Characterization of the mature form of a β-defensin-like peptide, Hoa-D1, in the lobster Homarus americanus

• Published in: Molecular immunology Vol. 101; pp. 329 - 343
• Main Authors: Vu, Giap H., Do, Daniel, Rivera, Cindy D., Dickinson, Patsy S., Christie, Andrew E., Stemmler, Elizabeth A.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.09.2018
• Subjects: Antimicrobial peptide; Chip-based nanoLC-QTOF-MS/MS; Defensin; Hemolymph; Homarus americanus; Transcriptomics; TSA; AMP; DTT; EST; IAA; Hemolymph; TIC; Homarus americanus; nanoESI; MWCO; QTOF-MS/MS; nanoLC; Transcriptomics; Defensin; Chip-based nanoLC-QTOF-MS/MS; EIC; CID; Antimicrobial peptide
• ISSN: 0161-5890; 1872-9142
• DOI: 10.1016/j.molimm.2018.07.004

•Homarus americanus defensin 1 (Hoa-D1) is identified in the lobster.•Hoa-D1 is related to β-defensin-like peptides from Panulirus species.•Hoa-D1 is localized to semigranulocytes and granulocytes.•NanoLC–MS analysis reveals processing details lost when using proteomic strategies.•Heat treatment of hemolymph provides a rapid approach for peptide isolation. We report on the characterization of the native form of an American lobster, Homarus americanus, β-defensin-like putative antimicrobial peptide, H. americanus defensin 1 (Hoa-D1), sequenced employing top-down and bottom-up peptidomic strategies using a sensitive, chip-based nanoLC-QTOF-MS/MS instrument. The sequence of Hoa-D1 was determined by mass spectrometry; it was found to contain three disulfide bonds and an amidated C-terminus. The sequence was further validated by searching publicly-accessible H. americanus expressed sequence tag (EST) and transcriptome shotgun assembly (TSA) datasets. Hoa-D1, SYVRScSSNGGDcVYRcYGNIINGAcSGSRVccRSGGGYamide (with c representing a cysteine participating in a disulfide bond), was shown to be related to β-defensin-like peptides previously reported from Panulirus japonicas and Panulirus argus. We found Hoa-D1 in H. americanus hemolymph, hemocytes, the supraoesophageal ganglion (brain), eyestalk ganglia, and pericardial organ extracts, as well as in the plasma of some hemolymph samples. Using discontinuous density gradient separations, we fractionatated hemocytes and localized Hoa-D1 to hemocyte sub-populations. While Hoa-D1 was detected in semigranulocytes and granulocytes using conventional proteomic strategies for analysis, the direct analysis of cell lysates exposed evidence of Hoa-D1 processing, including truncation of the C-terminal tyrosine residue, in the granulocytes, but not semigranulocytes. These measurements demonstrate the insights regarding post-translational modifications and peptide processing that can be revealed through the MS analysis of intact peptides. The identification of Hoa-D1 as a widely-distributed peptide in the lobster suggests the possibility that it may be pleiotropic, with functions in addition to its proposed role as an antimicrobial molecule in the innate immune system.