Proposed O-GalNAc/Gal glycosylation path- ways in blood group O and non-O blood groups during Plasmodium falciparum infections

The coevolution of species drives diversity in animals and plants and contributes to natural selection, while in host–parasite coevolution, a parasite may complete an incomplete evolutionary/developmental function by utilizing the host cell’s machinery. Analysis of related older data suggests that Plasmodium falciparum (P. falciparum), the pathogen of malaria tropica, cannot survive outside its human host because it is unable to perform the evolutionarily first protein glycosylation or blood group-independent (serologically A-like) O-GalNAcα1-Ser/Thr-R, Tn antigen (“T nouvelle”) formation owing to its inability for synthesizing the amino sugar N-acetyl-D-galactosamine (GalNAc). This parasite breaks the species barrier via hijacking the host's A-like/Tn formation by abundantly expressing serine residues and creating hybrid A-like/Tn structures. In the human blood group O(H), these hybrid structures are attacked by the germline-encoded nonimmune polyreactive immunoglobulin M (IgM), which physiologically regulates the expression of the syngeneic A-like/Tn antigen. In non-O blood groups, this antibody molecule has undergone the phenotypic accommodation of plasma proteins, which results in loss of blood group Aand B-corresponding anti-A and anti-B isoagglutinin activities. This loss allows the generation of human Aand B allele-connected hybrid epitopes and the development of lifethreatening disease almost exclusively in non-O blood groups. Although malaria infection occurs regardless of the blood group, the synthesis of the blood group AB enables the strongest contact with the pathogen, and molecularly precluding any isoagglutinin activity makes this group the least protected and the smallest among the ABO blood groups. In contrast, blood group O(H) individuals have the least contact with the pathogen; they maintain the isoagglutinins, rarely develop severe disease, and survive this coevolution in an immunological balance with the pathogen as the largest blood group worldwide.

Malaria-causing parasites interact with glycoprotein synthesis and form hybrid ABO blood types to complete their life cycle. 1 In an earlier article, I proposed that in malaria tropica infection the first step of pathogen transmission occurs regardless of the blood group across species barriers via molecular mimicry or formation of an intermediate hybrid Tn antigen [1], and showing another evolutionary function of this developmental structure. Meanwhile, my actual analysis of related older data suggests that the malaria-causing parasite Plasmodium falciparum, which cannot survive outside its human host, lacks complete protein synthesis. This protozoan parasite does not perform the metazoan host's evolutionarily first protein glycosylation, or blood group-independent, (serologically A-like) O-GalNAcα1-Ser/Thr-R, Tn antigen ("T nouvelle") formation owing to its inability for synthesizing the amino sugar N-acetyl-D-galactosamine (GalNAc): although some studies have reported traces of GalNAc in the parasite [2] [3], more recent research investigating the cytosol across multiple life cycles has not detected the molecule [4][5] [6]. Furthermore, glucosamine (Glc)-GalNAc epimerization does not occur in the parasite [7], nor does it possess genes required for mucin-type O-glycan synthesis [8]. When this parasite evokes life-threatening disease predominantly in people with blood group A, the host's proteoglycome plays a significant role in pathogenesis.
In fact, in host-parasite coevolution, parasites may complete their development using host cell machinery, with mucin-type O-glycans acting as a trans-species functional, molecular bridge. The human histo-blood group antigens are potential factors for rotavirus cross-species transmission; in particular, blood group A antigen and A-like GalNAc residues have been identified as receptors [9]. According to a glyco-evasion hypothesis, a pathogen can alter host immune systems by hijacking the glycosylation pathways [10]. Evidence shows that parasitic helminths (worms) coevolve with vertebrate immune systems [11].
The pathogen P. falciparum, which is transmitted to human hosts through the anthropophilic mosquito Anopheles gambiae [12], does not have the cellular machinery to complete protein glycosylation independently and hypothetically breaks the species barrier by abundantly expressing serine residues [13] for hijacking host glycosylation, specifically the (serologically A-like) O-GalNAcα1-Ser/Thr-R or "T nouvelle" (Tn) [14] antigen formation process. Although several glycans can be O-linked to Ser/Thr residues, O-GalNAc glycans appear to be especially relevant to mucosal sites and other locations of parasitic invasion [15]. The "bulky" [16] polyfunctional GalNAc molecule is a crucial component of glycosylation, an evolutionarily conserved pathway, which dominates critical functions during metazoan growth and reproduction and may even be the target of P. falciparum in a recently observed inverse relationship between global malaria incidence and cancer mortality [17].

