We found that mebipred is an acceptable one of the best machine learning-based method for identifying metal binding proteins from sequence-derived features. However, this data base is not completely user friendly because after feeding the canonical sequence of the proteins it stops several times and it needs to be restarted and often does not continue. Using the latest MetalPDB version from 2018, none of the El Bagre-EPF antigens proteins were found, and therefore we could not make any further comparisons.
In our patient population, large-scale environmental perturbations are present in the endemic area; in particular, multiple polluting metal ions are widely distributed in this zone. In the endemic area the metals are entering the complete food chain, starting with microorganisms. Variables such as the presence of specific metals in the earth's crust, their solubility in water, and both anaerobic and aerobic bacteria, plus acidic and nonacidic conditions can influence the impact of metal ions. It is essential to acknowledge that living cells are not at equilibrium with their external environment; therefore, kinetic considerations are also relevant (Putignano et al. 2018).
Metal ion exchanges in biology can be divided into three classes: a) ions in fast exchange with biological ligands such the alkali metals Na+ and K+, the alkali earth metals Ca2+ and Mg2+, plus H+; b) ions in intermediary exchange with biological ligands such as Fe2+ and Mn2+ and c) ions in slow exchange with biological ligands; these include those involved in the active sites of metalloenzymes such as Fe3+, Zn2+, Ni2+, and Cu2+. Metal-to-metal interactions and covalency predominate in the slow-exchange metals (Wood 1985) It is evident that environmental metals ions present in the endemic area, specifically mercury (mercuric sulfides/selenides and cyanide are used for gold extraction) may compete with physiologic ions. The physiologic ions are essential metals universal to human function: Na, K, Ca, Mg, Mn, Fe, Co, Ni, Cu, Mo, W and Zn (. Permyakov 2021). Inorganic ions compete for transportation paths in living organisms, and many of the ions such as Cd2+ and aluminum Al3+ effectively compete with Mg2+ and Ca2+ in proteins and biological membranes (McCall et al. 2000). To be functional, most proteins fold into a precise three-dimensional structure, representing their native conformation. Misfolded proteins are cytotoxic, as they may aggregate and/or interact inappropriately with other cellular components.
It is well known that all metal ions, including physiological metal ions, are toxic being in an overdose. Some metals and metalloids are xenobiotic and highly toxic at any concentration (arsenic, cadmium, lead, mercury). The xenobiotic metals can change the function of specific proteins, both 1) by forming a complex with functional side chain groups or 2) by thiol binding, and/or by 3) substituting for essential metal ions in metalloproteins (Permyakov 2009). Sometimes they create reactive oxygen and thus elicit oxidative stress. They also cause DNA damage and/or violate DNA repair mechanisms and disturb both membrane function and nutrient absorption (Permyakov 2009).
Most of the biotic chain in the El Bagre EPF endemic area is especially contaminated by mercury and cyanide; thus, microbe-mineral-metalloid interactions are consistently occurring diorite (González et al. 2010, Ayala et al. 2017, Luna Arcila et al. 2017). Evidence supports the concept that membrane transport of methylmercury occur via multiple mechanism. El Bagre and its surrounding municipalities are rich in micas (biotite and muscovite), potassium feldspar, Cu, Zn, Mn, Ni, Mo, Pb, plutonium (Pu), Au, quartz (QCM), rutile, granite, magnetite, almenite, sulfur, pyrite, chalcopyrite, and galena (González et al, 2010, Ayala et al. 2017, Luna Arcila et al. 2017). Others widespread minerals include altaite (TePb). calcite, malachite, chalcedony, and granite-tone-diorite (González et al, 2010, Ayala et al. 2017, Luna Arcila et al, 2017). Several large geological areas have oxidation of minerals. Mining in alluvial deposits is rich in K, feldspar, and plagioclase. Venilleum, salbanda and fine pyrite are also present (González et al, 2010). The endemic area also has very fine particle deposits of quartzdiorite, quartzdiorite with batholiths (a large mass of intrusive igneous rock, also called plutonic rock), larger than 100 km2 (40 sq mi) in area, that are formed from cooled magma, pyrite-calcopyrite-galena, hematite (alone as well as associated with quartz), and minerals created by meteorization and alterations manganese oxides (González et al, 2010).
