Christopher J. Coates

Diverse immune functions of hemocyanins

Substantial evidence gathered recently has revealed the multiple functionalities of hemocyanin. Contrary to previous claims that this ancient protein is involved solely in oxygen transport within the hemolymph of invertebrates, hemocyanin and hemocyanin-derived peptides have been linked to key aspects of innate immunity, in particular, antiviral and phenoloxidase-like activities. Both phenoloxidase and hemocyanin belong to the family of type-3 copper proteins and share a high degree of sequence homology. While the importance of phenoloxidase in immunity and development is well characterised, the contribution of hemocyanin to biological defence systems within invertebrates is not recognised widely. This review focusses on the conversion of hemocyanin into a phenoloxidase-like enzyme and the array of hemocyanin-derived immune responses documented to date.

Dose-dependent cellular and humoral responses in Galleria mellonella larvae following β-glucan inoculation

Galleria mellonella larvae were inoculated with different doses of beta-glucan by injection into the haemocoel. Those larvae that had received high doses of beta-glucan (15, 30 or 60microg/larva) demonstrated increased survival following infection with the yeast Candida albicans. High concentrations of glucan induced an increase in haemocyte density and a reduction in yeast proliferation within the haemocoel. Proteomic analysis of glucan-treated larvae revealed increased expression of a variety of peptides some of which may possess antimicrobial properties. Analysis of expression profiles revealed that low doses of beta-glucan (3.75microg/larva) triggered the increased expression of certain peptides (e.g. hemolin) while high dose inoculation was required before the increased expression of others (e.g. archaemetzincin) was evident. These results indicate that low doses of beta-glucan induce a limited immune response while high doses induce an immune response that has the potential to curtail the threat within the haemocoel but also withstand a subsequent infection. Immune priming gives insects the ability to withstand a potentially lethal infection if exposed to a low level of the pathogen 24-48h previously. Immune priming has resource implications and this work indicates that a graded immune response is initiated depending upon the amount of the immune priming agent encountered.

Possible role of phosphatidylserine–hemocyanin interaction in the innate immune response of Limulus polyphemus

Phenoloxidase enzymes and the associated pro-phenoloxidase activation cascade play an essential role in the immune response of arthropods. Phenoloxidase activity can be elicited in the oxygen carrier, hemocyanin, by the addition of the artificial inducer, SDS. There is some evidence to support hemocyanin acting as a phenoloxidase in vivo; however, the identity of natural activators remains unclear. This study explores the role of the phospholipid, phosphatidylserine, as a possible natural activator of hemocyanin-derived phenoloxidase activity. Characterisation of the structural changes associated with activation of hemocyanin-derived phenoloxidase suggests that phosphatidylserine induces similar conformational changes to those caused by the artificial inducer, SDS. We propose that anionic phospholipids, in particular phosphatidylserine, may act as natural activators of hemocyanin-derived phenoloxidase.

Immunological properties of Oxygen-Transport Proteins: Hemoglobin, Hemocyanin and Hemerythrin

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.

A putative link between phagocytosis-induced apoptosis and hemocyanin-derived phenoloxidase activation

Apoptosis and phagocytosis are crucial processes required for developmental morphogenesis, pathogen deterrence and immunomodulation in metazoans. We present data showing that amebocytes of the chelicerate, Limulus polyphemus, undergo phagocytosis-induced cell death after ingesting spores of the fungus, Beauveria bassiana, in vitro. The observed biochemical and morphological modifications associated with dying amebocytes are congruent with the hallmarks of apoptosis, including: extracellularisation of phosphatidylserine, intranucleosomal DNA fragmentation and an increase in caspase 3/7-like activities. Previous studies have demonstrated that phosphatidylserine is a putative endogenous activator of hemocyanin-derived phenoloxidase, inducing conformational changes that permit phenolic substrate access to the active site. Here, we observed extracellular hemocyanin-derived phenoloxidase activity levels increase in the presence of apoptotic amebocytes. Enzyme activity induced by phosphatidylserine or apoptotic amebocytes was reduced completely upon incubation with the phosphatidylserine binding protein, annexin V. We propose that phosphatidylserine redistributed to the outer plasma membrane of amebocytes undergoing phagocytosis-induced apoptosis could interact with hemocyanin, thus facilitating its conversion into a phenoloxidase-like enzyme, during immune challenge.

