ABO Blood Group Antigens as a Model of Studying Protein-Protein Interactions

2.1 Antigen A and its predicted biological activity

It is almost impossible to evaluate the specific properties of the terminal fragments of antigenic structures in an experiment, but with the method of computer simulation, one can predict the biological effects of substances.

Glycosylation of protein molecules significantly affects their ability to contact with other molecules, which is important for understanding the mechanisms of intermolecular interactions, signaling, and adhesion at the cellular and molecular levels. The group-specific antigens of the blood ABO system are formed by the glycosylation of transmembrane proteins presented on the surface of red blood cells.

A-Antigen terminal monosaccharide N-acetylgalactosamine contains in the position C2 NHCOCH3 group, and B-antigen terminal monosaccharide d-galactose, in position C2, contains OH group. Functional groups confer variability in the structure of antigens and provide specificity for binding ligands. The presence in the structure of the N-acetylgalactosamine acetyl group leads to the disappearance of the positive charge.

N-Acetylgalactosamine is an amino sugar found in almost all glycoproteins. The immediate precursor of N-acetylgalactosamine is fructose-6-phosphate. Amino sugar is further acetylated with acetyl-CoA.

Monosaccharides can take part in all reactions which are typical for hydroxyl-containing compounds: they form esters and ethers, acetals and ketals, undergo substitution and elimination reactions. An important property of monosaccharides is their ability to form glycosides due to hydroxyl at the first carbon atom. Elucidation of the potential biological activity of antigens, determined by the structural characteristic of the antigenic determinants of the ABO system, is an important task.

We used a program for computer modeling called PASS. Prediction of Activity Spectra for Substances (PASS) is a program designed for computer modeling created by a group of Russian scientists. This tool is based on the dependence between chemical formula of a random substance and its functional activity. Chemical formula is described using Multilevel Neighborhoods of Atom (MNA) descriptors, the combination of which is unique for each substance. The user gets the list of probable activities based on the program’s self-educating “training set,” which aggregates data on active compounds from databases, publications, and patents, marked with Pa (probability to be active) and Pi (probability to be inactive), placed in the order from the maximum Pa to the minimum one. The program uses the Bayesian approach with some modifications for calculating the Pa and Pi (for more detailed information, see [6, 7]). In our study we chose effects with Pa >0.5. Total number of biological activities in the database is 4130, 501 of them are pharmacological effects, 3295 are molecular mechanisms of action, 57 are toxic effects, 199 are mediated metabolic actions, and 29 are influences on gene expression.

We have identified a significant number of previously unknown properties and mechanisms of action for the antigenic determinant antigen A, namely, 99 out of 501 possible pharmacological effects, 304 out of 3295 possible molecular mechanisms of action, 17 out of 57 adverse and toxic effects, 12 of 199 metabolic-related activities, 2 of 29 effects regulating the expression of genes, and 5 of 49 effects associated with the transport of substances. We chose biological activities with a probability of Pa greater than 0.5. The PASS program allowed us to establish that the antigenic determinant of antigen A exhibits the following pharmacological effects (Table 1).

It is predicted that the antigenic determinant of antigen A exhibits antibacterial, immunostimulating, antifungal, antiviral, and pharmacological effects, as well as antibiotic properties.

ABO phenotypic analysis of blood groups is often used to detect the degree of susceptibility of a person to infectious diseases. In the literature, there are data in which it is noted that the interaction of certain parasites and bacteria with human cells depends on the presence of certain blood groups [8].

Thus, antigen A exhibits high adhesive activity against lactic acid bacteria. Some of the antigens affect the humoral and cellular response [9].

For the oligosaccharide of the antigen A, the antineoplastic effect on the cancer of various etiologies and localization is predicted: gastric and lung cancer, sarcoma, leukemia, and cancer of the brain and ovaries. Numerous studies have shown an association between ABO blood groups and the risk of developing various types of cancer [10].

