plasmodium falciparum malaria is a major human disease that accounts for 12 million deaths annually . the disease affects predominantly children under the age of five , as older children and adults living in malaria endemic areas become immune against severe forms of the disease after being repeatedly exposed to the parasite .   during blood - stage development , p. falciparum parasites invade red blood cells ( rbc ) and cause them to sequester from the blood circulation by adhering to host endothelial cells . both invasion and sequestration are central to the pathogenesis of malaria , and they require an adhesive cysteine - rich domain referred to as the duffy - binding - like ( dbl ) domain . the dbl domains are present in two different protein families ; the erythrocyte - binding - like ( ebl ) family of invasion proteins and the large and diverse p. falciparum erythrocyte membrane protein 1 ( pfemp1 ) family of cytoadherence proteins [ 2 , 3 ] . we recently reported the identification of a previously unknown domain in the p. falciparum antigen 332 with a high degree of similarity to the dbl domains of the ebl family . most ebl members are transmembrane proteins , which are expressed in schizonts and localize to merozoite micronemes from where they are released prior to or during host cell invasion . in contrast , pf332 is expressed in trophozoites and is later on cotransported together with the surface - destined pfemp1 in parasite - induced membrane structures , referred to as maurer 's clefts [ 6 , 7 ] . in schizonts , pf332 can be found in association with the rbc plasma membrane , although it is not clear whether the antigen becomes surface exposed or not [ 4 , 7 ] . pf332 is a large protein of approximately 700  kda consisting predominantly of degenerate repeats rich in glutamic acid ( glu ) [ 810 ] . pf332 further lacks the typical ebl gene structure with the tandem dbl domains and the cysteine - rich region adjacent to the transmembrane domain . in pf332 , the single dbl domain is located in the n - terminus of the protein and is followed by a putative transmembrane region and a large number of negatively charged glu - repeats , which make up a major part of the molecule ( figure 1(a ) ) . in addition , pf332 has a c - terminal tryptophan - rich region ( wrd ) with similarities to wrds found in surfins , pksicavar , and pfemp1 . although the function of pf332 is not clear , the antigen is present in all isolates investigated [ 4 , 10 ] , indicating that it is of importance for the parasite . previous studies on pf332 have primarily focused on eb200 , a 157 amino - acid - long glu - rich fragment , located in the central repeat region of the protein ( figure 1(a ) ) . individuals living in malaria endemic areas have been shown to have antibodies recognizing this region [ 12 , 13 ] and these antibodies were associated with fewer clinical malaria attacks . however , antibodies recognizing the repeat regions in pf332 are possibly cross - reactive with other glu - rich antigens in p. falciparum such as pf155/resa , making it difficult to ascertain the antibody specificity . the dbl domain of pf332 is conserved between different parasite strains but with a distinct sequence , as compared to the dbl domains of the ebl family . the conservation and distinct sequence of the dbl region makes it an attractive target for more detailed and specific studies on the molecule pf332 .   in the present study , we have expressed soluble pf332-dbl recombinant protein in escherichia coli and analyzed its folding and secondary structure content by circular dichroism ( cd ) spectroscopy . we have further created a homology model of pf332-dbl in order to assess structural similarities to the dbl domains of the ebl family . the predicted three - dimensional structure was found to have a fold similar to the dbl domains of p. falciparum eba-175 and p. knowlesi duffy - binding protein , which was in good agreement with the measured cd data . we have further examined naturally acquired antibodies towards the dbl domain of pf332 and a dbl1-domain from the variant surface antigen pfemp1 , in order to get a better insight into the immunogenicity of the dbl domain of pf332 . we here show that pf332 is widely recognized in distinct malaria endemic regions and that antibodies towards pf332-dbl do not display any cross - reactivity with dbl - containing ebl proteins . this makes the dbl domain an attractive region to use in further studies on the pf332 molecule . the dbl1-domain of var1fcr3s1.2 and the dbl domain of pf332 were amplified with oligonucleotide primers ( f - dblfcr3s1.2ccatgg cta ctt ccg gag ga , r - dblfcr3s1.2agatct gagacg gta ctt ctt ctg ctt atc ; f - dbl3d7pf332gcatgc gaa gca aca tca aca aca agg , r - dbl3d7pf332ggatcc