Metazoan protein O-glycosylation and human ABO blood group phenotype formation: A hypothetical basis of P. falciparum infection and malaria tropica disease.
In metazoan evolution, up to 20 distinct genetically undefined polypeptide O-GalNAc transferases catalyze the first addition of GalNAc to a protein [18] and synthesizes the above ( serologically A-like) O-GalNAcα1-Ser/Thr-R Tn [14], which results from the most complex and differentially regulated step in protein glycosylation [19] and represents a normal yet fleeting intermediate structure, characterizing stem cell fidelity [20] [21]. Hypothetically, it occurs as a reversible glycosylation and predetermined breaking point of the human polyreactive nonimmune immunoglobulin M (IgM), whose secretory version represents an O-glycan-depleted antibody molecule, exerting serine residues at its V and/or Fc immunoglobulin regions [22] [23] and acts as the complementary protein of the A-like/Tn antigen. The primarily pentameric structure of this mammalian antibody gives rise to speculation regarding an evolutionary relationship with the hexameric structure of the anti-A-reactive Helix pomatia agglutinin [24], a primitive invertebrate defense protein, emerging from the coat proteins of fertilized eggs and reflecting the snail-intrinsic, reversible O-GalNAc glycosylations [25]. The mammalian nonimmune IgM is considered the humoral spearhead of innate immunity, which is not only produced by B cells but also throughout epithelial tissues [26] [27] and demonstrates the serological profile of the human blood group O(H) via complement-mediated, cytotoxic anti-A and anti-B isoagglutinin activities [28][29] (Fig. 1).
An X chromosome-encoded T-synthetase appears to control the Tn antigen expression [30] through carbohydrate chain elongation and mucin-type glycoprotein formation [31][32] [33], which involves the subsequent synthesis of the disaccharide Galβ1-3GalNAcα1-O-Ser/Thr or "T" antigen (also known as the Thomsen-Friedenreich antigen) [34] and is completed by the ABO phenotype and Lewis-type generation. Intriguingly, serological data obtained as early as 1971 [35], indicated an X chromosome/germline-encoded origin for the murine nonimmune anti-A-reactive IgM, although the complementarity of this antibody protein with syngeneic Alike ovarian glycoproteins and/or glycolipids became evident in subsequent studies [36] [37].
Murine and human nonimmune IgM exhibit a serologically similar reaction with human red blood cells (RBCs) and human anti-A and anti-B isoagglutinin activities have been attributed to anti-A/Tn and anti-B/T antibodies owing to their cross-reactivity with Tn and T structures [38]. During the establishment of the human ABO blood group phenotype these innate isoagglutinin activities, exerted by the nonimmune IgM, are downregulated through phenotypic glycosidic accommodation of plasma proteins in the non-O blood groups A, B, and AB [28] [29]. Because of donor-substrate availability, protein glycosylation is thought to occur intracellularly in the Golgi cisternae, in which nonimmune immunoglobulin production may be adapted to ABO phenotype formation. Accordingly, during immunoglobulin secretion, the poorly glycosylated intracellular IgM molecule becomes loaded with L-fucose and D-galactose [39], and the subsequent expression of fucose and galactose residues by the extracellular IgM might form the basis of phenotypic accommodation. This process may also be performed and/or completed by soluble plasma glycotransferases: blood platelets, for example, have been detected as a rich source of both glycosyltransferases and energy-rich sugar and amino sugars that are released from activated platelets to function in the extracellular space [40]  The lack of any ABO blood group glycosylation or phenotype formation, as shown by the rare O(h) or Bombay type [42] [43], which originates from consanguinity, is associated with strongly elevated isoagglutinin levels and an unusual anti-H reactivity, acting over a wide range of temperatures, with a thermal amplitude at 37 °C. This overexpression of anti-glycan antibody activity might cause autoimmunological impairment of germ cell maturation or function, responsible for the male infertility of this group [43]. In contrast, the blood group AB constitutes (likely to a result of evolutionary selective diseases) the smallest among the human ABO blood groups, precluding any isoagglutinin formation and representing the other extreme of phenotype diversity. Thus, the O(h) or Bombay type and blood group AB appear to mark two opposite 7 directions of negative (natural) selection and demonstrate how phenotype and isoagglutinin production form evolutionary functional unity, wherein the degree of phenotype diversity and innate immunity are inversely proportional and show again the central evolutionary and immunological position of the human blood group O(H) [29][1] (Fig. 2).
