Dilutedtear samples of two individuals were incubated twice with PBS (±) orintact parasites (+), as described earlier (17). The IgA stainingpattern after the second extraction is shown. (B) Intact parasiteswere double stained by incubation with tears of V4, known to have abrisk anti-49-kDa titer, and a mAb speci®c for the major surfaceantigen of T. gondii, SAG1. Staining of parasites by IgA in tears andanti-SAG1 antibodies was analyzed using a laser scanningmicroscope. Tear IgA and anti-SAG1 patterns are superimposed in`Overlay'. Red in the tear anti-IgA±FITC and SAG1±Cy3 picturesindicated saturated pixels. In `Overlay', green indicates predominanttear IgA staining, red indicates predominant SAG1 staining, andyellow indicates co-localization of tear IgA and SAG1 staining. Tearsof V4 were diluted 25 times. Bar: 2 mm.




Dow

nloaded from


human cells. The other region of 8 amino acids usuallycontains at least six conserved residues, the VKVVVxxN motif.

This region is conserved between T. gondii and human PDIsequences (Fig. 4). The conformational ¯exibility of naturalantibodies could allow cross-reactivity with PDI that havemoderately diverged (34,35). That conserved regions within a

conserved protein are recognized, allowing interspeciescross-reactivity, establishes these anti-PDI antibodies as so-called natural antibodies and exponents of the innate immunesystem (see Fig. 5). In contrast, forced immunization with

recombinant T. gondii PDI did not generate antibodies that arecapable of cross-reaction, which indicates that these anti-bodies are primarily directed against species-speci®c regionsof PDI (`B' regions, Fig. 4).

Table 3 summarizes our ®ndings and those obtained byothers regarding characteristics of natural antibodies directedagainst protein antigens. Although analysis of polyreactivityhas been used convincingly to characterize sets of antibodies

(10), this is the ®rst report in which delineation of epitopes hasbeen used for characterization of a particular (natural)antibody.

PDI is a multi-functional enzyme. It belongs to the super-

family of thioredoxins, functioning primarily as an endoplasmicreticulum oxidoreductase of eukaryotic cells. It facilitates denovo formation of disul®de bridges between cysteines (oxi-dation) and/or rearranges existing disul®de bridges (isomer-

ization) in secretory and cell-surface proteins (19). All majorsurface antigens of T. gondii, SAG1-, SAG3- and SAG1-related proteins have high numbers of intramolecular disul®de

bonds (25), which might explain the high expression level of T.gondii PDI.

Although its intracellular localization suggests that PDI isunavailable for binding by sIgA antibodies in human mucosal

secretions, there are indications that PDI has functions apartfrom its role in the endoplasmic reticulum. Despite itsendoplasmic reticulum retention signal, PDI is found on thesurface of several cell types: on thyroid cells, where it is

involved in shedding of the thyroid stimulating hormonereceptor ectodomain (36); on platelets, where PDI mediatesadhesion to integrin (37); on lymphocytes, involved in adhe-sion of HIV (38); and on hepatocytes (39). Our results

demonstrate that T. gondii expresses PDI on its cell surfaceas well. Like PDI on the membrane of human cells, T. gondiiPDI may contribute to adhesion processes, e.g. to host cellsduring infection, being involved in maintenance of structure

and function of surface antigens, and/or the concomitantshedding of surface antigens, such as SAG1 (40). Importantly,this would make mucosal anti-PDI IgA function against T.gondii in the innate, ®rst-line defense immune reaction.

Although there are strong indications that tear IgA also

recognizes P. falciparum PDI, it is not known whether PDI is

exposed on the surface of this parasite as well.Natural antibodies are usually cross-reactive, and recognize

non-self antigens of pathogens and self-related antigens

(2,8,10,34), like the anti-PDI antibodies described here. It is

possible that the anti-PDI antibodies may be selected for their

ability to bind to self-PDI, as both the thioredoxin-like domain

and the VKVVVxxN motif are conserved between PDI. There

are several models to explain natural antibody repertoire

selection (30,31,41), but only one seems compatible with our

results: anti-PDI sIgA could represent the evolutionary con-

served germline repertoire of antibodies (10,30) and form part

of the `natural immune memory' (42,43) or `immunological

homunculus' (31). These antibodies might be selected for their

ability to bind to evolutionary conserved self-antigens having

the potential to be (ab)used by pathogens to adhere to cells

and initiate infection, e.g. as PDI by HIV in adherence to

lymphocytes (38). This implies that secretion of anti-PDI sIgA

could be due to their ability to bind to both `self' and `non-self'

PDI, including T. gondii PDI. The production of these

antibodies appears to be largely con®ned to mucosal surfaces

where dimeric IgA is rapidly secreted, thereby restricting their

autoreactive potential.T. gondii PDI was identi®ed as a major target for naturally

occurring human IgA antibodies secreted at mucosal sites.

