PII: S0925-4439(97)00005-7 Ž .Biochimica et Biophysica Acta 1360 1997 241–246 Association of malaria with inactivation of a 1,3-galactosyl transferase in catarrhines Ranjan Ramasamy ), Rupika Rajakaruna Molecular Biology and Immunology Laboratories, DiÕision of Life Sciences, Institute of Fundamental Studies, Hantana Road, Kandy, Sri Lanka Received 5 December 1996; accepted 10 January 1997 Abstract Ž . ŽPresent-day catarrhines old world monkeys and hominoids lack Gal a 1-3 Gal b 1-4 GlcNAc-R structures a-galactosyl . Ž . Ž .epitopes and produce the corresponding anti-galactosyl antibodies anti-gal , while platyrrhines new world monkeys and non-primate mammals possess a-galactosyl epitopes and lack anti-gal. Anti-gal is shown to inhibit Plasmodium falciparum growth in culture in a concentration dependent manner, probably by binding to a-galactosyl epitopes on merozoite surface molecules and causing complement mediated damage. A P. falciparum-like malaria parasite may therefore have selected for the inactivation of an a 1-3 galactosyl transferase in catarrhines. The implications of the results for the development of clinical immunity to falciparum malaria are briefly discussed. Ž .Keywords: Anti-galactosyl antibody; a-Galactosyl epitope; Galactosyl transferase; Malaria; Primate evolution; Plasmodium falciparum 1. Introduction Malaria due to Plasmodium falciparum has ex- erted selective pressure on human populations at the w xlevel of globin 1 , major histocompatibility complex Ž . w xMHC 2,3 and glucose 6-phosphate dehydrogenase w x4 genes. The MHC class I antigen HLA-Bw53, which is common among west and central Africans but rare in caucasians and orientals, affords 40% Abbreviations: anti-gal, anti-galactosyl antibodies; a-galacto- syl epitope, Gal a 1-3 Gal b 1-4 GlcNAC-R; a 1,3 GT, a 1,3 galactosyl transferase; MHC, major histocompatibility complex; mya, million years ago; rbc, red blood cells. ) Corresponding author. Fax: q94 8 232131; E-mail: ranjan@ifs.ac.lk protection against cerebral malaria or severe malaria- w xinduced anaemia 2 . The molecular basis for this protection has been ascribed to HLA-Bw53 restricted recognition of an epitope on a P. falciparum liver- w xstage specific antigen by cytotoxic T cells 3 . Gal Ža 1-3 Gal b 1-4 GlcNAc-R structures termed a- .galactosyl epitopes are absent in old world primates Ž .including man the catarrhines but common in gly- colipids and glycoproteins of new world monkeys Ž . w xthe platyrrhines and non-primate mammals 5,6 . Correspondingly, natural antibodies to the a- Ž .galactosyl epitopes termed anti-gal are present in catarrhines and absent in platyrrhines and non-primate ww xmammals 6 , Ramasamy, R. and Rajakaruna, R., xunpublished data . Inactivation of an a 1-3 galactosyl Ž .transferase a 1,3 GT in the Golgi of catarrhines is 0925-4439r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. Ž .PII S 0 9 2 5 - 4 4 3 9 9 7 0 0 0 0 5 - 7 ( )R. Ramasamy, R. Rajakaruna r Biochimica et Biophysica Acta 1360 1997 241–246242 w xresponsible for the difference 7 and it has been w xspeculated that a pathogen 7 , possibly a malaria w xparasite 8,9 , may have been the cause. We provide evidence that a P. falciparum-like parasite could have exerted the necessary selective pressure to inac- tivate a 1,3 GT. A preliminary report of this work was presented at the Biochemical SocietyrBritish Society for Immunology joint congress in Harrogate, UK in December 1996. 