gta ctt ctt ctc gaa cac c ) from genomic dna and cloned into the ncoi and bglii or sphi and bamhi restriction site of the pqe60 or pqe70 vector ( qiagen , dsseldorf , germany ) , respectively . his - tagged recombinant pf332-dbl and var1fcr3s1.2-dbl1 proteins were expressed according to standard procedures with modifications . briefly , sg13009 e. coli ( qiagen ) were grown in lb medium until od600 reached 0.81.0 , and the culture was subsequently induced with 100  m iptg and grown for an additional 3  h at room temperature . the bacteria were harvested by centrifugation at 990  g for 20  min , after which the pellet was resuspended in washing buffer ( 20  mm hepes ( ph 7.4 ) , 30  mm imidazole , and 500  mm nacl ) . 1  mg / ml lysozyme ( biosite , san diego , ca , usa ) was added , to the lysate , which was incubated for 30  min on ice . complete cocktail protease inhibitors ( roche , basel , switzerland ) were added and the cells were broken by sonication on ice . the lysate was clarified by centrifugation at 13250  g for 20  min , after which the supernatant was collected and filtered through a 0.45  m filter . for protein purification , a his gravitrap column ( ge healthcare , uppsala , sweden ) was used , according to the manufacturer 's instructions . the column was washed extensively with washing buffer and the bound protein eluted with elution buffer containing 20  mm hepes ( ph 7.4 ) , 500  mm imidazole , and 500  mm nacl . all eluted fractions of pf332-dbl were analyzed by sds - page using a 12% separation gel , and fractions containing the highest concentration of pure protein were pooled , dialyzed overnight against phosphate buffered saline ( pbs ) , and finally concentrated using centriprep centrifugal filter units ( millipore , billerica , ma , usa ) . possible aggregates of pf332-dbl were thereafter spun down ( 5  min at 16060  g ) and the supernatant was applied to a 16/60 superdex 200 size exclusion chromatography column ( ge healthcare ) , equilibrated with 20  mm hepes ( ph 7.5 ) , 300  mm nacl , and 10% glycerol at a flow rate of 0.5  ml / min . the elution was followed by uv absorbance at 280  nm , and the peak fractions were collected and analyzed by sds - page , as above . the identity of the purified protein was confirmed by mass spectrometry using the royal institute of technology ( kth , stockholm , sweden ) mass spectrometry core facility . circular dichroism ( cd ) spectroscopy measurements of pf332-dbl in pbs ( ph 7.4 ) were conducted on a jasco j-810 spectropolarimeter , using a cuvette with a 0.1  cm path length . the spectra were accumulated from eight scans at 20c , using a 0.1  nm data pitch , 1  s signal response time , and a 1  nm bandwidth . the average spectra were corrected by subtracting the background spectrum measured for the buffer . a homology model of 3d7 pf332-dbl ( pf11_0506 amino acids briefly , the hhpred method is specialized in detecting homology using hidden markow models built from psi - blast profiles and secondary structure . the crystal structure of the eba-175 f2-domain was used as a template ( protein data bank code 1zro   chain a . the alignment was manually adjusted at positions 180 and 254 to align two cysteines , which are in close proximity , to allow for the modeling of two disulphide bridges conserved in pfeba-175 and pk-dbp . the model was validated by using the procheck program , available in the swiss - model workspace   and verify3d , available in the hhpred server toolkit . plasma samples from adult donorsin uganda , plasma samples were collected from women over 18 years , taking part of a cross - sectional investigation in 2006 on malaria infection in pregnancy at mulago hospital , uganda 's national referral hospital in kampala . ethical permissions have been obtained both in sweden and uganda ( permit number 04 - 533/2 and mv922 ) . in mali , plasma samples were collected at the end of the malaria transmission season in november 2001 in the malaria mesoendemic mopti area . adult donors belonged to different sympatric ethnic groups ( fulani and dogon ) living 3001000  m apart . in burkina faso , plasma samples were collected during the peak of malaria transmission season in september and october 1999 in a malaria holoendemic area outside ouagadougou , the capital of burkina faso . studies in both mali and burkina faso have shown that the fulani tribe is less parasitized , has higher levels of antimalaria antibodies , and suffers from fewer malaria attacks compared to the mossi and dogon tribes despite the same level of malaria transmission [ 2628 ] . ethical permissions have been obtained both in sweden and mali ( permit number 03 - 536 and 1866/fmpos ) and burkina faso ( permit number 98 - 086 and 9800839/ms / sg / dep ) . 