With a high degree of probability, the antigen A is able to regulate angiogenesis and has a potential vasoprotective effect, as well as an effect of inhibitor of membrane permeability and integrity. The formation of new blood vessels in the organ or tissue is activated only when the damaged tissues are regenerated. Some factors, depending on the dose, can be both inducers of angiogenesis and inhibitors [11]. Many predicted effects of the antigenic determinant of antigen A are realized through the molecular mechanisms of its action (Table 2).

It is predicted that the oligosaccharide of the antigen A can act as an agonist of nerve growth factor, tumor necrosis factor, hyaluronic acid, α-interferon, interleukin-2, and tissue kallikrein inhibitor. The stimulating effect of the oligosaccharide antigen A on the activity of caspases 3, 8, and 9, participants in the apoptosis process, is predicted. As it is known, all caspases are synthesized in an inactive form and are activated when necessary by initiating caspases in the process of partial proteolysis. Probably, the antigenic determinant A, by activating caspases, can trigger a signal chain of programmed cell death.

Analyzing molecular mechanisms of action of antigenic determinants A, we paid attention to the inhibitory effect, to a number of carbohydrate metabolism enzymes, complex lipid metabolism, and protein biosynthesis process. Oligosaccharide antigen A may inhibit the activity of enzymes involved in the metabolism of simple carbohydrates, such as α-glucosidase, β-glucuronidase, and β-galactosidase, and in the exchange of complex carbohydrates, predominantly heteropolysaccharides—α-N-acetyl-glucosaminidase, dolichol glycosyltransferase, and GDP-mannose-6-dehydrogenase, which creates the possibility of inhibiting the metabolism of the components of the extracellular matrix of connective tissue—glycoproteins and proteoglycans. Dolichol glycosyltransferase plays a leading role in the glycosylation of membrane proteins.

It is predicted that the oligosaccharide of the antigen A can be an agonist of hyaluronic acid. Hyaluronic acid belongs to the innate immunity system and is involved in tissue regeneration, as evidenced by the likely manifestation of the pharmacological effects of tetrasaccharide A, as immunostimulating and vasoprotective.

Also, we predicted possible toxic effects (Table 3).

The effect of carbohydrate determinants of antigen A on the metabolism of complex lipids is predicted, and the molecular mechanism of action is inhibition of the activity of CDP-glycerol glycerophosphotransferase, ceramide glycosyltransferases, ganglioside galactosyltransferases, and galactosylglucosyl-ceramidase, involved in the synthesis of phospho- and glycolipids, necessary for the construction of cell membrane structures. Glycolipids play an important role in making cell-to-cell contacts; some serve as a kind of receptor for a number of bacterial toxins.

The possibility of an inhibitory effect on a gene expressing telomerase was predicted. About 85% of cancer cells acquire unlimited replicative potential due to the reactivation of a specific telomerase enzyme [12].

After analyzing the data obtained by computer prediction, we can conclude that the immunochemical specificity of the antigenic determinant of antigen A is realized by the characteristic and diverse biological activity and toxicity.

2.2 Antigen B and its biological activity

The antigenic determinant of antigen B contains terminal d-galactose, at position C2 where it has a hydroxyl group.

d-Galactose itself can enter into the reactions of alkylation, acylation, reduction, and oxidation. Analysis of the data of the probable biological activities of the antigenic determinant of antigen B showed 106 out of 501 possible pharmacological effects, 311 of the 3295 probable molecular mechanisms of action, 16 of 57 adverse and toxic effects, 15 of 199 metabolically mediated actions, 3 of 29 effects regulating gene expression, and 6 of 49 effects associated with transport of substances. We chose biological activities with a probability of Pa greater than 0.5. The pharmacological effects of the antigenic determinant of antigen B are shown in Table 4.

Many effects and mechanisms of action are common for both antigen A and antigen B, but they are characterized by different degrees of probability of manifestation (Pa value). It is predicted that the antigen B is able to exhibit antibacterial, antiviral, antifungal, and pharmacological effects. According to the literature, group-specific antigens A and B can play a direct role in the susceptibility of the infection, acting as receptors or co-receptors for microorganisms, parasites, and viruses (Table 5).