2. Materials and methods 2.1. Preparation of anti-gal antibodies and anti-gal deficient serum Thirty ml AB serum from donors with no known Žmalaria exposure and 7 ml pools of sera of unknown .blood groups from malaria-endemic Weheragala vil- lage with antibodies to P. falciparum merozoite sur- w xface proteins 10 were sequentially passed through a 2 ml control silica column and a 2 ml Gal a 1-3 ŽGal b 1-4 GlcNAc-R Synsorb column Chembiomed, .Edmonton, Canada for affinity purifying anti-gal antibodies. The columns were washed extensively in Ž .0.01 M phosphate buffered saline, pH 7.2 PBS . Proteins bound to the control silica and Gal a 1-3 Gal b 1-4 GlcNAc-R columns were then eluted with 3 ml 0.1 M glycine-HCl buffer, pH 3.0 to yield the control w xand anti-gal eluates respectively 11,12 . The two parallel eluates were neutralised with 0.1 M NaOH, dialysed extensively against the same batch of RPMI 1640 and sterilised by filtration through 0.22 mm filters. Anti-gal activity in the two eluates were deter- mined by direct haemagglutination of 5% haematocrit w xrabbit red blood cells 11 . Different preparations of anti-gal and control eluates had haemagglutination titres of 32–2000 and 2–64 respectively, with the anti-gal titre being always greater than that of the corresponding control. Analysis of anti-gal by SDS- PAGE revealed mainly IgG in anti-gal with traces of IgM and other serum proteins and a total protein concentration of 32–172 mg mly1 in different prepa- rations. Control eluates contained mainly IgM with traces of IgG and other serum proteins. AB serum that had been sequentially passed through the silica and oligosaccharide columns was used as anti-gal deficient serum for parasite cultures. [ 3 ]2.2. H hypoxanthine incorporation assay for deter- mining parasite growth inhibition Ž .P. falciparum 3D7 isolate was routinely main- Ž .tained in culture in 0 q red cells rbc and RPMI 1640 supplemented with 10% AB q human serum, 22 mM Hepes, 24 mM sodium bicarbonate and 10 mg mly1 gentamicin in an atmosphere of 5% 0 , 5%2 w xCO , 90% N at 378C 13,14 . P. falciparum was2 2 adapted to grow in anti-gal deficient AB serum for parasite growth-inhibition experiments. Late stage parasites were obtained from cultures by gelatine w xsedimentation 15 . For a given experiment, different eluates in RPMI 1640 were used to prepare complete medium containing 10% of the same anti-gal defi- cient serum to which the parasites were culture- adapted. Parasites at 0.5%–1.0% parasitaemia in dif- ferent experiments were then added to the medium to yield 5% haematocrit, and 100 ml aliquots dispensed into sterile 96 well culture plates in 4–8 replicates and incubated in an atmosphere of 5% CO , 95% air2 w 3 xat 378C. Twenty four hours later, 5 mCi H hypo- Ž .xanthine Amersham in 100 ml RPMI 1640 was w xadded to each well as described 14 . At 48 h, the cells were harvested onto Millipore glass fibre filters, the DNA precipitated with ice-cold 5% trichloro- acetic acid and washed in 70% ethanol, and radioac- tivity in the DNA determined by liquid scintillation counting. 2.3. Microscopic determination of parasite growth inhibition Late stage 3D7 parasites were cultured as de- scribed above in the presence of anti-gal and control column eluates isolated from immune serum from w xanother malaria endemic village, Nikawehera 16 . Smears of cultures were obtained at 24h and 48h after initiation of culture, stained with Giemsa and the proportions of different parasite stages deter- mined by microscopy. The anti-gal used in this exper- Ž .iment produced 41% inhibition P - 0.001 when compared to the corresponding control antibodies, in w 3 xa parallel H hypoxanthine incorporation assay. 2.4. Requirement for complement An aliquot of the anti-gal deficient AB serum used to grow parasites was heated to 568C for 30 min to heat inactivate complement. Parallel cultures were ( )R. Ramasamy, R. Rajakaruna r Biochimica et Biophysica Acta 1360 1997 241–246 243 then set up as described using the complement inacti- vated and normal anti-gal deficient sera to each of which were added the same preparation of anti-gal and control antibodies. 