The epitopes on PDI recognized by these natural antibodies

are evolutionarily conserved, suggesting they have evolved to

participate in the innate immunity against protozoan parasites

and perhaps other eukaryotic pathogens as well. Whether PDI

can be regarded as a primitive invasion protein that can be

neutralized by primitive (natural) IgA remains to be investi-

gated. Our results suggest that the human lacrimal gland,

along with the mammary gland, is one of the few mucosal sites

with predominant secretion of natural antibodies. This should

be con®rmed by detailed analysis of plasma cells present at

these sites.

Abbreviations

IB immunoblotIF immuno¯uorescenceMIS mucosal immune systemMLN mesenteric lymph nodePDI protein disul®de isomeraseSAG surface antigensIgA secretory IgASC secretory componentSer serum

Table 3. Are the anti-PDI IgA conventional or natural antibodies?

Protein function (10,16,29,44±46) Species-speci®c function conventional antibodyconserved function natural antibody

Age (20,29,30,47) relation with age conventional antibodyno relation with age natural antibody

Epitope-containing regions species speci®c stretches conventional antibodyconserved stretches natural antibody

Our ®ndings are shown in bold.




Dow

nloaded from


References

1 Brandtzaeg, P. 1995. Molecular and cellular aspects of thesecretory immunoglobulin system. APMIS 103:1.

2 Bouvet, J. P. and Fischetti, V. A. 1999. Diversity of antibody-mediated immunity at the mucosal barrier. Infect. Immun.67:2687.

3 Mazanec, M. B., Nedrud, J. G., Kaetzel, C. S. and Lamm, M. E.1993. A three-tiered view of the role of IgA in mucosal defense.Immunol. Today 14:430.

4 Ochsenbein, A. F. and Zinkernagel, R. M. 2000. Naturalantibodies and complement link innate and acquired immunity.Immunol. Today 21:624.

5 Quiding-Jarbrink, M., Eriksson, K., Lakew, M., Butcher, E.,Banchereau, J., Lazarovits, A., Holmgren, J. and Czerkinsky, C.1996. Generalized and compartmentalized mucosal immuneresponses in humans: cellular and molecular aspects. InKagnoff,M.F. and Kiyono,H., eds. Essentials of MucosalImmunology, p. 477. Academic Press, San Diego, CA.

6 McGhee, J. R. and Kiyono, H. 1993. New perspectives in vaccinedevelopment: mucosal immunity to infections. Infect. Agents Dis.2:55.

7 Czerkinsky, C., Prince, S. J., Michalek, S. M., Jackson, S., Russell,M. W., Moldoveanu, Z., McGhee, J. R. and Mestecky, J. 1987. IgAantibody-producing cells in peripheral blood after antigeningestion: evidence for a common mucosal immune system inhumans. Proc. Natl Acad. Sci. USA 84:2449.

8 Avrameas, S. 1991. Natural autoantibodies: from `horrorautotoxicus' to `gnothi seauton'. Immunol. Today 12:154.

9 Murakami, M. and Honjo, T. 1995. Involvement of B-1 cells inmucosal immunity and autoimmunity. Immunol. Today 16:534.

10 Quan, C. P., Berneman, A., Pires, R., Avrameas, S. and Bouvet, J.P. 1997. Natural polyreactive secretory immunoglobulin Aautoantibodies as a possible barrier to infection in humans.Infect. Immun. 65:3997.

11 Bos, N. A., Bun, J. C., Popma, S. H., Cebra, E. R., Deenen, G. J.,van der Cammen, M. J., Kroese, F. G. and Cebra, J. J. 1996.Monoclonal immunoglobulin A derived from peritoneal B cells isencoded by both germ line and somatically mutated VH genesand is reactive with commensal bacteria. Infect. Immun. 64:616.

12 Fischer, M. and Kuppers, R. 1998. Human IgA- and IgM-secretingintestinal plasma cells carry heavily mutated VH region genes. Eur.J. Immunol. 28:2971.

