Styelin D, an Extensively Modified Antimicrobial Peptide from Ascidian Hemocytes

Abstract

We isolated styelin D, a 32-residue, C-terminally amidated antimicrobial peptide, from the blood cells (hemocytes) of the solitary ascidian, Styela clava. Styelin D had remarkably extensive post-translational modifications, containing two novel amino acids, dihydroxyarginine and dihydroxylysine, and two distinctly unusual ones, 6-bromotryptophan and 3,4-dihydroxyphenylalanine. In addition, the peptide exhibited microheterogeneity because of differential mono-and dihydroxylation of several lysine residues. Gram-negative and Gram-positive bacteria, and this activity was retained in 200 mM NaCl. The role of the extensive modifications may be to preserve activity at low pH and/or high salinity because, under these conditions, the native peptide was considerably more active against the Gram-positive bacterial strains than its unmodified synthetic analogue. The peptide was also hemolytic and quite cytotoxic to eukaryotic cells. These broad ranging activities, combined with its relative abundance in ascidian hemocytes, suggest that styelin D plays a significant role in the innate immune mechanisms of S. clava.As primitive chordates, ascidians are excellent subjects for research into the evolution of vertebrate innate immunity. Although ascidians lack the immunoglobins and T cell receptors found in higher vertebrates, their hemocytes can recognize and destroy foreign materials such as antigens or non-selftissues that penetrate their tunic or contact their internal tissues (1). In certain ascidian species, vacuolated cells (including the berry-shaped morula cells) contain hemagglutinin (2, 3) and proteases (4, 5). The morula cells of Halocynthia roretzi contain halocyamines (tetrapeptides containing L-3,4-dihydroxyphenylalanine (DOPA) 1 and a 6-bromoindole) that are active against Gram-positive bacteria, fungi, marine bacteria, and some fish RNA viruses (6, 7). Phenoloxidase, perhaps acting in concert with DOPA-derived metabolites and proteins (8 -10), may also be a critical player in cellular defenses. Two families of antimicrobial polypeptides, clavanins and styelins, were identified from hemocytes of the stolidobranch ascidian, Styela clava. The former are histidine-rich, ␣-helical polypeptides with 23 amino acid residues and C-terminal amidation (11). The latter are phenylalanine-rich peptides with 32 amino acid residues (12). Whereas styelins demonstrated a broad pH optimum in killing bacteria, the clavanins showed optimal antibacterial activity at pH 5.5. The initial 20 amino acid residues of styelins A and B were sequenced and indicated an unusual structure consisting of several unidentified amino acid residues and hydroxylysine residues that are common only in collagen-like polypeptides. Although they were not specifically identified, the absorbancy of styelins at 280 nm suggested that methylated or otherwise modified tyrosine residues were present.Amino acid sequences corresponding to both the clavanins and styelins were identified using partial sequences and a cDNA library prepared from hemopoietic pharyngeal t...

Antimicrobial Peptides from Marine Invertebrates

“…In addition, styelin D exhibits microheterogeneity due to differential hydroxylation of several lysine residues. Styelin D displayed activity against gram-negative and -positive bacteria in 200 mM NaCl, the role of the extensive posttranslational modifications possibly being to preserve activity under low-pH or high-salt conditions in which an unmodified synthetic analogue was considerably less active (84). Plicatamide (Phe-Phe-His-Leu-His-Phe-His-dc⌬DOPA), in which dc⌬DOPA is decarboxy-(E)-␣,␤-dehydro-3,4-dihydroxyphenylalanine, is a potently antimicrobial octapeptide from the blood cells of the ascidian Styela plicata (86,87).…”

On the evolutionary significance and metal-binding characteristics of a monolobal transferrin fromCiona intestinalis

“…NicaTf is able to retain a significant amount of bound Fe(III) in the presence of high concentrations of the strong affinity chelator tiron (59). Iron-accumulating ascidians typically have high concentrations of phenolic metabolites (62) and DOPAcontaining polypeptides (63), and tiron is a good model of these molecules. That Fe(III)-nicaTf is not readily labile suggests that the protein would be an efficacious delivery agent for Fe(III) to target receptors.…”

Discovery and Characterization of Two Isoforms of Moronecidin, a Novel Antimicrobial Peptide from Hybrid Striped Bass

“…These include the cysteine-rich peptides of mussels (myticin) (9) and horseshoe crab (tachyplesins and polyphemusins) (10), the proline-and cysteine-rich peptides from shrimp (penaeidins) (11), and the ␣-helical peptides from ascidians (clavanins and styelins) (12)(13)(14) and fish (misgurin, pleurocidin, paradaxins, hagfish intestinal antimicrobial peptides, and parasin I) (15)(16)(17)(18)(19).…”