3. Results Anti-gal isolated from immune and non-immune human sera inhibited the re-invasion and growth of P. falciparum in cultures grown in AB serum de- pleted of anti-gal. The results of two experiments with seven different preparations of anti-gal are given in Table 1. Pooling results from several experiments, w 3 xit was observed that anti-gal inhibited H hypo- xanthine incorporation in 19 out of 22 tests when compared to the corresponding controls. In 11 out of 19 tests the inhibitions were statistically significant at P F 0.05 by the t test. Microscopic determination of the numbers of rings and trophozoitesrschizonts present at 24 and 48 h Žafter adding antibodies to cultures i.e. upto one .complete cycle of rbc invasion and growth indicated that inhibition of merozoite invasion was primarily Ž .responsible for this effect Fig. 1 . The binding of anti-gal but not the control antibodies to P. falci- parum merozoites was demonstrable by immunofluo- rescence on acetone fixed late stage parasites ŽRamasamy, R. and Rajakaruna, R., unpublished .data . Varying the concentration of anti-gal by serial dilution of added antibodies in RPMI 1640 showed that the anti-gal eluate inhibited parasite growth at lower protein concentrations than the control eluate Ž .Table 2 . In a related experiment, the addition of a Ž .human IgG1rkappa myeloma protein Sigma, USA at a concentration of 100 mg mly1 in RPMI 1640 did w 3 xnot significantly inhibit H hypoxanthine incorpora- tion into parasites in comparison to the addition of control eluate from immune serum at a protein con- y1 Žcentration of 88 mg ml 19 486 " 1490 vs 19 820 ." 2075 cpm " S.D. respectively . Table 1 Inhibition of P. falciparum growth by anti-galactosyl antibodies 3Ž . w xAddition protein concn H hypoxanthine Inhibition P incorporation Ž .mean cpm " S.D. Experiment 1 Ž .A i Control medium 9 450 " 316 – – Ž .B i Control eluate from immune serum – pool A 9 991 " 395 – – Ž .ii Anti-gal eluate from pool A 7 644 " 395 23% 0.002 Ž .C i Anti-gal eluate from immune serum –pool B 6 117 " 399 35% 0.0001 Ž .ii Anti-gal eluate from immune serum –pool C 6 894 " 1 403 27% 0.01 Experiment 2 y1Ž . Ž .A i Control eluate from immune serum –pool D 88 mg ml 35 961 " 2 800 – – y1Ž . Ž .ii Anti-gal eluate from pool D 144 mg ml 26 696 " 2 275 17% 0.001 y1Ž . Ž .B i Control eluate from non-immune AB serum –pool E 64 mg ml 34 580 " 3 741 – – y1Ž . Ž .ii Anti-gal eluate from pool E 160 mg ml 31 284 " 2 261 10% 0.05 y1Ž . Ž .C i Control eluate from immune serum –pool F 104 mg ml 39 813 " 2 127 – – y1Ž . Ž .ii Anti-gal eluate from pool F 160 mg ml 34 613 " 596 13% 0.001 y1Ž . Ž .D i Control eluate from non-immune AB serum – pool G 72 mg ml 32 106 " 2 251 – – Ž . Ž y1.ii Anti-gal eluate from pool G 120 mg ml 32 266 " 1 758 0% n.s. w 3 xThe effects of anti-gal and control eluates on P. falciparum growth determined by H hypoxanthine incorporation. Different pools of w 3 xsera used to prepare eluates are labelled A–G. Th immune sera were obtained from Weheragala village. The H hypoxanthine was used at a specific activity of 6 GBq mmoly1 in Exp. 1 and at 200 GBq mmoly1 in Exp. 2 and subsequent experiments. Protein concentrations Ž . w 3 xof eluates are given where available. Significance P of the differences between H hypoxanthine incorporated in the presence of anti-gal and the corresponding control eluates were determined by Student’s t test, except in 1C where anti-gal treatment was compared Ž .to parasites cultured in RPMI 1640 medium containing anti-gal deficient serum without added antibodies control medium 1A . Data represent mean " standard deviation for four or more determinations. n.s. s not significant, P ) 0.05. ( )R. Ramasamy, R. Rajakaruna r Biochimica et Biophysica Acta 1360 1997 241–246244 Table 2 Effect of anti-gal concentration on inhibition of P. falciparum growth 3Ž . w xAntibody protein concn H hypoxanthine Inhibition P incorporation Ž .mean cpm " S.D. y1Ž .1. Anti-gal from immune serum – pool H 108 mg ml 49 821 " 2200 9.5% 0.001 y1Ž .2. Anti-gal from immune serum – pool H 10.8 mg ml 52 052 " 3169 5.5% 0.05 y1Ž .3. Anti-gal from immune serum – pool H 1.1 mg ml 54 256 " 2416 1.5% n.s. y1Ž .4. Control eluate from immune serum – pool H 88 mg ml 55 059 " 998 – Ž .The effect of 10-fold dilutions of anti-gal immune Weheragala sera pool H on P. falciparum growth. The anti-gal was diluted in fresh RPMI 1640 and 100 ml aliquots of the antibodies were added to