Let us consider some properties of the polluting metals Cu, Zn, Mn, Ni, Mo, Pb, plutonium (Pu), Au and Hg. Cu, Zn, Mn, Ni, Mo ions are natural ions, which have special binding sites in human proteins. Strong excess of these metal ions can lead to a situation where these ions, in addition to the binding to the correct binding sites, will displace some other metal cations from their binding sites. For example, copper ions can successfully compete with zinc ions for some zinc binding sites in proteins. The opposite situation is also possible. Copper-binding proteins take part in various biological processes from electron transfer to oxidation of various substrates. Copper is an essential element for all living organisms. Copper ligand in Cu binding sites are usually His, Cys, Met residues in tetrahedron or axially distorted tetrahedron configuration (Permyakov 2009).
Acidic conditions alter the metals and metalloids mobility (Wood 1985). The El Bagre geographic are very acidic).
Zinc serves as a co-factor for more than 300 various enzymes, including representatives of all the six main classes of enzymes. Zinc is used as a structural factor in many proteins including DNA-binding proteins containing the domain called “zinc finger”. The most common amino acids that provide ligands to Zn binding sites are His, Glu, Asp and Cys (usually four ligands in square configuration) (Permyakov 2021). A feature common to all zinc sites is that Zn2+ is surrounded by a shell of hydrophilic groups that is embedded within a larger shell of hydrophobic groups (Michael et al. 1992. Zinc and cobalt ions compete for the same binding sites in peptides and proteins, but zinc ions usually have about three orders of magnitude higher affinity (McCall et al. 2000). Zinc dissociation constants for zinc fingers are very low and lie in the range 10-11 – 10-9 М (Berg et al. 1989).
Manganese (Mn2+) ions prefer “hard” ligands such as oxygen atoms of aspartates and glutamates, as well as oxygen atoms of water molecules (Christianson et al. 1999). Sometimes nitrogen atoms of histidines also can be manganese ligands in metalloenzymes. Coordination geometry of manganese in manganese enzymes is usually squire pyramid or trigonal pyramid. Mn2+ competes with Ca2+ and Mg2+ for their binding sites in proteins. Nickel (Ni2+) is known to occur naturally in only a few classes of proteins: urease, carbon monoxide dehydrogenase, S-methyl coenzyme-M reductase and hydrogenase (Volbeda et al. 1996). Nickel and cobalt ions are necessary for cellular function but require strict regulation to hinder toxic side reactions or misincorporation into non-native binding sites. An uptake of too large quantities of nickel has the following consequences: higher chances of development of lung cancer, nose cancer, larynx cancer and prostate cancer; sickness and dizziness after exposure to nickel gas; lung embolism; respiratory failure; birth defects; asthma and chronic bronchitis; allergic reactions such as skin rashes; heart disorders. Ni2+ binding sites usually contain His, Asp and Glu residues.
More than 50 enzymes are known to contain Mo and most of them occur in bacteria while in eukaryotes only six were found (Mendel et al. 2006). Molybdenum (Mo) occurs in a wide range of metalloenzymes in bacteria, fungi, algae, plants, and animals where it forms part of the active sites of these enzymes. In order to gain biological activity, Mo has to be complexed by a pterin compound thereby forming the molybdenum cofactor (Moco). In general, reactions catalyzed by Mo-enzymes are characterized by a transfer of an oxygen atom in a two-electron redox reaction in which the oxidation state of Mo changes between IV and VI.
Pb, Hg, Au, and plutonium (Pu) do not needed for living organisms, they can cause serious disturbances in the functioning of biological systems.