Identification of candidate antimicrobial peptides derived from abalone hemocyanin

Hemocyanins present in invertebrate hemolymph are multifunctional proteins, responsible for oxygen transport and contributing to innate immunity through phenoloxidase-like activity. In arthropods, hemocyanin has been identified as a source of broad-spectrum antimicrobial peptides during infection. Conversely, no hemocyanin-derived antimicrobial peptides have been reported for molluscs. The present study describes a putative antimicrobial region, termed haliotisin, located within the linking sequence between the α-helical domain and β-sheet domain of abalone (Haliotis tuberculata) hemocyanin functional unit E. A series of synthetic peptides based on overlapping fragments of the haliotisin region were tested for their bactericidal potential. Incubating Gram-positive and Gram-negative bacteria in the presence of certain haliotisin peptides, notably peptides 3-4-5 (DTFDYKKFGYRYDSLELEGRSISRIDELIQQRQEKDRTFAGFLLKGFGTSAS) led to reductions in microbial growth. Furthermore, transmission electron micrographs of haliotisin-treated bacteria revealed damages to the microbial cell wall. Data discussed here provides the first evidence to suggest that molluscan hemocyanin may act as a source of anti-infective peptides.

Hemocyanin-derived phenoloxidase activity: A contributing factor to hyperpigmentation in Nephrops norvegicus

The phenomenon of hyperpigmentation (melanosis) in shellfish has long been attributed to phenoloxidase enzymes. Over the last number of years, the oxygen carrier hemocyanin, has demonstrated several immune- and physiological functionalities, most notably, inducible phenoloxidase activity. In this study, hemocyanin purified from the hemolymph of Nephrops norvegicus displays diphenoloxidase activity in the presence of a number of elicitors and retains structural and functional integrity throughout the process of freeze-thawing (at -25 °C). Conversely, cellular phenoloxidase activity (present in cell-lysates), demonstrates >98% reduction in activity after freeze-thawing. We present evidence that hemocyanin may act as a causative agent of hyperpigmentation in N. norvegicus. The inhibition of hemocyanin-derived phenoloxidase activity is discussed, and for the first time, the biophysical interactions of shellfish hemocyanin with known phenoloxidase inhibitors are presented.

Effects of known phenoloxidase inhibitors on hemocyanin-derived phenoloxidase from Limulus polyphemus

Inhibitors of phenoloxidase are used routinely to characterise the structural and functional properties of phenoloxidases. Hemocyanin-derived phenoloxidase activity is also sensitive to standard phenoloxidase inhibitors. In this study, we characterise the effects of a number of phenoloxidase inhibitors on hemocyanin-derived phenoloxidase activity from the chelicerate, Limulus polyphemus. Both inhibition type and K(i) values were similar to those observed for hemocyanin-derived phenoloxidase from another chelicerate, Eurypelma californicum. In addition, substrate inhibition was observed at concentrations above 2mM dopamine. The conformation in which two of the inhibitors, namely tropolone and kojic acid, would bind near the Cu(II) centre of hemocyanin is proposed.

Phagocytic activity of Limulus polyphemus amebocytes in vitro.

Phagocytosis of invading microorganisms is a fundamental component of innate immunity. The Atlantic horseshoe crab, Limulus polyphemus, possesses a single immune cell type, the granular amebocyte. Amebocytes release a repertoire of potent immune effectors in the presence of pathogens, and function in hemostasis. In contrast to other arthropod immunocytes, the properties of amebocyte phagocytosis remain poorly characterised, restricted by the technical challenges associated with handling these labile cells. We have addressed these challenges and observed the internalisation of microbial and synthetic targets by amebocytes in vitro. Confirmation of target internalisation was achieved using a combination of fluorescent quenching and lipophilic membrane probes: R18 and FM 1-43. Viability, morphological integrity and functionality of extracted amebocytes appeared to be retained in vitro. The phagocytic properties of L. polyphemus amebocytes described here, in the absence of endotoxin, are similar to those observed for arthropod immunocytes and mammalian neutrophils.

Re-evaluation of insect melanogenesis research: Views from the dark side

Melanins (eumelanin and pheomelanin) are synthesized in insects for several purposes including cuticle sclerotization and color patterning, clot formation, organogenesis, and innate immunity. Traditional views of insect immunity detail the storage of pro‐phenoloxidases inside specialized blood cells (hemocytes) and their release upon recognition of foreign bodies. Activated phenoloxidases convert monophenols into reactive quinones in a two‐step enzymatic reaction, and until recently, the mechanism of tyrosine hydroxylation remained a mystery. Herein, we present our interpretations of these enzyme–substrate complexes. The resultant melanins are deposited onto the surface of microbes to immobilize, agglutinate, and suffocate them. Phenoloxidase activity and melanin production are not limited to the blood (hemolymph) or cuticle, as recent evidence points to more diverse, sophisticated interactions in the gut and with the resident symbionts. This review offers insight into the somewhat neglected areas of insect melanogenesis research, particularly in innate immunity, its role in beneficial insects such as pollinators, the functional versatility of phenoloxidases, and the limitations of common experimental approaches that may impede progress inadvertently.