cultures. Data represent mean " standard deviation of eight replicate cultures. Statistical analyses were done as described in Table 1. Table 3 Role of complement in anti-gal mediated inhibition of parasite growth 3Ž . w xAddition protein concn Serum H hypoxanthine Inhibition P incorporation Ž .mean cpm " S.D. y1Ž .1. Anti-gal from immune serum – pool H 108 mg ml Normal 51 639 " 4 191 17% 0.001 y1Ž .2. Control eluate from immune serum – pool H 88 mg ml Normal 62 158 " 3 776 – y1Ž .3. Anti-gal from immune serum – pool H 108 mg ml Heat inactivated 65 563 " 3 756 0% n.s. y1Ž .4. Control eluate from immune serum – pool H 88 mg ml Heat inactivated 65 280 " 1 756 – The effect of heat inactivating complement on anti-gal mediated inhibition of parasite growth. The anti-gal was prepared from immune Weheragala serum. Data represent mean " S.D. of eight replicate cultures. Statistical analyses were done as described in Table 1 for differences between samples 1 vs. 2 and 3 vs. 4. Ž . Ž .Fig. 1. Effect of control A and anti-gal B eluates from immune Nikawehera serum on growth of P. falciparum deter- mined from Giemsa stained films. Inactivating complement components by heating the anti-gal deficient serum used for culturing the parasites, abolished growth inhibition mediated by Ž .added anti-gal Table 3 . 4. Discussion Terminal a-galactose residues are present on the w x w x185 kDa 17 and 45 kDa 18 P. falciparum mero- zoite surface proteins in O-linked oligosaccharides w x19 or glycophosphatidyl inositol membrane anchors w x18 . The binding of Bandeiraea simplicifolia IB4 lectin to parasite membranes shows that the terminal w xgalactose residues are present in a 1-3 linkages 17 . w x w xAntibodies in non-immune 8 and immune 17,20 human sera have been previously shown to recognise a-galactosyl epitopes in P. falciparum by ELISA or immunofluorescence. Indeed, the binding of natural antibodies in non-immune human sera to P. falci- parum can be inhibited by 0.5 M a-methyl galacto- w xside 8 . The data presented here show that the bind- ( )R. Ramasamy, R. Rajakaruna r Biochimica et Biophysica Acta 1360 1997 241–246 245 ing of anti-gal to merozoite surface proteins inhibits parasite growth through complement mediated lysis of merozoites. It is probable that the in vitro growth inhibition assay underestimates the capacity of anti- bodies to inhibit parasite growth in vivo. Merozoites probably traverse greater distances between rbc in vivo than in the settled layer of rbc in 96 well plates and can therefore be exposed to antibodies for a longer time. Complement mediated lysis is likely to be more efficient in plasma in vivo than in culture medium containing 10% serum in vitro. There is also evidence that monocytes, macrophages and neu- trophils, that were not present in the cultures, can promote antibody-dependent, cell-mediated immunity w xin vivo 21 . The degree of growth inhibition varied between different anti-gal preparations of similar protein con- centration. Differential loss of antibody activity dur- ing preparation and storage and differences in IgG isotype composition and consequently complement fixing ability, may be some of the factors responsible for this variability. The maximum growth inhibition observed with anti-gal is less than the 96% inhibition reported with 121 mg mly1 of an IgG monoclonal antibody directed against a peptide epitope on the w x45kDa merozoite surface antigen 22 . The difference may partly reflect higher affinity and faster binding of anti-peptide antibodies to merozoites. Since mero- zoites invade rbc rapidly, the rate of antibody binding is likely to a critical factor in invasion inhibition. It is possible that a proportion of merozoites that express fewer a-galactosyl epitopes avoid complement medi- ated lysis and that this process can give rise to P. falciparum mutants lacking a-galactosyl epitopes. However the presence of a-galactosyl epitopes in w x w x w xFVO 17 , Honduras 1 8,17 , FC27 8 and 3D7 isolates P. falciparum suggests that the