Lead (Pb) is one out of four metals that have the most damaging effects on human health. Elevated Pb2+ level in human body has been associated with hypertension in adults. In children, Pb2+ can cause neurobehavioral and endocrine alterations, delayed puberty, and cognitive deficits. Pb2+ and Ca2+ compete for transport systems of the plasma membrane, such as Ca2+ channels, and the Ca2+ pump (Simons 1993). Pb2+ disturbs intracellular Ca2+ homeostasis. The interaction of lead with proteins represents a fundamental mechanism by which Pb exerts toxicity (Goering 1993). Pb2+ interacts with a number of Ca2+-binding proteins, such as calmodulin, protein kinase C, Ca2+-dependent K+ channels in the plasma membrane and so on. The actions of Pb2+ on neurotransmission may cause human neuropathy and encephalopathy; it was identified 23 lead targets (de Souza et al.2018, Wani et al. 2019) including proteins involved in heme synthesis, calcium metabolism, and neurotransmission. Moreover, the lead damages bio-membranes and disturbs the normal process of DNA replication and transcription [32].
Some authors (Katti et al. 2018) found that synaptotagmin I (SytI), a regulator of Ca2+-evoked neurotransmitter release, has two high-affinity Pb2+ binding sites in its cytosolic C2A and C2B domains. The protein binding Pb2+ sites have holodirected coordination geometries and all-oxygen coordination spheres. The on-rate constants of Pb2+ binding to the C2 domains of SytI are diffusion-limited and comparable to those of Ca2+. In contrast, the off-rate constants are at least two orders of magnitude lower. The authors suggested that Pb2+ can serve as a thermodynamic and kinetic trap for the C2 domains.
It has been found that lead sulfur-rich sites prefer trigonal pyramidal Pb(II)-S3 or Pb(II)-S5-8 geometry (Magyar et al. 2005). In the case of structural zinc-binding protein sites, lead binds in a three-coordinate mode in a geometry that is fundamentally different from the natural coordination of zinc in these sites. This explains why lead disrupts the structure of these peptides and thus provides a detailed molecular understanding of the toxicity of lead.
Other authors, (Zawia et al. 2000), found that zinc fingers of transcription factors could be potential targets for perturbation by Pb. Pb changes the DNA-binding properties of various transcription factors both in vivo and in vitro. The action of Pb on Zn-fingers suggests that this protein domain can serve as a potential mediator for Pb-induced alterations in protein function.
Lead interacts with metallothioneins thru the biosphere, from the lowest organism such bacteria, to algae, to fish, to humans, is important in influencing pathways for lead to enter and harm physiologically important protein function, and thus its toxicity (Won et al. 2017).
Since antigenic proteins studied in the present work possess Cys residues (see Supplement 1) they can be readily attacked by Pb ions, which can change their local or even total conformation.
Metallothionein (MT) is a ubiquitous, cysteine-rich protein that is involved in homeostatic metal response for the essential metals zinc and copper. It was shown (Fitsanakis et al. 2005) that affinity of the metal-binding cysteine residues of MT follows that of metal binding to thiols: Zn(II) < Pb(II) < Cd (II) < Cu(I) < Ag(I) < Hg(II) < Bi(III). It means that lead easily substitute zinc ions in the Zn binding sites.
Pb demonstrates a wide range of effects, from modulation of gene regulation, second messenger systems and other signal transduction pathways, to alterations in the catecholaminergic, opioid, GABAergic and glutamatergic systems (Reddy et al. 2007). Pb has high affinity for free SH groups in enzymes and proteins and its binding can alter their function. This may be the reason for the observed inhibition of acetylcholinesterase activity and consequent increase in acetylcholine level (Reddy et al. 2007).
All forms of mercury (Hg) cause toxic effects in a number of tissues and organs. The kidneys are the primary target organ where inorganic mercury is taken up, accumulated, and expresses toxicity. Within the kidney, the pars recta of the proximal tubule is the most vulnerable segment of the nephron to the toxic effects of mercury. Mercury causes the following main effects (Balali-Mood et al. 2021): disruption of the nervous system; damage to brain functions; DNA damage and chromosomal damage; allergic reactions, tiredness, and headaches; negative reproductive effects.
Mercuric ions bind to various nucleophilic groups, the greatest affinity they have to reduced sulfur atoms, especially those on endogenous thiol-containing molecules, such as glutathione, cysteine, homocysteine, N-acetylcysteine, metallothionein, and proteins (Permyakov 2009). The affinity constant for mercury bonding to thiolate anions is of the order of 1015 to 1020 M-1 (Arino et al . 2013). In contrast, the affinity constants for mercury binding to carbonyl or amino groups are about 10 orders of magnitude lower.