  in uganda , plasma samples were collected from women over 18 years , taking part of a cross - sectional investigation in 2006 on malaria infection in pregnancy at mulago hospital , uganda 's national referral hospital in kampala . ethical permissions have been obtained both in sweden and uganda ( permit number 04 - 533/2 and mv922 ) . in mali , plasma samples were collected at the end of the malaria transmission season in november 2001 in the malaria mesoendemic mopti area . adult donors belonged to different sympatric ethnic groups ( fulani and dogon ) living 3001000  m apart . in burkina faso , plasma samples were collected during the peak of malaria transmission season in september and october 1999 in a malaria holoendemic area outside ouagadougou , the capital of burkina faso . studies in both mali and burkina faso have shown that the fulani tribe is less parasitized , has higher levels of antimalaria antibodies , and suffers from fewer malaria attacks compared to the mossi and dogon tribes despite the same level of malaria transmission [ 2628 ] . ethical permissions have been obtained both in sweden and mali ( permit number 03 - 536 and 1866/fmpos ) and burkina faso ( permit number 98 - 086 and 9800839/ms / sg / dep ) . plasma samples from childrenplasma samples were collected in 2002 from children under the age of five with an ongoing p. falciparum infection in apac , a malaria holoendemic area   250  km north of kampala , uganda . children were identified with mild or severe malaria according to who guidelines   and the modified blantyre scale . ethical permissions for the study have been obtained both in sweden and uganda ( permit number 03/095 and mv717 ) . plasma samples were collected in 2002 from children under the age of five with an ongoing p. falciparum infection in apac , a malaria holoendemic area   250  km north of kampala , uganda . children were identified with mild or severe malaria according to who guidelines   and the modified blantyre scale . ethical permissions for the study have been obtained both in sweden and uganda ( permit number 03/095 and mv717 ) . maxisorp plates ( nunc , roskilde , denmark ) were coated overnight at 4c with antigens dissolved in 15  mm na2co3 and 35  mm nahco3 ( ph 9.6 ) at a concentration of 10  g / ml . the plates were blocked with 1% bovine serum albumin ( bsa ) in pbs containing 0.1% tween 20 ( pbs - t ) for 1  h at room temperature and subsequently washed three times with pbs - t . all plasma samples were titrated six - fold by serial dilutions in pbs - t to determine concentration dependency , added in duplicates and allowed to bind for 1  h at 37c . the plates were washed and bound immunoglobulin g ( igg ) was detected by incubation for 1  h at 37c with alkaline phosphatase - conjugated goat anti - human igg ( sigma , st . louis , mo , usa ) diluted 1  :  1000 in pbs - t . the plates were washed and the assay developed by adding p - nitrophenyl phosphate ( sigma ) as a substrate for 15  min . the optical density ( od ) was read at 405  nm in an elisa plate reader ( multiskan ex version 1.0 , labsystems , stockholm , sweden ) . on each plate , a pool of plasma from 11 nonmalaria exposed swedish donors was assayed as well as control wells without plasma ( background level ) . cutoff threshold for seropositivity was determined as the mean od405 value plus three standard deviations from the 11 swedish donors . amount of igg was expressed as od ratio and calculated as follows : ( od sample  od background)/(od swedish nonimmune pool  od background ) . the presented results and data used for statistical analyses were obtained with plasma samples diluted 1  :  500 . the dbl domain of pf332 was coupled to cnbr - activated sepharose ( ge healthcare ) according to manufacturer 's instructions and the coupled medium slurry was thereafter degassed and packed into an empty polystyrene column ( thermo fisher scientific inc . , rockford , il , usa ) . for purification , 20  ml of plasma from a pool of adult donors ( reactive to pf332-dbl in elisa - assays ) were diluted 1  :  4 in degassed pbs , sterile filtered , and loaded onto the column and allowed to pass three times . the column was washed extensively with pbs and antibodies were eluted with 150  mm glycine ( ph 2.5 ) in 10  1 ml fractions and immediately neutralized with 100  l 1  m tris buffer ( ph 8.6 ) . antibody concentration was determined spectrometrically , using the molar extinction coefficient of 280 = 210 000  m  cm for immunoglobulins . fractions containing antibodies were pooled , dialyzed against pbs , and finally concentrated using centriprep centrifugal filter units ( millipore , billerica , ma , usa ) . slides with p. falciparum peptide epitopes were manufactured by jpt ( berlin germany ) and consisted of three identical subarrays . each subarray had a set of 52 overlapping peptides of 15 amino acids shifted by four residues covering 3d7 pf332-dbl ( pf11_0506 , spanning amino acids 1219 ) and 148 overlapping peptides