Styelin D, an Extensively Modified Antimicrobial Peptide from Ascidian Hemocytes

Steven W Taylor,A Grey Craig,Wolfgang H Fischer,Minkyu Park,Robert I Lehrer

doi:10.1074/jbc.m006762200

Steven W Taylor, A Grey Craig + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m006762200

Copy DOI

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. The primary sequence of one variant was: GW(*)LR(**)K(**)AAK(**)SVGK(**)FY(*)Y(*)K(**)HK(*)Y(*) Y(*)IK(*)AAWQIG KHAL-NH(2), where W(*) is 6-bromotryptophan, R(**) is dihydroxyarginine, Y(*) is 3,4-dihydroxyphenylalanine, K(*) is 5-hydroxylysine, and K(**) is dihydroxylysine. Styelin D exhibited activity against 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.

Highlights

As primitive chordates, ascidians are excellent subjects for research into the evolution of vertebrate innate immunity
Amino acid sequences corresponding to both the clavanins and styelins were identified using partial sequences and a cDNA library prepared from hemopoietic pharyngeal tissue [13, 14]
These studies established that styelins are highly basic polypeptides whose prepropeptides have a signal sequence and a polyanionic C-terminal extension whose charge counter balances the cationic residues in the mature peptide domain

Summary

EXPERIMENTAL PROCEDURES

Purification—S. clava specimens were collected from docks in local marinas in San Diego. Further purification of the polypeptides was carried out by a second HPLC step on an analytical Phenomenex 250 ϫ 4.6 mm Jupiter C-18 column at a flow rate of 1 ml/min column using linear gradients (see Fig. 2). The reaction was terminated with 10 ␮l of 20% trifluoroacetic acid, and the mixture was directly loaded onto a semi-preparative C-18 column and eluted with a 0 – 40% gradient of mobile phase over 60 min (see Fig. 3). GC-MS Characterization of Volatile Derivatives of the Amino Acids— The styelin D (1.0 mg) obtained after the first HPLC step was hydrolyzed using the fast acid hydrolysis method (5 M HCl with 8% trifluoroacetic acid and 8% phenol in vacuo at 155 °C for 45 min) of Tsugita and co-workers [27].

TABLE I The styelins

RESULTS

DISCUSSION

Average mass calculated