Serrulin: A Glycine-Rich Bioactive Peptide from the Hemolymph of the Yellow Tityus serrulatus Scorpion

Abstract

Antimicrobial peptides (AMPs) are small molecules, which have a potential use as antibiotic or pharmacological tools. In chelicerate organisms, such as scorpions, these molecules constitute an alternative defense system against microorganisms. The aim of this work was to identify AMPs in the hemolymph of the Tityus serrulatus scorpion. Fractions of plasma and hemocytes were subjected to high-performance liquid chromatography (HPLC) and then analyzed to determine their activity in inhibiting microbial growth. One of the fractions from the hemocytes presents antimicrobial activity against microorganisms, such as Gram-negative and Gram-positive bacteria, fungi, and yeast. These fractions were analyzed by mass spectrometry, and a fragment of 3564 Da. was identified. The peptide was called serrulin, because it is derived from the species T. serrulatus. A comparison of the amino acid sequence of serrulin with databases shows that it has a similarity to the glycine-rich peptides described in Cupienius salai and Acanthoscurria gomesiana (spiders). Furthermore, serrulin has no hemolytic activity against human erythrocytes. While the presence of AMPs in T. serrulatus venom has been described in other works, this is the first work to characterize the presence of these molecules in the hemolymph (hemocytes) of this species and show its potential use as an alternative to conventional antibiotics against different species of microorganisms.

Keywords: Tityus serrulatus; antimicrobial peptide; glycine-rich peptide; innate immune system; scorpions.

Figure 1

Reverse-phase high-performance liquid chromatography (RP-HPLC) of the hemocyte samples eluted at 5% acetonitrile (ACN). After the fractionation step using a Sep-Pak^® C18 column, the samples eluted in 5% ACN were subjected to a Jupiter^® C18 semi-preparative column, with a linear gradient from 0% to 20% ACN (dotted line) in acidified water for 60 min (1.5 mL/min). The fractions were collected manually and submitted to a test of the inhibition of microbial growth in a liquid medium. Peaks with squares indicate the antimicrobial activities against Aspergillus niger (□).

Figure 2

Reverse-phase high-performance liquid chromatography (RP-HPLC) of the hemocyte samples eluted at 40% acetonitrile (ACN). After the fractionation step using a Sep-Pak^® C18 column, the samples eluted in 40% ACN were subjected to a Jupiter^® C18 semi-preparative column, with a linear gradient from 2% to 60% ACN (dotted line) in acidified water for 60 min. The fractions were collected manually and submitted to a test of the inhibition of microbial growth in a liquid medium. Peaks with squares and circles indicate antimicrobial activities against the tested microorganisms: Escherichia coli SBS 363 (●), Microccocus luteus A270 (○), Candida albicans MDM 8 (■), and Aspergillus niger (□) (A). Serrulin, indicated with an arrow, was submitted to RP-HPLC using a Jupiter^® C18 analytic-column, with a linear gradient from 13% to 43% ACN (dotted line) in 0.05% TFA, for 60 min. The fractions were collected manually and submitted to a test of the inhibition of microbial growth in a liquid medium and mass analyses (B).

Figure 3

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis 20% (SDS-PAGE-20%), stained with Coomasie-R Blue. Line 1, molecular weight marker (invitrogen SeeBlue^®), expressed in kDa; line 2, serrulin in a non-reducing condition (20 µg).

Figure 4

Serrulin spectrum. Mass spectrometry analyses (LC-MS/MS, coupled with a LTQ-Orbitrap Velos) of the peptide serrulin revealed an m/z of 3564.0 Da. Ions were submitted to MagTran^® software.

Figure 5

Collision-induced dissociation (CID) spectrum of the novo sequence from serrulin. The ions belonging to the -y (red) and -b (blue) series, indicated in the spectrum, correspond to the amino acid sequence of the peptide: GFGGGRGGFGGGRGGFGGGGIGGGGFGGGYGGGKIKG. The fragments of the sequenced peptide are represented by standard amino acid code letters.

Figure 6

Alignment of the amino acid sequence of serrulin with other antimicrobial glycine-rich peptides. Using the UNIProt alignment, the serrulin sequence was aligned with AMPs: Acanthoscurrin 1, Acanthoscurrin 2, and Ctenidin. Amino acids that were identical in all sequences are shown in blue (or *), and minor modifications are shown in yellow or (:).