13 Holtmeier, W., Hennemann, A. and Caspary, W. F. 2000. IgA andIgM VH repertoires in human colon: evidence for clonallyexpanded B cells that are widely disseminated.Gastroenterology 119:1253.

14 Dunn-Walters, D. K., Isaacson, P. G. and Spencer, J. 1997.Sequence analysis of human IgVH genes indicates that ileallamina propria plasma cells are derived from Peyer's patches.Eur. J. Immunol. 27:463.

15 Dunn-Walters, D. K., Hackett, M., Boursier, L., Ciclitira, P. J.,Morgan, P., Challacombe, S. J. and Spencer, J. 2000.Characteristics of human IgA and IgM genes used by plasmacells in the salivary gland resemble those used in duodenum butnot those used in the spleen. J. Immunol. 164:1595.

16 Vassilev, T. L. and Veleva, K. V. 1996. Natural polyreactive IgAand IgM autoantibodies in human colostrum. Scand. J. Immunol.44:535.

17 Meek, B., Klaren, V. N., van Haeringen, N. J., Kijlstra, A. and Peek,R. 2000. IgA antibodies to Toxoplasma gondii in human tears.Invest. Ophthalmol. Vis. Sci. 41:2584.

18 Meek, B., Back, J. W., Klaren, V. N., Speijer, D. and Peek, R. 2002.Protein disul®de isomerase of Toxoplasma gondii is targeted bymucosal IgA antibodies in humans. FEBS Lett. 522:104.

19 Frand, A. R., Cuozzo, J. W. and Kaiser, C. A. 2000. Pathways forprotein disulphide bond formation. Trends Cell Biol. 10:203.

20 Beaman, M. H., McCabe, R. E., Wong, S. Y. and Remington, J. S.1995. Toxoplasma gondii. In Mandell, G. L., Bennett, J. E. andDolin, R., eds, Mandell, Douglas and Bennett's Principles andPractice of Infectious Diseases, p. 2455. Churchill Livingstone,New York.

21 Kortbeek, L. M. 1999. Toxoplasmose in Nederland. Ned. Tijdschr.Klin. Chemie 24:65.

22 Mack, D. G. and McLeod, R. 1992. Human Toxoplasma gondii-speci®c secretory immunoglobulin A reduces T. gondii infection ofenterocytes in vitro. J. Clin. Invest. 90:2585.

23 Chardes, T., Bourguin, I., Mevelec, M. N., Dubremetz, J. F. andBout, D. 1990. Antibody responses to Toxoplasma gondii in sera,intestinal secretions, and milk from orally infected mice andcharacterization of target antigens. Infect. Immun. 58:1240.

24 Coyle, P. K. and Sibony, P. A. 1986. Tear immunoglobulinsmeasured by ELISA. Invest. Ophthalmol. Vis. Sci. 27:622.

25 Manger, I. D., Hehl, A. B. and Boothroyd, J. C. 1998. The surfaceof Toxoplasma tachyzoites is dominated by a family ofglycosylphosphatidylinositol-anchored antigens related to SAG1.Infect. Immun. 66:2237.

26 Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227:680.

27 Sambrook, J. and Russel, D. W. 2001. Molecular Cloning, 3 edn.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

28 Cole, M. F., Bryan, S., Evans, M. K., Pearce, C. L., Sheridan, M. J.,Sura, P. A., Wientzen, R. L. and Bowden, G. H. 1999. Humoralimmunity to commensal oral bacteria in human infants: salivarysecretory immunoglobulin A antibodies reactive withStreptococcus mitis biovar 1, Streptococcus oralis,Streptococcus mutans, and Enterococcus faecalis during the®rst two years of life. Infect. Immun. 67:1878.

29 Abbas, A. K., Lichtman, A. H. and Pober, J. S. 2002. Cellular andMolecular Immunology, 4 edn. Saunders, Philadelphia, PA.

30 Mouthon, L., Nobrega, A., Nicolas, N., Kaveri, S. V., Barreau, C.,Coutinho, A. and Kazatchkine, M. D. 1995. Invariance andrestriction toward a limited set of self-antigens characterizeneonatal IgM antibody repertoires and prevail in autoreactiverepertoires of healthy adults. Proc. Natl Acad. Sci. USA 92:3839.

31 Nobrega, A., Haury, M., Grandien, A., Malanchere, E., Sundblad,A. and Coutinho, A. 1993. Global analysis of antibody repertoires.II. Evidence for speci®city, self-selection and the immunological`homunculus' of antibodies in normal serum. Eur. J. Immunol.23:2851.

