Crustins: Enigmatic WAP domain-containing antibacterial proteins from crustaceans

Abstract

Crustins are antibacterial proteins of ca 7-14 kDa with a characteristic four disulphide corecontaining whey acidic protein (WAP) domain, expressed by the circulating haemocytes of crustaceans.Over 50 crustin sequences have been now reported from a variety of decapods, including crabs, lobsters, shrimp and crayfish. Three main types seem to occur but all possess a signal sequence at the amino terminus and a WAP domain at the carboxyl end. Differences between types lie in the structure of the central region. Those crustins purified as the native protein or expressed recombinantly all kill Gram-positive bacteria, and gene studies have shown that they are constitutively expressed, often at high levels, but show no consistent patterns of change in expression following injection of bacteria. This variable response to infection is enigmatic but indicates that these proteins could perform additional functions, perhaps as immune regulators in recovery from wounding, trauma or physiological stress.Prof Kenneth Soderhall Co-Editor in Chief Developmental and Comparative Immunology Dear Kenneth I attach the revised manuscript for the invited review, Crustins: enigmatic WAP domaincontaining antibacterial proteins from crustaceans, for which all the referees comments have been addressed. Below is a summary of changes made.1. Two of the reviewers have issues with the SS tree (formerly Fig 2), with reviewer 2 making the point that the functional WAP domain will more usefully indicate relationships rather than the SS. We do not concur with the comment that the signal sequence (SS) tree is unnecessary but do accept that its placement in the article could be better and that it could be more fully discussed. Importantly, the SS radiation tree did not drive the 3-type classification, rather it was led by the overall domain organisation of the various types that we judged, from our starting definition, to be within the crustin 'family'. The region of variation is mainly in the 'central' region of the molecule, ie between the SS and WAP domain. We constructed the SS and WAP domain trees to test if the suggested classification was supported, independently by phylogenies within these regions. We believe that they do and the inclusion of both trees, rather than just a tree for the WAP domain, makes the case for the classification of crustins into Types I, II or III, even stronger. We have therefore retained the SS radiation tree but discuss it more fully in the section on 'Relationships' (new pages 11 & 12) rather than its former position. We prefer not to add species identifiers to the as these will make it very 'busy' and untidy. We feel that Accession numbers are more appropriate than species names. Please note we have slightly modified the annotations by leaving ambiguous sequences out of the clusters marked by dotted or dashed lines.2. Referee 1 correctly points out that the argument (beginning in the last paragraph on pg 6 and continued on pg 7in the original m/s) that the signal sequence is probably not involved in crust...

Molecular characterization of a crustin-like antimicrobial peptide in the giant tiger shrimp, Penaeus monodon, and its expression profile in response to various immunostimulants and challenge with WSSV

“…Crustins are cationic, cysteine-rich AMPs with a molecular weight of 7-14 kDa and isoelectric point of 7.0-8.7 and one whey-acidic protein (WAP) domain at the carboxy terminus (Smith et al 2008). Several isoforms of crustins have been described in a wide range of penaeid prawns including Litopenaeus vannamei (Bartlett et al 2002), Litopenaeus setiferus (Bartlett et al 2002), Penaeus monodon (Supungul et al 2004;Chen et al 2004;Amparyup et al 2008), Marsupenaeus japonicus (Rattanachai et al 2004), Litopenaeus schmitti (Rosa et al 2007), Fenneropenaeus chinensis (Zhang et al 2007), Farfantepenaeus brasiliensis (Rosa et al 2007), Farfantepenaeus paulensis (Rosa et al 2007), Farfantepenaeus subtilis (Rosa et al 2007) and Fenneropenaeus indicus (Antony et al 2010).…”

Purification and Characterization of a Cysteine-Rich 14-kDa Antibacterial Peptide from the Granular Hemocytes of Mangrove Crab Episesarma tetragonum and Its Antibiofilm Activity

“…Among the crustacean AMPs, crustins are cationic, rich in cysteines, with a molecular weight of 7-14 kDa, and have an amphipathic secondary structure and one whey acidic protein (WAP) domain at the carboxy terminus [13]. Due to its negative nature, crustins interact with positively charged bacterial membranes by electrostatic interaction and exert a wide spectrum of antimicrobial activity by permeabilizing cell membranes and/or inhibiting intracellular macromolecule synthesis after penetration across cell membranes [14].…”

Hemolymph proteins in marine crustaceans

“…Most of these proteins are small cationic molecules exhibiting a broad spectrum of activity against gram-positive and/or gram-negative bacteria. There are some antibacterial proteins that are not inducible such as lysozyme [5] and andropin [6] . Over the past several years, many antimicrobial peptides have been found and characterized in crab species.…”

“…Crustins are antibacterial proteins of 7-14 kDa with a characteristic four-disulphide core-containing whey acidic protein (WAP) domain, expressed by the circulating haemocytes of crustaceans [5] . Many immune proteins are released from crustacean haemocytes by exocytosis so it is plausible that crustins might be liberated into the body fluids in the same way.…”