Proteolytic activation and function of the cytokine Spätzle in the innate immune response of a lepidopteran insect, Manduca sexta

Abstract

The innate immune response of insects includes induced expression of genes encoding a variety of antimicrobial peptides. The signaling pathways that stimulate this gene expression have been well characterized by genetic analysis in Drosophila melanogaster, but are not well understood in most other insect species. One such pathway involves proteolytic activation of a cytokine called Spätzle, which functions in dorsal-ventral patterning in early embryonic development and in the antimicrobial immune response in larvae and adults. We have investigated the function of Spätzle in a lepidopteran insect, Manduca sexta, in which hemolymph proteinases activated during immune responses have been characterized biochemically. Two cDNA isoforms for M. sexta Spätzle-1 differ because of alternative splicing, resulting in a 10 amino acid residue insertion in the pro-region of proSpätzle-1B that is not present in proSpätzle-1A. The proSpätzle-1A cDNA encodes a 32.7 kDa polypeptide that is 23% and 44% identical to D. melanogaster and Bombyx mori Spätzle-1, respectively. Recombinant proSpätzle-1A was a disulfide-linked homodimer. M. sexta hemolymph proteinase 8 cleaved proSpätzle-1A to release Spätzle-C108, a dimer of the C-terminal 108 residue cystine-knot domain. Injection of Spätzle-C108, but not proSpätzle-1A, into larvae stimulated expression of several antimicrobial peptides and proteins, including attacin-1, cecropin-6, moricin, lysozyme, and the immunoglobulin domain protein hemolin, but did not significantly affect the expression of two bacteria-inducible pattern recognition proteins, immulectin-2 and beta-1,3-glucan recognition protein-2. The results of this and other recent studies support a model for a pathway in which the clip-domain proteinase pro-hemolymph proteinase 6 becomes activated in plasma upon exposure to Gram-negative or Gram-positive bacteria or to beta-1,3-glucan. Hemolymph proteinase 6 then activates pro-hemolymph proteinase 8, which in turn activates Spätzle-1. The resulting Spätzle-C108 dimer is likely to function as a ligand to activate a Toll pathway in M. sexta as a response to a wide variety of microbial challenges, stimulating a broad response to infection. Structured digital abstract * MINT-7295125: Spätzle 1A (uniprotkb:C8BMD1) and Spätzle 1A (uniprotkb:C8BMD1) bind (MI:0407) by comigration in gel electrophoresis (MI:0807).

Fig. 1

(A) cDNA and deduced amino acid sequences of M. sexta proSpätzle. The one-letter code for each amino acid residue is aligned with the second nucleotide of the corresponding codon. The stop codon is marked with “*”. The predicted secretion signal peptide is underlined. The proteolytic activation site is indicated with “‖”. The amino terminal sequence, determined by Edman degradation, of the activated form of Spätzle (C108) after cleavage by HP8, is shown in bold. Putative N- and O- linked glycosylation sites are shaded. AATAAA sequences (double-underlined) near the end of the 3′ untranslated region are potential polydenylation signals. Intron positions identified within the ORF are indicated by “◊”, with a filled symbol “♦” showing the position of an intron conserved in the orthologous Spätzle genes from D. melanogaster, B. mori, and T. castaneum. (B) The alternative splicing boundaries leading to two proSpätzle isoforms (accession numbers GQ249944, GQ249945).

Fig. 2

Alignment of full length of M. sexta proSpätzle-1A (Ms_Spz), B. mori Spätzle-1 (Bm_Spz) and D. melanogater Spätzle (Dm_Spz). Completely conserved amino acid residues are indicated by “*”, and conservative substitutions by “:” below the sequences. The P1 residue at the activation cleavage site is shown in bold, and the scissile bond is indicated with an arrow.. Absolutely conserved cysteine residues are shaded and numbered. The paired numbers (1-1, 2-2, 3-3) indicate the intrachain disulfide linkage in Dm_Spz [10]. Cys-4 forms an intermolecular disulfide bond with its counterpart in another subunit. The GenBank accession numbers are: Ms_Spz, GQ249944; Bm_Spz, NM_001114594; Dm_Spz, NM_079802.

