The ubiquitin-like protein SUMO-1 is conjugated to a variety of proteins including Ran GTPase-activating protein 1 (RanGAP1), IB␣, and PML. SUMO-1-modified proteins display altered subcellular targeting and/or stability. We have purified the SUMO-1-activating enzyme from human cells and shown that it contains two subunits of 38 and 72 kDa. Isolation of cDNAs for each subunit indicates that they are homologous to ubiquitinactivating enzymes and to the Saccharomyces cerevisiae enzymes responsible for conjugation of Smt3p and Rub1p. In vitro, recombinant SAE1/SAE2 (SUMO-1-activating enzyme) was capable of catalyzing the ATP-dependent formation of a thioester linkage between SUMO-1 and SAE2. The addition of the SUMO-1-conjugating enzyme Ubch9 resulted in efficient transfer of the thioester-linked SUMO-1 from SAE2 to Ubch9. In the presence of SAE1/SAE2, Ubch9, and ATP, SUMO-1 was efficiently conjugated to the protein substrate IB␣. As SAE1/SAE2, Ubch9, SUMO-1, and IB␣ are all homogeneous, recombinant proteins, it appears that SUMO-1 conjugation of IB␣ in vitro does not require the equivalent of an E3 ubiquitin protein ligase activity.

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...

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Extracts of the granular haemocytes of Carcinus maenas were subjected to ion-exchange chromatography and reverse-phase (RP)-HPLC to investigate the presence of an antibacterial protein of < 11 kDa. This protein was isolated, characterized and subjected to partial amino acid sequence analysis. It was found by mass spectrometry to have a molecular mass of 11 534 Da, to be cationic and hydrophobic and active only against marine Grampositive bacteria. In addition its activity is stable after heating to 100 8C and is retained at concentrations as low as 10 mg´mL 21 . It has an unusual amino acid sequence, unlike any known antibacterial peptide described in the literature but bears a consensus disulphide domain signature, indicating that it might be a member of the fourdisulphide core proteins. Partial cDNA sequence data has been obtained.Keywords: antibacterial protein; Carcinus; crustacean; haemocytes; innate immunity.Antimicrobial peptides are important in the first line of the host defence system of many animal species [1]. Their value in innate immunity lies in their ability to function without either high specificity or memory. Moreover their small size makes them easy to synthesize without dedicated cells or tissues and they rapidly diffuse to the point of infection. They have been defined as molecules of less than 10 kDa which show stoichiometric, as opposed to enzymatic, antimicrobial properties [1]. Many antibacterial peptides show a remarkable specificity for prokaryotes with low toxicity for eukaryotic cells; a phenomenon which has favoured their investigation and exploitation as potential new antibiotics [2]. Aside from their commercial or chemotherapeutic value, antibacterial peptides provide a useful way of assessing and studying innate immunity at the biochemical and molecular levels. This is especially appropriate for invertebrates, which lack acquired memory-type immunity based on clonally derived immunoglobulins or T lymphocyte subsets. They have been isolated from a wide variety of invertebrate phyla, including insects [1], ascidians [3,4], chelicerates [5], annelids [6] and molluscs [7,8]. Whilst there have been many reports of crustacean defence strategies, such as phagocytosis, nodule formation and phenoloxidase activation [9], only two studies have been made of the presence, variety and biological function of antibacterial peptides in this group of arthropods [10,11]. There are good reasons for obtaining a greater understanding of the biochemical and molecular underpinnings of the host defences of this important group of invertebrates. Certainly, crustaceans are of enormous commercial significance in many parts of the world. The global value of the industry is in excess of £9.5 million per year [12] and as losses due to disease can be enormous there is a great need to find ways of enhancing natural immunity to infectious agents in these animals.The first antibacterial protein to be isolated and partially characterized from a marine decapod was a 6.5-kDa prolinerich peptide with sequence similar...

Skin exudates of rainbow trout contain a potent 13.6 kDa anti-microbial protein which, from partial internal amino acid sequencing, peptide mass fingerprinting, matrix-associated laser desorption/ionization MS and amino acid analysis, seems to be histone H2A, acetylated at the N-terminus. The protein, purified to homogeneity by ion-exchange and reversed-phase chromatography, exhibits powerful anti-bacterial activity against Gram-positive bacteria, with minimal inhibitory concentrations in the submicromolar range. Kinetic analysis revealed that at a concentration of 0.3 microM all test bacteria lose viability after 30 min incubation. Weaker activity is also displayed against the yeast Saccharomyces cerevisiae. The protein is salt-sensitive and has no haemolytic activity towards trout erythrocytes at concentrations below 0.3 microM. Reconstitution of the protein in a planar lipid bilayer strongly disturbs the membrane but does not form stable ion channels, indicating that its anti-bacterial activity is probably not due to pore-forming properties. This is the first report to show that, in addition to its classical function in the cell, histone H2A has extremely strong anti-microbial properties and could therefore help contribute to protection against bacterial invasion.