Immunological properties of oxygen-transport proteins: hemoglobin, hemocyanin and hemerythrin

Abstract

It is now well documented that peptides with enhanced or alternative functionality (termed cryptides) can be liberated from larger, and sometimes inactive, proteins. A primary example of this phenomenon is the oxygen-transport protein hemoglobin. Aside from respiration, hemoglobin and hemoglobin-derived peptides have been associated with immune modulation, hematopoiesis, signal transduction and microbicidal activities in metazoans. Likewise, the functional equivalents to hemoglobin in invertebrates, namely hemocyanin and hemerythrin, act as potent immune effectors under certain physiological conditions. The purpose of this review is to evaluate the true extent of oxygen-transport protein dynamics in innate immunity, and to impress upon the reader the multi-functionality of these ancient proteins on the basis of their structures. In this context, erythrocyte-pathogen antibiosis and the immune competences of various erythroid cells are compared across diverse taxa.

Keywords: Antimicrobial peptides; Enzyme promiscuity; Erythrocytes; Innate immunity; Metabolism; Myoglobin; Phenoloxidase; Redox.

Fig. 1

Three major classes of oxygen-transport proteins. Each of the 4 subunits (α1, α2, β1, β2) making up human Hb (and monomeric myoglobin) contain heme cofactors (Fe²⁺—protoporphyrin IX) that bind O₂. Each heme group is indicated by an arrow head. The proximal histidine forms a direct bond with the iron atom, while the distal histidine is suggested to form a hydrogen bond with O₂. The distal His hinders the energetically favoured straight binding of O–O. Arthropod hemocyanin subunits consist of three domains (green, blue, orange) and mollusc hemocyanin FUs consist of two domains (blue, orange). The blue domains possess two copper atoms, each is coordinated by three highly conserved histidine residues. O₂ is bound in a ‘side on’ (μ–η²:η²) bridging formation between CuA and CuB. Hemerythrin (and myo-hemerythrin) secure O₂ between two iron atoms (Fe₁ and Fe₂) which are held in place by five histidines residues, one aspartic acid and one glutamic acid. In the process, a hydroperoxide (OOH–) complex is formed. Images were produced using UCSF Chimera [202] and crystal structures from the Protein Data Bank: human hemoglobin tetramer 1GZX (α₂β₂, ~64 kDa), arthropod hemocyanin subunit 1OXY (~72 kDa), mollusc hemocyanin functional unit 1JS8 (~50 kDa) and sipunculid hemerythrin homo-octamer 1I4Y (~108 kDa). Inset oxygen is coloured grey; iron is coloured red; copper is coloured orange; histidines are coloured blue: aspartic acid is pink and glutamic acid is green

Fig. 2

Schematic representation of hemoglobin functionality beyond oxygen transport. Hemolysis, whether it is caused by microbes or physical trauma, leads to the uncontrolled release of hemoglobins (Hbs). Extracellular Hb inflicts damages by producing reactive oxygen/nitrogen species (1) and interfering with hepatic, splenic and renal physiologies (2). Inflammation can be avoided/controlled by Hb-scavenging glycoproteins [haptoglobin (Hp), hemopexin (Hx) and apoplipoprotein A-I (apoAI)] and soluble receptors (CD163). These proteins intercept Hb, neutralise its oxidant properties, and direct it towards immune cells for degradation and to promote anti-inflammatory responses. Chemical (glutathione) and enzymatic antioxidants (superoxide dismutase, catalase) are recruited also. Anti-infective responses are triggered when Hb binds to PAMPs/DAMPs (3). Microbial ligand (PAMPs)–Hb complexes are recognised by immune cells whereupon pro-inflammatory molecules are released, and Hb is converted into a pseudoperoxidase (POX). If Hb has been enzymatically processed prior to erythrocyte rupture, then hemocidins (antimicrobial peptides) will also been disseminated (4). This scheme was produced by summarising information presented in the following manuscripts: [61, 62, 64, 68, 80]

Fig. 3

Hemoglobins and the locations of hemocidins. a Human (PDB 1GZX red), rabbit (PDB 2RAO grey), fish (PDB 3BJ2 blue) and mollusc (PDB 4HRR brown) β-globin structures have been aligned and superimposed to highlight the conserved helical structures. The protoporphyrin ring is coloured black. B Secondary structural features of the oxy-hemoglobin tetramer (PDB 1GZX) are presented as ribbons. Alpha and beta chains are coloured light yellow and grey, respectively. Coils are coloured black. The space-filling models of Hb were used to highlight the location of various antimicrobial peptides (black arrows). The α-chain peptides consist of residues 1–29 (red), 12–29 (red), 33–76 (yellow) and 77–141 (purple). The β-chain peptides consist of residues 1–21 (blue), 9–21(blue), 56–72 (orange) and 111–146 (green). It is worth noting that the Hb-β peptide 111–146 was detected in the placenta and in the cytosol of erythrocytes [94]

Fig. 4

Hemoglobin-derived antimicrobial peptides from fish. The overlapping encrypted peptides (HbβP-1, 2 and 3) of fish (Ictalurus punctatus; GI:318171215) hemoglobin are presented using the crystal structure of rainbow trout hemoglobin (3BOM). The helical structures of each peptide are presented as ribbons, and their locations are indicated by black arrows. It has not been confirmed whether the peptides retain these structural features upon detachment from the Hb

Fig. 5

Arthropod hemocyanin-derived antimicrobial peptides. The crystal structure of Panulirus interruptus hemocyanin (PDB 1HC1) is used to illustrate the location of the encrypted peptides: PvHCt (FEDLPNFGHIQVKVFNHGEHIHH: blue), astacidin 1 (FKVQNQHGQVVKIFHH: blue) from crayfish, and PsHCt2 (LVVAVTDGDADSAVPNLHENTEYNHYGSHGVY: orange) from shrimp on the hemocyanin hexamer (~420 kDa) and corresponding subunit (~70 kDa). Hemocyanin subunit domains I and II are coloured green and purple, respectively. Both peptides are located on the C-terminal subunit (III) of the hemocyanin where they can be liberated through proteolysis. The shrimp peptide (PvHCt) is linear, α-helical and amphipathic, with an overall net-negative charge (theoretical pI = 6.1) as revealed by NMR. The electrostatic surface potential was calculated using UCSF Chimera [202]. PvHCt was isolated from Litopenaeus vannamei hemolymph and displays strict fungicidal activity. Both peptides are exposed to the environment even in the hexameric aggregation state. See also [21, 183]