In human milk, mercury is mainly bound to caseins and albumin, while in bovine milk, mercury is associated with caseins and β-lactoglobulin(Mata L et al. 1997). In human blood, mercury is bound mostly by serum albumin and interacts with cysteine and cysteine, which seriously perturbs the protein structure (Song S et al. 2021). Moreover, Hg2+ exposure of blood induces appearance of mercurized tetrameric form of hemoglobin resistant to urea denaturation and which is only partially dissociated by dithiothreitol, likely due to additional protein–Hg interactions involved in the aggregate formation (Piscopo et al 2020). In addition, Hg2+ binds to specific membrane proteins, including band 3 and cytoskeletal proteins 4.1 and 4.2. Interestingly, some forms of Se are effective in reducing Hg toxicity by forming inert Se–Hg complexes in biological tissues (Fang SC 1997, Tinggi U et al. 2022).
Since all the antigenic proteins studied in the present work have thiolate ions of Cis residues (see Supplement 1) they can also be readily attacked by Hg ions, which can change their local or even total conformation.
Elemental form Hg(0) is relatively nontoxic, other forms such as Hg2+ and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on humans (Parks JM et al. 2016). Inorganic Hg2+ can be methylated by certain bacteria and archaea to form methylmercury. Metallic mercury volatilizes at room temperature to form a vapour that is well absorbed by the lungs (Rooney JP 2007). This form of mercury is lipid soluble and it can cross the blood brain barrier and placenta and can be oxidized by catalase and hydrogen peroxide into inorganic mercury (Hg2+), which is retained in the brain for years.
Chelation agents such as the dithiols sodium 2,3-dimercaptopropanesulfate (DMPS) and meso-2,3-dimercaptosuccinic acid (DMSA) are used for the treatment of mercury toxicity (Rooney JP 2007). Zinc and selenium exert protective effects against mercury toxicity, most likely due to induction of the metal binding metallothionein and selenoprotein-P.
Gold (Au) can exist in multiple oxidation states I, 0, I, II, III, IV, and V, but only Au 0, I. and III are stable in aqueous systems (Kumar A et al. 2013). The cytotoxicity of Au(III) complexes is strictly related to the presence of the Au(III) center. The most adverse cases of Au toxicity is restricted to skin and mucous membranes.
Some gold complexes can bind to proteins and change their properties. For example, some authors (Talib J et al 2006), have found interactions of the clinically used antiarthritic agent [Au(S2O3)2]3-, and AuPEt3Cl, a derivative of another clinically used agent auranofin, with human serum albumin. It has been found that both gold compounds interact with Cys34 in human serum albumin. The intact gold molecules are non-covalently bound in these complexes.
The reactions of gold(III) complexes -[Au(ethylenediamine)2]Cl3, [Au(diethylentriamine)Cl]Cl2, [Au(1,4,8,11-tetraazacyclotetra-decane)](ClO4)2Cl, [Au(2,2',2'-terpyridine)Cl]Cl2, [Au(2,2'-bipyridine)(OH)2][PF6] and the organometallic compound [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine-H)(OH)][PF6]- with bovine serum albumin were investigated by various spectroscopic methods and separation techniques (Marcon G et al. 2003). Weak metal-protein interactions were revealed for the [Au(ethylenediamine)2]3+ and [Au(1,4,8,11-tetraazacyclotetradecane)]3+ species. In contrast, tight metal-protein adducts are formed when bovine serum albumin is reacted with either [Au(diethylentriamine)Cl]2+ and [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine-H)(OH)]+. It has been shown that adduct formation for both of these Au(III) complexes occurs through coordination by surface histidines. Metal binding to the protein was tight: complete detachment of the metal from the protein has been achieved only after the addition of excess potassium cyanide.