Fig. 3

Phylogenetic analysis of the cystine-knot domains in Spätzle from M. sexta and other insect species. The tree was derived from an alignment that can be found in supplementary Figure S2. Numbers at the nodes are bootstrap values as percentage. The nodes signifying Spz2, Spz3, Spz4, Spz5, and Spz6 specific branches are denoted by “•”. The circled bootstrap value indicates that M. sexta Spätzle-1A probably belongs to the Spz1 group. The scale bar indicates the number of substitutions per site. Abbreviations used are: Aa, Aedes aegypti; Ag, Anopheles gambiae; Bm, Bombyx mori; Dm, Drosophila melanogaster; Ms, Manduca sexta; Nv, Nasonia vitripennis; Tc, Tribolium castaneum.

Fig. 4

M. sexta Spätzle gene expression is up-regulated after injection of microbial elicitors. Quantitative RT-PCR was used to assess the transcript level of Spätzle-1, with ribosomal protein S3 (rpS3) as in internal standard to indicate consistent total mRNA amount. Day 2, fifth instar larvae were injected with water, E. coli, M. luteus, or curdlan. RNA was extracted from hemocytes and fat body collected 24 h after injection. The bars represent mean ± S.D. (n=3). Bars labeled with different letters are significantly different (one-way ANOVA followed by the Newman-Keuls test, P < 0.05).

Fig. 5

SDS-PAGE analysis of purified recombinant proteins. (A) Purified proSpätzle-1A (0.1 µg) was treated with SDS sample buffer in the absence or presence of 0.14 M β-mercaptoethanol (β-ME) at 95°C for 5 min and separated by SDS-PAGE followed by silver staining. (B) Purified proHP8_Xa (75 ng) was analyzed by SDS-PAGE under reducing conditions followed by silver staining. The sizes and positions of the molecular weight markers are indicated on the left side of each gel.

Fig. 6

Activation of purified recombinant proHP8_Xa by Factor Xa. (A) Purified recombinant proHP8_Xa (50 ng) and Factor Xa (100 ng) were incubated separately or mixed together in the presence of 0.005% Tween-20 at 95°C for 5 min, and the mixtures were separated by SDS-PAGE, followed by immunoblot analysis using antiserum against M. sexta HP8. Bands representing the proHP8_Xa zymogen, a truncated form of proHP8_Xa, and the catalytic domain of active HP8 are marked by arrowheads. The size and position of molecular weight standards are indicated on the left. (B) Catalytic activity of activated HP8_Xa (50 ng) was detected by spectrophotometric assay using IEARpNA as a substrate, as described in Experimental Procedures. The bars represent mean ± S.D. (n=3). Bars labeled with different letters are significantly different (one-way ANOVA followed by the Newman-Keuls test, P < 0.05).

Fig. 7

(A) Proteolytic activation of proSpätzle by HP8_Xa. ProHP8_Xa (25 ng) was activated by bovine Factor Xa (50 ng) and then incubated with proSpätzle (100 ng) at 37°C for 1 h. The mixtures were subjected to SDS-PAGE and immunoblotting using Spätzle antibodies. The sizes and positions of molecular weight standards are indicated on the left. Bands representing proSpätzle precursors, cysteine-knot domain (Spätzle-C108), and Spätzle-C108 dimer are marked by arrows. (B) SDS-PAGE analysis of Spätzle-C108. Spätzle-C108 (40 ng) purified after activation by HP8_Xa, was treated with SDS sample buffer in the absence or presence of 0.14 M β-mercaptoethanol (β-ME) at 95°C for 5 min and separated by SDS-PAGE followed by silver staining. Sizes and positions of the molecular weight markers are indicated on the left.

Fig. 8

Effects of Spätzle injection on the humoral immune response. Fifth instar, day 0 larvae were injected with buffer, proSpätzle-1A, or activated Spätzle-C108. Twenty h later, hemolymph was collected, and fat body RNA samples were prepared from each insect. (A) Antimicrobial activity of plasma assayed against E. coli and identification of antimicrobial plasma proteins by SDS-PAGE and peptide mass fingerprinting or immunoblotting. Sizes and positions of molecular weight standards are indicated on the left. (B) Relative transcript levels for indicated genes were assayed by quantitative RT-PCR as described in Experimental Procedures. Symbols represent mean ± S.D. (n=3). Lack of error bars indicates that S.D. was smaller than the size of the symbol. Asterisks indicate means that are significantly different from the buffer-injected control (one-way ANOVA followed by the Newman-Keuls test, P < 0.05).