Diversity in penaeidin antimicrobial peptide form and function

Abstract

Penaeidins are a diverse family of two-domain antimicrobial peptides expressed in shrimp. Variation in penaeidin sequence results in functional diversity, which was discovered using synthetic reproductions of native penaeidins. An isoform of penaeidin class 3 from Litopenaeus setiferus (Litset Pen3-4) was synthesized using native ligation and compared directly with the synthetic penaeidin class 4 known to be expressed in the same organism. New antimicrobial activity data are included in this review that emphasize differences in effectiveness that are apparent from a direct comparison of two classes. A novel approach to intact penaeidin analysis is presented in the form of Fourier Transform Ion-Cyclotron Resonance Mass Spectrometry, which has implications for the identification of individual penaeidin isoforms without chemical modification or enzymatic cleavage. The new information included in this review helps gather the perspective on relevance of penaeidin diversity to antimicrobial function, the use of synthetic peptides as tools to evaluate specific immune functions and the application of high mass resolution, top-down sequencing methods to the intact analysis of individual penaeidin isoforms.

Figure 1

Penaeidin class 4 (Litset Pen4−1) abbreviated as Pen4 aligned with selected proline-rich and cysteine-rich antimicrobial peptides. (A) Here Bactenecin 5 (Bac5) from Bos taurus (#B36589) is aligned with the proline-rich domain of Pen4 (PRD Pen4−1), and human β-defensin 1 (Def1) (#CAA90650) is aligned with the cysteine-rich domain of Pen4 (CRD Pen4−1). Considerable identity is observed between Bac5 and PRD Pen4−1 with 53% identity in a specific region (boxed), containing shared Pro, Arg, Ile and Phe residues (bold text). (B) Alignment of a segment of a 6.5kDa proline-rich peptide (6,5k) (#P82964) isolated from a crab [74], hypothesized to be a penaeidin family member [40], compared to PRD Pen4−1; several residues are shared (bold text). (C) Heliomicin (Heli) (#P81544), a defensin-like antimicrobial peptide from budworm [75, 76], is aligned with CRD Pen4−1 emphasizing, in addition to similarities in amino acid sequence, the relative location of the known helix structure for both peptides [38, 75]. In this case the placement of the helix overlaps considerably in the aligned region. The italicized numbers above the alignments indicate the amino acid position within the mature peptide sequence. Accession numbers for each protein sequences are listed here in parentheses and highlighted by the pound (#) symbol.

Figure 2

Synthesis of L. setiferus penaeidins. Class 4 (left) was the product of one native ligation step combining two peptide segments [53]. The synthesis of class 3 (right) required two steps of native ligation; one to make the complete CRD followed by a second step to produce the full-length molecule [52]. Both full-length peptides were folded in aqueous buffer at neutral pH using dimethylsulfoxide to promote disulfide formation. The purified, folded products were thoroughly analyzed for purity, uniformity of disulfide bonding and synthesis accuracy [38, 52, 53]. Included is a MALDI-TOF spectrum for each finished product emphasizing synthesis accuracy and purity (shown with singly and multiply charged ions labeled). The final folded products are rendered here as ribbon images with Pro (green) and Arg (red) side chains shown in each PRD and disulfide bonds (yellow) arranged according to experimentally determined patterns [38, 52]. The ribbon structure shown for Litset Pen4−1 was determined experimentally [38], and the image included for Litset Pen3−4 is a homology model described in this manuscript that is included for discussion purposes. More detailed renderings are shown in Fig. 6 and 7. The amino acid position is noted at the top of each peptide segment by italic numbers.

Figure 3

MALDI-TOF spectra from hemocyte extracts of two L. setiferus individuals. Hemocytes were lysed in acidified water (0.1% trifluoroacetic acid) and the total lysate was subjected to MALDI-TOF MS analysis [53]. These two spectra illustrate the different masses that can be observed when looking at ions within the full-length penaeidin mass range from different L. setiferus individuals. Insight into the regulation of penaeidin expression might be gained from such extracts under controlled conditions if the identity of the proteins that represent the peaks in this mass range could be conclusively identified. The new FTICR MS/MS data presented in Fig. 5 for Litset Pen3−4 has implications for penaeidin identification in mixed samples.

Figure 4

MALDI-TOF MS analysis of a pool of hemocyte extracts from many L. setiferus animals [53] is shown in the top panel. The middle and bottom panels show spectra obtained from two fractions that were obtained by immunoprecipitation followed by two reversed phase separation steps [53]. Another fraction that contained Litset Pen4−1 was subject to TOF/TOF analysis and was detailed previously [53], so it is not presented here. The two fractions that produced the spectra in the middle and bottom panels of this figure contained prominent ions that were co-immunoprecipitated with rabbit anti-Litset Pen4−1 serum. These results emphasize the potential for cross-reactivity by an antiserum from an individual rabbit generated against synthetic Litset Pen4−1, and might reflect a common antigenic determinant on penaeidins. The conclusive identification of peptides that potentially represent penaeidins within this mass range would have provided information about the specificity of this antiserum for penaeidins.