The pathogenesis of malaria will be discussed in view of these complex developmental and evolutionary/immunological conditions, under which P. falciparum reaches every tissue during its life cycle progression and accesses the proteoglycome of the host.  group AB appear to mark two opposite directions of negative (natural) selection and demonstrate how phenotype and isoagglutinin production form evolutionary functional unity, in which the degree of phenotype diversity and innate immunity are inversely proportional. Because statistical data are not available from the Bombay type due to its small population size, the isoagglutinin levels are estimated according to existing reports and the hypothetical serological profile of the classic Bombay type (h/h; se/se) [42], which is characterized by the complete lack of ABO blood group glycosylations.

Why life-threatening malaria is especially prominent in non-O blood groups.
Brooks and Mclennan (1992) [45] argued that P. falciparum, transmitted to humans by Anopheles gambiae, has no alternative vertebrate reservoir hosts. We know that this parasite does not have the cellular machinery to complete protein glycosylation independently because it lacks the ability to synthesize the amino sugar GalNAc and to glycosate O-GalNAc, while intriguingly expressing abundant serine residues. Moreover, considering that malaria tropica infections occur regardless of the ABO blood group but life-threatening disease develops almost exclusively in non-O blood groups, the human glycoproteome must play a critical role in these infections. Indeed, these infections occur through two genetically distinct glycosylation pathways of the amino sugar GalNAc: 1) non-somatic,

blood group-independent, serologically A-like or intermediate Tn-reactive O-GalNAc-glycosylation
and 2) somatic glycosylation and formation of human mucin-type ABO-allelic structures or phenotypes.
As explained earlier, after the transmission of infectious P. falciparum sporozoites, their characteristic and abundantly synthesized serine residues and/or phosphorylated serine residues [13], arising throughout the parasite ' s life -cycle, access the human A-like intermediate Tn-reactive O-Gal-NAc-glycosylations. P. falciparum reaches every tissue, with the liver, bone marrow, and erythrocytic stages being the most critical stages during the life cycle progression. However when syngeneic glycosylation of peptides mainly occurs intracellularly, hybrid glycosylation or hybrid, mucin-type ABO blood group antigen formation may occur extracellularly, enabled by abundant, soluble glycotransferases and energy-rich monosaccharides released from activated platelets [41], which play a dominant role in malaria pathogenesis [46].
The complex invasion mechanism utilized by P. falciparum has been comprehensively explained in a recent study by Belachew (2018) [47]. In humans, this invasion implicates a subsequent molec-  (Figs. 3, 4); however, other observations suggest that only additional crosslinking functions of functionally synergistic α2-macroglobulin makes this adhesion possible [61].
While the details of the involvement of these macromolecules in rosette formation are thus still unknown, this hypothetically occurs according to the principle of glycosidic exclusion or phenotypic accommodation of plasma proteins, as published by this author [28] [29]: in the case of malaria infection, the phenotype-determining glycotransferases catalyze the binding of IgM and alpha-2 macroglobulin to both infected erythrocyte surfaces as well as the serine-rich peptides of the parasite.   (Fig. 4).
Furthermore, ABO mucin-type generation [65] involves fucosylation (Fig. 3), which protects from autoreactive anti-H activity during phenotypic accommodation of plasma proteins [28]. This mechanism may also enable the hybrid fucosylation between the parasite and blood group O(H) RBCs, which thus have the least contact with the parasite when compared with those of non-O blood groups (Fig. 3). Moreover, without A and B blood group-determining glycotransferases, O(H) can maintain nonimmune IgM and adaptive secondary IgG against syngeneic and hybrid A and B antigens, making it the most protected blood group (Fig. 3). This special immunological position of the O(H) phenotype was hinted at in an early study [66]. It has been discussed recently [1] and again becomes evident: innate IgM, which physiologically regulates the expression of the syngeneic inter- cross-reactive hybrid glycoproteins. The blood group AB enables the strongest contact with the pathogen, and molecularly precluding any isoagglutinin activity, makes this group the least protected and smallest among the ABO-blood groups (Fig. 3).