covering 3d7 pfeba-175 ( mal7p1.176 , spanning amino acids 145747 ) . the peptides were bound chemoselectively to the microarray surface by coupling of an active amine ( from the peptide ) to an epoxy - group ( from the slide surface ) . the slides were hybridized with 5  g / ml human igg or human affinity - purified pf332 antibodies diluted in pbs containing 3% fetal calf serum and 0.5% tween 20 ( pbs - t ) in a humid chamber for 16  h at 4c . next , the microarray slides were washed five times : two times with pbs - t and three times with distilled water . the slides were subsequently incubated with a secondary cy5-coupled goat anti - human antibody ( 1  :  500 ; jackson immunoresearch , suffolk , united kingdom ) for 2  h at room temperature in a humid chamber , followed by washing steps . , foster city , ca , usa ) at a wavelength of 635  nm and images were saved in tiff and jpg formats . image analysis was performed utilizing the circular feature alignment of the genepix pro 6.0 software ( axon instruments inc . ) and the genepix array list ( gal ) files supplied by jpt . to measure antibody response , the mean fluorescence intensity given by the difference between the foreground and the local background of the circular features briefly , parasites were cultivated at a 5% hematocrit in malaria culture medium supplemented with 10% swedish nonimmune a serum . parasites were synchronized with sorbitol treatment and synchronous parasites were harvested at different time - points for indirect immunofluorescence assays and western blot . for immunofluorescence assays , monolayers of synchronous fcr3s1.2 infected rbc ( irbc ) were prepared as previously described . monolayers were blocked for 1  h with 3% bsa in pbs and subsequently incubated for 1  h with affinity - purified human anti - pf332-dbl ( 1  :  2000 ) , polyclonal rabbit anti - pf332-eb200 ( 1  :  500 ) , polyclonal rabbit anti - mahrp1 ( 1  :  10 , kind gift from hans - peter beck ) , monoclonal mouse anti - pf332-dbl ( 1  :  800 ) , or polyclonal rabbit anti - eba 175 rvi ( 1  :  1600 , kind gift from john h. adams ) , all diluted in blocking buffer . slides were thereafter washed three times with pbs and primary antibodies were detected by incubation for 1  h with secondary alexa - fluor 594-conjugated anti - human igg , alexa - fluor 488-conjugated anti - rabbit igg or alexa - fluor 594-conjugated anti - mouse igg ( dilution 1 : 1000 ; invitrogen , carlsbad , ca , usa ) . to avoid photobleaching , cells were mounted with vectashield ( vector laboratories , burlingame , ca , usa ) containing 1.5  g / ml 4,6-diamidino-2-phenylindole ( dapi ) to stain for parasite dna . cells were viewed with a 100  oil immersion objective in a uv equipped nikon eclipse 90i/80i microscope ( nikon corporation , japan ) and images were captured by a hamamatsu orca - er ccd digital camera ( hamamatsu photonics system , japan ) and micromanager software version 1.3 ( developed by arthur edelstein , nico stuurman and nenad amodaj , university of california , san francisco , usa ) . all images were finally processed using imagej software version 1.43 ( national institutes of health , bethesda , usa ) . were enriched by magnetic cell sorting ( miltenyi biotec , bergisch gladbach , germany ) and returned into culture to reach approximately 3640  h p.i . samples were lysed in sds - loading buffer , separated on a 6% sds - page ( 2.5  10  irbc / lane ) and transferred onto nitrocellulose membranes ( bio - rad , hercules , ca , usa ) . membranes were probed with affinity - purified human anti - pf332-dbl ( 1  :  3000 ) , anti - eb200 ( 1  :  1000 ) , and monoclonal anti - pf332-dbl ( 1  :  100 ) . detection by enhanced chemiluminescence ( ecl plus western blotting detection reagents , ge healthcare ) was performed after a secondary probe of hrp - coupled anti - human igg , antirabbit igg , or anti - mouse igg ( 1  :  5000 , ge healthcare ) were added . statistical analyses were performed with prism version 5.0 ( graphpad software , san diego , usa ) . student 's paired t - test or wilcoxon signed ranked test ( when samples were not normally distributed ) was used when comparing antibody levels within the same group . student 's unpaired t - test or mann - whitney test ( when samples were not normally distributed ) was used when comparing antibody levels between different groups . the dbl domain of pf332 was expressed in e. coli as a his - tagged recombinant protein and purified by ni - affinity chromatography , followed by size exclusion chromatography . a band migrating at approximately 27  kda could be visualized on the coomassie - stained sds - polyacrylamide gel from the soluble e. coli lysate , as well as from the eluted fractions after affinity purification and size exclusion chromatography ( figure 1(b ) ) . the protein eluted from the size exclusion column at an elution volume of 90  ml , which corresponds to the size of a monomer . to verify the molecular mass of the purified protein , analysis