correspond- ing oligosaccharide structures may be conserved be- cause they have a vital function in parasite biology. Analysis of gene sequences indicates that inactiva- tion of a 1,3 GT occurred as two independent events in the two groups of catarrhines viz. old world mon- keys and hominoids, no more than 17–25 million Ž . w xyears ago mya 7 . Plasmodium genus is about 150 w xmillion years old 23 . Analysis of the sequences of small subunit ribosomal RNA and circumsporozoite protein genes show that P. falciparum is closely related to a chimpanzee parasite P. reichenowi w x24,25 . These two malaria parasites form a clade that is more closely related to avian and reptilian malaria Žparasites P. gallinaceum, P. lophurae and P. mexi- . Žcanum than to other primate and human e.g. P. .knowlesi, P. ÕiÕax, P. malariae and P. oÕale or Ž . w xmurine e.g. P. berghei parasites 23–25 . Catar- w xrhines diverged from platyrrhines 30–35 mya 26 . We postulate that during the Oligocene or early ŽMiocene, a P. falciparum-like, non-primate prob- .ably avian or reptilian parasite adapted itself to infect primates in the old world which later led to the inactivation of a 1,3 GT in the hosts. The driving force for inactivation of a 1,3 GT may have been the ability to produce antibodies reactive with a- galactosyl epitopes of the parasite which then con- ferred significant protection against falciparum-like malaria without inducing autoimmunity. It is not possible to extrapolate easily from human anti-gal inhibition of P. falciparum growth in cultures to the selective advantage afforded by anti-gal in early catarrhines, which may have been considerably greater. Since P. falciparum was probably introduced w xinto the Americas after Christopher Columbus 27 , there would have been no similar selective pressure to inactivate a 1,3 GT in platyrrhines. A prediction of this hypothesis viz. that P. falciparum infects platyrrhines more readily than old world monkeys, is w xalready established 28 . Natural anti-gal in non-im- mune humans, like antibodies to blood group A and B antigens, are probably produced as a result of stimulation of the immune system by cross-reactive epitopes on the cell walls of common intestinal bacte- w xria 29 . There is no evidence at present to support an alternative possibility that selection due to a common bacterial pathogen prevalent in the old world, but not in the new world, could have caused the inactivation of a 1,3 GT in catarrhines. The natural anti-gal clearly provides only partial protection against human falciparum malaria. How- ever partial protection against malaria afforded by MHC molecules have produced significant genetic w xchanges in human populations 2,3 . The relevance of anti-gal to strain transcending clinical immunity to falciparum malaria observed in adults living in highly malaria-endemic areas, and the detailed structure of parasite a-galactosyl epitopes and its relationship to the specificity of human anti-gal need to be deter- mined. In this context it is relevant to note that higher ( )R. Ramasamy, R. Rajakaruna r Biochimica et Biophysica Acta 1360 1997 241–246246 levels of anti-gal have been reported in persons living in malaria-endemic areas and in patients with acute w xfalciparum malaria 30 . The human blood group B Ž .epitope has the structure Gal a 1-3 Fuc a 1-2 Gal b 1-4 GlcNAc-R and a subset of anti-B antibodies are w xreported to have anti-gal activity 31 . Hence the possibility that A and O blood groups afford some degree of protection against falciparum malaria re- quires investigation. P. falciparum proteins are presently being investi- gated as the basis of a vaccine against falciparum malaria. Our results suggest that oligosaccharide moi- eties of the parasite may also be useful for this purpose. Acknowledgements We are grateful to Dr. U. Galili for the Synsorb columns and V. Udawatte for secretarial assistance. References w x Ž .1 Allison, A.C. 1954 Trans. R. Soc. Trop. Med. 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