In the study, Kato shows that carbohydrates can act not only as receptors for various microbes but also function as a barrier to infection [13].

Numerous molecular mechanisms of the action of antigen B tetrasaccharide have been predicted, in particular the inhibitory effect on the activity of a number of enzymes involved in the metabolism and stimulating effects on various bioregulators.

The stimulating effect of oligosaccharide B on the activity of caspases 3 and 8 is predicted; it can act as an agonist of hyaluronic acid, nerve growth factor, interleukin-2, and interferon antagonist. It has been established that galactooligosaccharides selectively increase the content of useful intestinal microbes, as well as C-reactive protein and interleukins [14].

The probable effect of oligosaccharide B on the expression of the telomerase gene (Pa 0.785) and the transport of electrons in mitochondria (Ra 0.504) is shown. Oligosaccharide B is highly likely to be a substrate for cytochrome P-450 2J2 (Pa 0.980), glutathione-S-transferase (0.907), and cyclooxygenase (Pa 0.759).

Using PASS we also identified possible toxic effects (Table 6).

The PASS program revealed that the antigenic determinant of antigen B can exhibit a variety of biological effects and molecular mechanisms of action that regulate various physiological and metabolic processes in the body.

The role of carbohydrates as key biological ligands is well known. This is due to the high degree of isomerism, possible within individual carbohydrate units, in a variety of ways to combine monosaccharides, among themselves, different variations of substituents (acetyl, sulfate) and flexibility of carbohydrate chains.

With the development of computational methods for studying protein-ligand interactions, it became possible to determine the type of bonds and the most important positions of atoms “C” in monosaccharides for the formation of an antigen-antibody complex. Using the molecular docking method, Stanca-Kaposta with a group of scientists found that hydrogen bonds and hydrophobic and van der Waals interactions are involved in the formation of an antigen-antibody association [15]. Monosaccharides of antigens can act as acceptors of hydrogen, and amino acid residues of paratope antibodies can serve as hydrogen donors. Most commonly, hydrogen bonds form atoms “C” at positions 3 and 5 in terminal galactose (antigen B) and atoms “C” at position 5 in N-acetylgalactosamine (antigen A). Accessibility of the nitrogen atom in the GalNAc epitope to participate in the formation hydrogen bonds are hampered by the presence of an acetyl group. The “C” atoms in position 6 are involved in hydrophobic and van der Waals interactions, both in galactose and in N-acetylgalactosamine [16].

In studies by J. Milland, it was found that in the N-acetylated version of the epitope of antigen A, the interaction of the acetyl group of the epitope with the tyrosine 35 of the immunoglobulin heavy chain precludes further penetration of the antigen into the antibody’s binding site. In contrast, the Gal epitope of antigen B penetrates deeper into the antibody’s binding site, and the second galactose antigenic determinant participates in hydrophobic interactions with tryptophan at position 36 of the immunoglobulin heavy chain [17]. ABO antigens, like other glycoconjugates, are important intercellular adhesion mediators and participants in signal transduction. Due to the diversity of biological effects manifested, oligosaccharides A and B are evaluated from a new perspective side as biologically active compounds and not only blood group antigens that protect blood cells [18].

Computer modeling exists as a way of combining the microscopic world of molecules and experimental results, which helps to confirm our understanding of metabolic processes and propose new directions for research. For many of the predicted types of activity of compounds in the available literature, experimental evidence has not been found, since these organic compounds are difficult for conformational analysis. Computational methodology makes it possible to obtain structures of compounds at the atomic level and information about activity with an accuracy equivalent to or greater than can be obtained in an experiment. The prognostic and interpretative program PASS has helped to better present the mechanisms of action of the studied metabolites and carbohydrate determinants of antigens A and B in relation to the main body systems.