by mass spectrometry the mass spectra showed one major ion at m / z 26935 , which corresponds to the theoretical weight of pf332-dbl ( figure 1(c ) ) . to evaluate the folding state and secondary structure content of the recombinant protein , the spectra indicate that the protein is folded , and the shape of the spectra , with a positive band at 195  nm and two negative bands at 208 and 220  nm , respectively , is typical for proteins with a high -helical content ( figure 1(d ) ) . crystal structures of two different ebl - dbl domains have been determined , namely , the tandem dbl domains of p. falciparum eba-175 ( f1 and f2 )   and the single dbl domain of p. knowlesi duffy - binding protein ( pk-dbl ) . using the crystal structure of eba-175 f2 as a template ( pdb code 1zro ) , a three - dimensional ( 3d ) model of pf332-dbl was constructed by comparative modeling on the basis of the 3d7 sequence ( pf11_0506 , amino acids 1255 ) in modeller 9v3 . the hhpred probability score for eba-175 f2 to be used as a template for pf332-dbl modeling was 100% , although the sequence identity between the two domains was only 29% ( figure 2(a ) ) . the procheck ramachandran plot showed that 90.1% of the residues in the model were in the core region , 9.1% in the allowed region , 0.4% in the generously allowed region , and 0.4% in the disallowed region , proving that the 3d model was of good quality . the homology model of pf332-dbl consists predominantly of -helices , which is in accordance with our cd - spectrum ( figure 2(b ) ) . the model further adopts a basic fold similar to both the dbl domains of eba-175   and pk-dbl . of the twelve cysteine residues present in pf332-dbl , ten correspond in location to cysteines in eba-175 f2 and eight to cysteines shared between eba-175 f1 , f2 , and pk-dbl ( figure 2(a ) ) . five disulphide bridges were further identified in the model and four of them were found to be conserved in eba-175 f1 , f2 , and pk-dbp whilst the fifth was only found in eba-175 f2 . since our homology model of pf332-dbl is structurally similar to ebl - dbls , we used the singh et al . nomenclature   and divided the pf332-dbl domain into three subdomains ( figure 2(c ) ) . in the model , all dbl domains crystallized to date have four invariant cysteine residues forming two conserved disulphide bridges in this subdomain . pf332-dbl , however , lacking two of these cysteines ( cys 1 and 4 ; figure 2(a ) ) , only has one disulphide bridge in this region ( cys 23 ; figure 2(c ) ) . one conserved disulphide bridge could be identified in the subdomain ( cys 56 ; figure 2(c ) ) as well as two unpaired cysteines ( cys 5 * and 6 * ) , which were found to be unique for pf332-dbl ( figure 2(a ) ) . subdomain 3 has one short stretch of -helices but the subdomain is dominated by its two long antiparallel -helices that are connected by a loop . the third subdomain is predicted to be held together by three disulphide bridges , two of which are conserved among all dbl domains studied here ( cys 810 and cys 1113 ) and one that is shared with only eba-175 f2 ( cys 712 ; figure 2(c ) ) . the third subdomain further lacks two cysteines that are present in all other dbl domains studied here ( cys 9 and 14 ; figure 2(a ) ) . in order to examine whether antibodies towards the dbl domain of pf332 were elicited during natural infection with p. falciparum , the recombinant pf332-dbl protein was examined together with a dbl1-domain from the variant surface antigen pfemp1 in elisa assays . seroprevalence and antibody levels were analyzed in plasma samples collected from semi - immune adults in kampala ( uganda ) and children with either mild or severe malaria in apac ( uganda ) . we further analyzed plasma samples from asymptomatic individuals with different susceptibility to malaria , belonging to sympatric ethnic groups in burkina faso ( the fulani and the mossi tribe ) and mali ( the fulani and the dogon tribe ) . as summarized in table 1 , the seroprevalence of antibodies directed against pf332-dbl and pfemp1-dbl1 was consistently high in the distinct malaria endemic regions studied here ( 6098% and 5087% , resp . ) , demonstrating the immunogenicity of the two antigens .   in kampala , over 60% of the individuals were seropositive for pf332-dbl and pfemp1-dbl1 ( table 1 ) and they had acquired comparable levels of antigen - specific antibodies ( median od ratio of immune plasma / swedish nonimmune plasma were 2.7 and 3.7 , resp . ; figure 3(a ) ) . in the highly endemic area of apac , over 71% of the children with mild malaria and 84% of the children with severe malaria were seropositive for the two antigens already at a very young age ( 7 months to 4 years ; table 1 ) . moreover , all children had acquired comparable levels of antigen - specific antibodies , regardless of whether they had mild or severe malaria ( median od ratio ranging from 4.0 to 4.8 ; figure 3(b ) ) . adults living in burkina faso had the highest pf332-dbl seroprevalence with over 95% positive