32 Florent, I., Mouray, E., Dali, A. F., Drobecq, H., Girault, S.,Schrevel, J., Sergheraert, C. and Grellier, P. 2000. Cloning ofPlasmodium falciparum protein disul®de isomerase homologue byaf®nity puri®cation using the antiplasmodial inhibitor 1,4-bis[3-[N-(cyclohexyl methyl)amino]propyl]piperazine. FEBS Lett. 484:246.

33 Freedman, R. B., Hirst, T. R. and Tuite, M. F. 1994. Proteindisulphide isomerase: building bridges in protein folding. TrendsBiochem. Sci. 19:331.

34 Bouvet, J. P. and Dighiero, G. 2000. Cross-reactivity andpolyreactivity: the two sides of a coin. Immunol. Today 21:411.

35 Wedemayer, G. J., Patten, P. A., Wang, L. H., Schultz, P. G. andStevens, R. C. 1997. Structural insights into the evolution of anantibody combining site. Science 276:1665.

36 Delom, F., Mallet, B., Carayon, P. and Lejeune, P. J. 2001. Role ofextracellular molecular chaperones in the folding of oxidizedproteins. Refolding of colloidal thyroglobulin by protein disul®deisomerase and immunoglobulin heavy chain-binding protein.J. Biol. Chem. 276:21337±21342.

37 Lahav, J., Gofer-Dadosh, N., Luboshitz, J., Hess, O. and Shaklai,M. 2000. Protein disul®de isomerase mediates integrin-dependentadhesion. FEBS Lett. 475:89.

38 Ryser, H. J., Levy, E. M., Mandel, R. and DiSciullo, G. J. 1994.Inhibition of human immunode®ciency virus infection by agentsthat interfere with thiol-disul®de interchange upon virus±receptorinteraction. Proc. Natl Acad. Sci. USA 91:4559.

39 Terada, K., Manchikalapudi, P., Noiva, R., Jauregui, H. O.,Stockert, R. J. and Schilsky, M. L. 1995. Secretion, surfacelocalization, turnover, and steady state expression of proteindisul®de isomerase in rat hepatocytes. J. Biol. Chem. 270:20410.

40 Dubremetz, J. F., Rodriguez, C. and Ferreira, E. 1985.Toxoplasma gondii: redistribution of monoclonal antibodies ontachyzoites during host cell invasion. Exp. Parasitol. 59:24.

41 Cohen, I. R. and Young, D. B. 1991. Autoimmunity, microbialimmunity and the immunological homunculus. Immunol. Today12:105.




Dow

nloaded from


42 Martin, F. and Kearney, J. F. 2000. B-cell subsets and the maturepreimmune repertoire. Marginal zone and B1 B cells as part of a`natural immune memory'. Immunol Rev. 175:70.

43 Berland, R. and Wortis, H. H. 2002. Origins and functions of B-1cells with notes on the role of CD5. Annu. Rev. Immunol 20:253.

44 Lydyard, P. M., Quartey-Papa®o, R., Broker, B., Mackenzie, L.,Jouquan, J., Blaschek, M. A., Steele, J., Petrou, M., Collins, P.,Isenberg, D. and Youinou, P. Y. 1990. The antibody repertoire ofearly human B cells. I. High frequency of autoreactivity andpolyreactivity. Scand. J. Immunol. 31:33.

45 Guilbert, B., Dighiero, G. and Avrameas, S. 1982. Naturallyoccurring antibodies against nine common antigens in human

sera. I. Detection, isolation and characterization. J. Immunol.128:2779.

46 Dighiero, G., Guilbert, B., Fermand, J. P., Lymberi, P., Danon, F.and Avrameas, S. 1983. Thirty-six human monoclonalimmunoglobulins with antibody activity against cytoskeletonproteins, thyroglobulin, and native DNA: immunologic studiesand clinical correlations. Blood 62:264.

47 Weksler, M. E. 2000. Changes in the B-cell repertoire with age.Vaccine 18:1624.

48 Ferrari, D. M. and Soling, H. D. 1999. The protein disulphide-isomerase family: unravelling a string of folds. Biochem. J. 339:1.




Dow

nloaded from


Conserved regions of protein disulfide isomerase are targeted by natural IgA antibodies in humans

Documents

Transcript of Conserved regions of protein disulfide isomerase are targeted by natural IgA antibodies in humans