Hemocyanin-derived phenoloxidase reaction products display anti-infective properties

Abstract

Hemocyanin is a multi-functional protein located in the hemolymph (blood) of certain arthropods and molluscs. In addition to its well-defined role in oxygen transport, hemocyanin can be converted into a phenoloxidase-like enzyme. Herein, we tested the antimicrobial properties of horseshoe crab (Limulus polyphemus) hemocyanin-derived phenoloxidase reaction products using broad ranges of phenolic substrates (e.g. -DOPA) and microbial targets (Gram-positive/negative bacteria, yeast). The enzyme-catalysed turnover of several substrates generated (by)products that reduced significantly the number of colony forming units. Microbicidal effects of hemocyanin-derived phenoloxidase were thwarted by the inhibitor phenylthiourea. Data presented here further support a role for hemocyanin in invertebrate innate immunity.

Discovery of Novel Hemocyanin-Like Genes in Metazoans

“…There are records of Hrs molecules involved in other functions besides oxygen loading, such as in the storage of iron atoms, detoxification of heavy metals, and metabolic pathways related to the innate immunity of some species of annelids (e.g., Theromyzon tessulatum, Hirudo medicinalis, and Neanthes diversicolor; Baert et al, 1992;Demuynck et al, 1993;Vergote et al, 2004; reviewed by Coates and Decker, 2017). Moreover, Hcs are known for contributing in many ways to immune defenses, such as the inhibition of viral replication, precursor of antimicrobial peptides, and the conformational switch to a phenoloxidase-like enzyme (Coates and Nairn, 2014;Coates and Talbot, 2018).…”

The stress–immunity axis in shellfish

“…The proPhenoloxidase (proPO) activation cascade is emblematic of the innate immune response of many aquatic and terrestrial invertebrates, as it facilitates the early steps of melanisation (reviewed by Cerenius et al, 2008). Melanotic polymers are employed by the host to immobilise microbial intruders (microbiostatic), and the toxic by-products of catalytic activities inflict damage to microbes and parasitesas determined in crustaceans (Cerenius et al, 2010a), chelicerates (Coates and Talbot, 2018), and gastropods (Quinn et al, 2020). The proPO cascade is a striking example of the intersection between humoral and cellular defences.…”

“…The term 'phenoloxidase' (PO) may incorporate four functionally distinct proteins, namely catecholoxidase, tyrosinase, laccase, and haemocyanin (Coates and Costa-Paiva, 2020). All have demonstrated a capacity to oxidise L-DOPA and dopamine into quinones (DOPAchromes), with haemocyanin-derived phenoloxidase (HC-d PO) also able to generate products from (nor)epinephrine (Jaenicke and Decker, 2008;Coates and Talbot, 2018). Catecholamines are generated from amino acids, phenylalanine  L-tyrosine  L-DOPA  dopamine, norepinephrine and epinephrine.…”

The ARM repeat domain of hemocyanin interacts with MKK4 to modulate antimicrobial peptides expression

“…The respiratory glycoprotein hemocyanin also possess many immune-related functions ( Coates and Talbot, 2018 ; Jiang et al., 2007 ; Laino et al., 2015 ; Nagai et al., 2001 ; Siddiqui et al., 2006 ; Yao et al., 2019 ) and can act as a pattern recognition receptor (PRR) to recognize pathogen-associated molecular patterns (PAMPs) in antibacterial response ( Zhang et al., 2017 ). Moreover, hemocyanin undergoes proteolytic degradation to generate antimicrobial peptides in response to different pathogenic microorganisms such as viruses, bacteria, and fungi ( Destoumieux-Garzon et al., 2001 ; Lee et al., 2003 ; Wen et al., 2016 ; Zhan et al., 2019 ; Zhang et al., 2004a ).…”