Other authors (Marcon G et al. 2003), studied interactions of metallothionein, a small metal-binding protein, with aurothiomalate, an anti-arthritic gold compound. Mass spectrometry measurements revealed gold atoms bound to individual metallothionein molecules. Under certain conditions, mass spectra show gold binding ratios of greater than 1:1 with the cysteine residues of metallothionein.
Gold may play a key pathogenic role in our patients since it can easily interact with other metals and metalloids in the body. Gold degradation has been described to be mediated by NADPH oxidase; NADPH oxidase produces highly reactive oxygen species (ROS) in lysosomes linked to cell-protective expression of the erythroid 2 nuclear factor therapy (Balfourier A et al. 2019). A gold recrystallization process occurs within aurosome structures, sometimes combined with sulfur and phosphorus. The recrystallization process was found in people and animals that received gold therapy (Balfourier A et al. 2019). Aurosomes have been found in combination with melanosomes and other lysosomes within dermal macrophages in people receiving gold therapy.
Plutonium (Pu) is produced in nuclear reactors when uranium 238 absorbs neutrons forming uranium 239, which is a beta emitter with a short half-life; its daughter neptunium 239 also emits an electron to form plutonium 239. Plutonium 239 (half-life 2.4×104 years) emits alpha particles. It undergoes fission with the emission of several neutrons and can maintain a chain reaction (OECD/NEA 2023).
Five oxidation states of Pu are known: Pu3+, Pu4+, PuO2+, PuO22+, and PuO5-. Ethylenediaminetetraacetic acid (EDTA) forms complexes with plutonium cations of all valences and is often used to decontaminate surfaces or to wash out ingested plutonium. Plutonium shares some similarities with biologically important trivalent transition metals, especially iron. Plutonium ingested by or injected into humans is transported in the transferrin-based iron(III) transport system and then is stored in the liver in the iron store ferritin (Sauge-Merle et al. 2017). Plutonium that is inhaled by humans lodges in the lungs and is slowly translocated to the lymph nodes. Inhaled plutonium has been shown to result in lung cancer in experimental animals. Pu effects on cells and organisms depend not only on alpha particle emission, but also on behavior of Pu as a heavy metal. Some researchers (Daronnat L et al. 2023) studied the complexation of Pu(IV) to the EF-hand motif of calmodulin. Three different species of Pu were found with formation of 1:1 Pu(IV):calmodulin peptide complexes, Pu(IV) reduction, and formation of peptide-mediated Pu(IV) hexanuclear cluster. Other scientists (Aryal BP et al. 2012) used a metalloproteomic approach to identify proteins interacting with Pu(IV). They found that several proteins from PC12 cells show affinity towards Pu(IV)-NTA (plutonium bound to nitrilotriacetic acid). The identified proteins in their experiments are known to bind calcium, magnesium, or divalent transition metal ions.
The amalgamation of metal ions is a process of extracting metals (as native gold and silver) from their ores by the addition of small quantities of mercury to the stamping or grinding unit so that the resulting amalgam is caught on mercury-coated copper plates from which it is then scraped.
We do not know if similar processes can proceed inside the cells and/or cell junctions, and such interactions of metals could change primary, secondary, or tertiary protein and/or lipid structures, as well as their junctions to other molecules.
Several of these antigens have both cytoplasmic and nuclear locations. Thus, these altered by interactions with metals structures could affect the manner that the host immune system may identify them. If they are identified as stranger molecules, the immune system could attack them, triggering an autoimmune phenomenon.
We conclude based on our current findings as well as previous ones (Abreu Velez, Warfvinge et al. 2003), that the presence of metals, metal ions, metalloids, and trace elements in the skin biopsies of patients affected by this orphan disease may produce different types of antigenic determinants on the El Bagre-EPF antigens metals-metalloids binding-altered proteins causing antibodies to be generated against the altered proteins through different mechanisms. The most dangerous metals from the polluting ones in El Bagre, Colombia, which can most seriously change the structure of proteins are mercury, lead, gold, and plutonium. Mercury and lead can not only bind to the calcium and zinc binding sites in proteins, but effectively interact with separate SH groups in proteins. All these interactions can seriously change structure and properties of almost any protein including the proteins of the desmoglein family. Experimental studies of these proteins are needed to confirm these predictions.