individuals in both the fulani and the mossi tribe ( table 1 ) . furthermore , individuals in both tribes had acquired similarly high levels of pf332-dbl specific antibodies ( median od ratio 7.4 in the fulani and 8.1 in the mossi ; figure 3(c ) ) . the seroprevalence of antibodies directed against pfemp1-dbl1 differed between the two ethnic groups and a significantly higher proportion of individuals of the mossi tribe were found to be seropositive ( 73% of the mossi compared to 52% of the fulani , p < 0.05 ) . the amount of antibodies specific for pfemp1-dbl1 was , however , similar in both tribes ( median od ratio 2.6 in the fulani and 3.4 in the mossi ; figure 3(c ) ) . when comparing antibody levels within each ethnic group , levels of antibodies directed against pf332-dbl were found to be significantly higher than levels of pfemp1-dbl1 antibodies in both the fulani and the mossi tribe ( p < 0.001 ) . in mali , the seroprevalence of pf332-dbl antibodies was over 93% in both the fulani and the dogon group ( table 1 ) and igg levels were similar and very high ( median od ratio 7.6 in the fulani and 7.2 in the dogon ; figure 3(d ) ) . opposite to what was observed in burkina faso , more fulani than dogon were seropositive for antibodies directed against pfemp1-dbl1 ( 71% of the fulani compared to 50% of the dogon ) , although this difference was not significant ( table 1 ) . the fulani tribe , which is less susceptible to malaria , had acquired significantly higher ( p < 0.01 ) levels of antibodies towards pfemp1-dbl1 than their sympatric tribe dogon ( median od ratio 3.2 in the fulani and 2.7 in the dogon , figure 3(d ) ) . similar to what was observed in burkina faso , levels of pf332-dbl antibodies were found to be significantly higher than levels of pfemp1-dbl1 antibodies in both the fulani and the dogon tribe ( p < 0.001 ) . previous studies on pf332 have been hampered by the potential cross - reactivity of anti - pf332 antibodies with other repetitive antigens . in an effort to evaluate the fine specificity of naturally acquired human antibodies to pf332-dbl , we affinity - purified human antibodies specific for the pf332 dbl domain from adult plasma displaying reactivity towards the region in elisa - assays . these antibodies were subsequently probed on a peptide array of 52 overlapping 15-mers ( spanning amino acids 1219 of pf332 ) . the pf332-dbl antibodies recognized two overlapping peptides , having the sequence kkdeyidiqsrv in common ( figure 4(a ) ) . when mapped on the 3d model of pf332-dbl , the epitope was found to be located on one of the two antiparallel -helices in subdomain 3 ( figure 4(b ) ) . to rule out the possibility of cross - reactivity with epitopes in the dbl domains of the ebl family of invasion proteins , we also probed a peptide array of 15-mers spanning amino acids 145747 covering both the f1 and f2-domain of eba-175 , with the human antibodies . the anti - pf332 antibodies did not bind to any of the eba-175 peptides , indicating that they do not cross - react with linear epitopes of the ebl - dbls ( see supplementary figure 1 available online at doi : 10.4061/2011/671439 ) . in order to analyze the ability of the naturally acquired antibodies to detect native pf332 protein , we performed immunofluorescence assays with the affinity - purified human anti - pf332-dbl antibodies on air - dried monolayers of rbc infected with the fcr3s1.2 parasite strain . at 2832  h p.i . , the human anti - pf332-dbl antibody reacted with maurer 's cleft like structures in the irbc cytosol ( figure 5(a ) ) . the antibody colocalized with an antibody specific for the pf332 repeat region eb200 , confirming the specificity of the affinity - purified human anti - pf332-dbl antibody ( figure 5(a ) ) . to further investigate antibody specificity , we carried out immunofluorescence assays using a monoclonal antibody against pf332-dbl and antibodies targeting the pf332 repeats , maurer 's clefts , and the ebl family of invasion proteins . at 3034 h p.i . , the anti - pf332-dbl antibody showed colocalization with anti - pf332-eb200 , confirming that the monoclonal anti - dbl antibody was able to detect native pf332 ( figure 5(b ) , upper panel ) . anti - pf332-dbl further colocalized with mahrp1 , a known maurer 's clefts marker in p. falciparum , which is in accordance with what has previously been described for pf332 [ 6 , 7 ] ( figure 5(b ) , middle panel ) . at 40  h p.i . , we could detect pf332 in maurer 's cleft like structures in close proximity to the rbc plasma membrane . these antibodies did not co - localize with anti - eba-175 antibodies , which gave a punctuate fluorescence pattern typical for antigens localized within the merozoite apical complex ( figure 5(b ) , bottom panel ) , demonstrating the lack of cross - reactivity between the two antigens . to confirm the specificity of the anti - pf332-dbl antibodies , pf332 could be detected as a high - molecular - weight band migrating well above the 170  kda marker