Although the formation of hybrid carbohydrates is accomplished by circumventing innate immunity, the adhesion of these structures to parasite proteins ultimately makes non-O blood groups an immunological target. The physiological lack of innate anti-A and anti-B antibodies poses the following immunological dilemma in these blood groups: on one side, it protects these blood groups from self-reactivity, but on the other side, it cannot prevent the formation of autoantigenic targets in subsequent pathogenic steps during malaria infection. This is evident in recent observations [67][68], wherein the induction of autoimmune processes contributes to the development of severe malaria disease, most likely even dominated by autoimmune inflammations.

Conclusions
My previous paper proposed self-destructive biological altruism of the host [1], which enables the survival of the malaria parasite P. falciparum. This phenomenon has now been identified as a defined molecular biological or biochemical event, involving two genetically different GalNAc gly- Cancer mortality may statistically interfere with malaria rates to some extent. In recent retrospective studies, performed on two different Asian populations, cancer mortality was highest in blood group A [77] [78]. It is not surprising that non-O blood groups are the ideal target of P.
falciparum owing to comprehensive presentation of both A-like/Tn and A/B allelic glycosylations, whereas the most ideal target should be the early cancer cell, generating a surplus of genetically undefined A-like O-GalNAc glycans, which might explain the above cited, recently observed inverse relationship between global malaria incidence and cancer mortality [17]. According to this report, cancer mortality was significantly reduced in malaria-endemic regions. This phenomenon awaits validation by studying the survival patterns of human tumors transplanted into non-primate animals with insufficient innate immunity or severe combined immunodeficiency, for example the castaneous mouse (CAST) [79] or SCID mouse [80], infected with P. falciparum. The insight into the interaction mechanisms of this parasite with cancer metabolism might provoke speculations on completely new, future therapeutic strategies for cancer. Intriguingly, a glycosaminoglycan binding malaria protein, targeting human cancer has been discovered by Salanti et al. [81] Although P. falciparum cannot survive in non-primate animals, most likely resulting from innate immunity, in humans, infections occur regardless of the blood group. P. falciparum para- Clearly, blood group phenotypes determine innate immunity: over the decades several concepts have been developed, in which the pathogen evades host immunity, and depending on the experimental approach and/or the interpretation of existing data, different cellular and molecular aspects of blood group A-favored infection become evident: in a model by Moll et al (2015) [87], the patient's RBC surrounds itself with normal RBC, which makes the pathogen's antigen inaccessible to yet undefined antibody activity and clearance by the immune system, whereas in my concept the pathogen utilizes the physiological absence of the corresponding antibody or phenotype-specific isoagglutinin, which has been neutralized by A, B or AB phenotypic glycosylation. ABO epitopes and innate isoagglutinin activities arise molecularly together during the same enzymatic process and represent a developmental and evolutionary unity. The overexpression of isoagglutinins in the Bombay type and their complete downregulation in blood group AB mark the opposite ends of the spectrum of ABO phenotype diversity in natural selection, whereas the central immunological position of the human blood group O(H) guarantees the maintenance of species and phenotype diversity via a mechanism of immunologically-controlled anti-self-reactivity during growth and reproduction [42]. Consequently, the blood group AB is the least protected and remains the smallest among the ABO blood groups. In contrast, O(H) individuals have the least molecular contact with the pathogen, rarely develop severe disease, and survive this coevolution in an immunological balance with the pathogen as the largest blood group worldwide. Although the significance of host -parasite relationships such as host identification of foreignness, host susceptibility to parasites, and antibody responses have been discussed for decades as causes of the present-day world distribution of human ABO blood groups [88] [89], this distribution may be explained through the coevolution of humans and P. falciparum [74] on a molecular and immunological basis and demonstrate how augmenting phenotype diversity is associated with decreasing immunity.