Figure 5

FTICR MS/MS data for Litset Pen3−4 were acquired on an IonSpec quadrupole Fourier Transform ICR mass spectrometer with a 9.4 Tesla actively shielded magnet, equipped with a Waters Z-spray electrospray interface. For these analyses, ions were accumulated for 500 milliseconds in the hexapole region and then pulsed into the ICR cell and then fragmented by electron capture dissociation (ECD). Electrons were generated with a cathode filament operated at 7 volts. Dissociation was performed by using a 250 ms pulse of electrons from an initial voltage of +5 V to a voltage of −1.00 V. For the MS/MS analyses, the ICR transient was measured at 1 MHz with 512k data points (∼0.5 sec transient) and 20 scans were accumulated. Each transient was processed with a Blackman windowing function, zero-filled once, and Fourier transformed to the frequency domain. The resultant c and z ions (asterisks) are labeled and noted on the Litset Pen3−4 sequence. M+5H, M+6H and M+7H ions represent multiply charged versions of the full-length Litset Pen3−4 peptide. This approach provided sufficient information about the isoform specific PRD of Litset Pen3−4 to result in conclusive identification. Such an approach could be used to distinguish closely related penaeidin isoforms from one another in mixed samples, and from un-related peptides of a similar mass.

Figure 6

(A) The PRD sequences for Litset Pen3−4, Litvan Pen3−1 and Litset Pen4−1 are aligned. Amino acid types presented in Fig. 6A and 6B are colored according to the following classification: basic (blue), proline (green), polar (yellow), non-polar (grey). Note the Pro-Arg repeats that are characteristic of this domain and the differences in organization between each isoform. The variability that is characteristic of the PRD is highlighted by the differences in length and composition. A Tyr-Thr and three Pro-Arg repeats are conserved between isoforms and across different classes. Numbers above the alignment indicate relative position across consensus. (B) Amino acid side chains along each of the PRDs are emphasized by space filling representation, while an orange ribbon is used to illustrate the peptide backbone. PRD Pen4−1 is an active antimicrobial peptide that shares the range of activity with its full-length congener. PRD Pen3−4 exhibited highly selective activity against yeasts that are pathogenic to humans. A unique Pro-Pro-Pro motif is noted in PRD Pen3−1, the only penaeidin PRD that has been tested in antimicrobial assays for which no level of antimicrobial activity has been observed.

Figure 7

(A) The CRD sequences for Litset Pen3−4, Litvan Pen3−1 and Litset Pen4−1 are aligned. Amino acid types presented in Fig. 7A and 7B are colored according to the following classification: basic (blue), acidic (red), proline (green), polar (yellow), non-polar (grey). Numbers above the alignment indicate relative position across consensus of this domain only, and do not reflect the exact residue number, which are used in Fig. 7B and 7C. (B) Exposed surface residues of the conserved alpha-helix are highlighted. The orientation is N-terminal to C-terminal, bottom to top for the helices, indicated by the directional arrow (top). In addition to the conserved Cys residue pair that is part of each helix (not shown, found on opposing side of helix) each helix contains a cluster of polar residues at the N-terminus and two Arg residues, including a canonical Arg residue that is found mid-way into the helix and is conserved across all penaeidin sequences [36]. (C) A ribbon image is shown to illustrate the conserved penaeidin CRD fold including the disulfide arrangement, which has been determined experimentally for all three proteins either by NMR or chemical means [37, 38, 52]. The rendering of Litset Pen3−4 is based on a homology model generated for conceptualization and discussion purposes. Numbering in Fig. 7B and 7C is indicative of exact residue position in each mature peptide.

Figure 7

(A) The CRD sequences for Litset Pen3−4, Litvan Pen3−1 and Litset Pen4−1 are aligned. Amino acid types presented in Fig. 7A and 7B are colored according to the following classification: basic (blue), acidic (red), proline (green), polar (yellow), non-polar (grey). Numbers above the alignment indicate relative position across consensus of this domain only, and do not reflect the exact residue number, which are used in Fig. 7B and 7C. (B) Exposed surface residues of the conserved alpha-helix are highlighted. The orientation is N-terminal to C-terminal, bottom to top for the helices, indicated by the directional arrow (top). In addition to the conserved Cys residue pair that is part of each helix (not shown, found on opposing side of helix) each helix contains a cluster of polar residues at the N-terminus and two Arg residues, including a canonical Arg residue that is found mid-way into the helix and is conserved across all penaeidin sequences [36]. (C) A ribbon image is shown to illustrate the conserved penaeidin CRD fold including the disulfide arrangement, which has been determined experimentally for all three proteins either by NMR or chemical means [37, 38, 52]. The rendering of Litset Pen3−4 is based on a homology model generated for conceptualization and discussion purposes. Numbering in Fig. 7B and 7C is indicative of exact residue position in each mature peptide.