in fcr3s1.2 parasite extract , when probing with affinity - purified human anti - pf332-dbl , anti - eb200 , and monoclonal anti - pf332-dbl antibodies ( figure 5(c ) ) . some smaller bands , which are likely processed protein or proteolytic degradation products of full - length pf332 , were also observed . although pf332 was identified two decades ago [ 810 ] , the function of this giant protein still remains elusive . a number of possible functions have , however , been described for pf332 and among them are involvement in rigidity modifications of irbc [ 38 , 39 ] , indirect adhesion to uninfected rbc by assisting in transportation of pfemp1 to the irbc surface , dbl - mediated adhesion to uninfected rbc , and involvement in parasite growth [ 4 , 4043 ] . pf332 is present in all isolates investigated [ 4 , 10 ] indicating that the molecule is of importance for the parasite . however , pf332 does not seem to be essential as parasites with a disrupted pf332 gene are capable of replicating normally in vitro although the irbc appears to be more rigid [ 38 , 39 ] . to date , two different ebl - dbl crystal structures are known ; the tandem dbl domains of p. falciparum eba-175 ( referred to as f1 and f2 ) and the single dbl domain of p. knowlesi duffy - binding protein ( pk-dbl ) . despite low sequence identity , all three share several invariant cysteine residues , which are involved in conserved disulphide bridges , demonstrating the importance of the cysteines in maintaining the dbl fold . it is interesting that pf332-dbl , having only approximately 2429% sequence identity with the dbl domains of pk-dbl and eba-175 but having the majority of its cysteine residues at conserved locations , shares this basic dbl - fold according to our model ( figure 2 ) . however , there are some differences between the pf332 model and the crystallized dbl domains . first , pf332-dbl lacks two cysteine residues in subdomain 1 , which form a conserved disulphide bridge in all dbl domains crystallized to date . this disulphide bridge holds two loops together in pk-dbl and eba-175 and the lack of a disulphide bridge in our model is likely to allow more flexibility in this region of pf332-dbl , which could be of functional importance . second , pf332-dbl has two unique cysteines in subdomain 2 , which are not involved in forming a disulphide bridge in our model , but have been predicted to do so by others . the addition of a unique disulphide bridge further allows for speculations about structural differences in the dbl domain of pf332 compared to the dbl domains of the ebl members . third , pf332-dbl is missing two cysteines in subdomain 3 , which form a disulphide bridge in all three dbl domain crystal structures analyzed here . it seems clear that the overall structure of pf332-dbl is similar to the dbl domains of the ebl family . however , the structural differences observed imply that pf332-dbl could have a different dynamical behavior , leading to differences in its receptor specificity or binding preferences . it is intriguing why pf332 has a dbl domain homologous to the ebl family , although it lacks the classic ebl gene structure and the typical ebl expression profile . in contrast to the ebl members , pf332 contains predominantly degenerate repeats , which in previous studies has been shown to bind actin and as a result , modulate the rigidity of the irbc [ 38 , 39 , 44 ] . the f1-domain of eba-175 is related to the single dbl domain of p. vivax and p. knowlesi [ 3 , 18 ] , and the domain is common to all ebls . in contrast , the f2-domain of eba-175 has so far only been found in p. falciparum and p. reichenowi and the domain is suggested to be the progenitor of var dbl . it is therefore interesting to note that the dbl domain of pf332 has more similarities to f2 than to f1 of eba-175 , as the dbl domain of antigen 332 so far only has been identified in p. falciparum , although antigen 332 orthologues are present in species such as p. berghei and p. yoelii . our data show that antibodies to pf332-dbl are frequent in plasma from individuals residing in distinct malaria endemic regions , demonstrating that it is immunogenic . the frequent seropositivity is also in accordance with the domain being conserved and having very few polymorphisms observed . the lack of variation in the dbl domain of pf332 suggests that the protein is concealed from the immune system and therefore is not under any selective pressure . as a result , it is likely that antibodies towards the antigen are generated upon schizont rupture , at which time the protein is released into the extracellular environment . previous studies using recombinant protein of the pf332 regions eb200 and c231 ( a c - terminally located region of pf332 , less rich in degenerate - repeats ) , have reported that children lack antibodies against both parts of the molecule . however , antibodies were frequently found in older individuals , indicating a slow acquisition of pf332 antibodies [ 14 , 48 ] . our results show that children living in apac , a highly endemic region in uganda experiencing the highest entomological inoculation rate reported in africa , had developed antibodies to both pf332-dbl and pfemp1-dbl already at a very young age . due to conservation of pf332-dbl , antibodies should be acquired early in life in nonimmune children and particularly in areas of high malaria endemicity , which is confirmed by our study . it must , however , be taken into consideration that plasma samples were collected from children with an ongoing p. falciparum infection where the host immune system had been recently boosted . high levels of antibodies against pfemp1-dbl1 were also frequently found among children in the apac area , although the sequence diversity of this domain is great . antibodies against the dbl domain of pf332 were highly prevalent among individuals in both burkina faso and mali regardless of their ethnic group and antibodies occurred at very high titers . one explanation might be that although living close to each other geographically and experiencing the same malaria transmission rate , the different tribes had encountered parasites presenting different pfemp1 molecules on the irbc surface . whilst the pf332 gene is present as a single copy gene in most parasite isolates studied so far , the var genes encoding the pfemp1 molecule are present in approximately 60 copies per genome [ 49 , 50 ] . in mali , the fulani who are less susceptible to malaria were found to have significantly higher antibody levels to pfemp1-dbl1 than their sympatric tribe dogon . further , the fulani were more seropositive , indicating either that they more frequently had encountered parasites expressing this particular or similar subtypes of pfemp1 to which they had acquired antibodies , or that they more easily develop high levels of antibodies , which could be an explanation for their lower susceptibility to malaria .   studies on pf332 have been hampered by the cross - reactive nature of the antigen due to its high content of negatively charged repeats , which are present in several malaria antigens . the dbl domain of pf332 is conserved between different parasite isolates , thus enabling more antigen specific studies . in an effort to look at antibody specificity and to exclude the possibility of cross - reactivity to dbl domains of the ebl family of invasion proteins , we affinity - purified human antibodies using the dbl domain of pf332 and analyzed them in peptide arrays , immunofluorescence microscopy assays , and western blotting . from the peptide microarray it was evident that the antibodies only reacted with epitopes found in pf332 and not in dbl domains of the ebl family of invasion proteins , here represented by the tandem dbl domains of eba-175 ( figure 4 and supplementary figure 1 ) . this method only takes linear epitopes into consideration ; however , it gives a strong indication that there is very little or no cross - reactivity between the dbl domains of pf332 and the ebl family . the lack of cross - reactivity when using native protein was confirmed by immunofluorescence microscopy and western blotting ( figure 5 ) , which concludes that antibodies raised against the dbl domain of pf332 do not cross - react with the dbl domains of the ebl family . in summary , the dbl domain represents a conserved region of the pf332 molecule , with a similar structure to the dbl domains of eba-175 and pk-dbp according to our 3d predictions . antibodies towards the domain are acquired early in life in areas of high endemicity and antibodies are commonly found in semi - immune adults . moreover , antibodies towards pf332-dbl are antigen specific and do not display cross - reactivity with dbl - containing ebl proteins . hence , the dbl domain of pf332 is expected to greatly facilitate further investigations of pf332 ; including its function and potential role in immunity to malaria .
studies on pf332 , a major plasmodium falciparum blood - stage antigen , have largely been hampered by the cross - reactive nature of antibodies generated against the molecule due to its high content of repeats , which are present in other malaria antigens . 
 we previously reported the identification of a conserved domain in pf332 with a high degree of similarity to the duffy - binding - like ( dbl ) domains of the erythrocyte - binding - like ( ebl ) family . 
 we here describe that antibodies towards pf332-dbl are induced after repeated exposure to p. falciparum and that they are acquired early in life in areas of intense malaria transmission . 
 furthermore , a homology model of pf332-dbl was found to be similar to the structure of the ebl - dbls . despite their similarities , antibodies towards pf332-dbl 
 did not display any cross - reactivity with ebl - proteins as demonstrated by immunofluorescence microscopy , western blotting , and peptide microarray . 
 thus the dbl domain is an attractive region to use in further studies on the giant pf332 molecule .