Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor

Upon wounding or infection, a serine proteinase cascade in insect hemolymph leads to prophenoloxidase (proPO) activation and melanization, a defense response against invading microbes. In the tobacco hornworm Manduca sexta, this response is initiated via hemolymph proteinase 14 (HP14), a mosaic protein that interacts with bacterial peptidoglycan or fungal beta-1,3-glucan to autoactivate. In this paper, we report the expression, purification, and functional analysis of M. sexta HP21 precursor, an HP14 substrate similar to Drosophila snake. The recombinant proHP21 is a 51.1 kDa glycoprotein with an amino-terminal clip domain, a linker region, and a carboxyl-terminal serine proteinase domain. HP14, generated by incubating proHP14 with beta-1,3-glucan and beta-1,3-glucan recognition protein-2, activated proHP21 by limited proteolysis between Leu(152) and Ile(153). Active HP21 formed an SDS-stable complex with M. sexta serpin-4, a physiological regulator of the proPO activation system. We determined the P1 site of serpin-4 to be Arg(355) and, thus, confirmed our prediction that HP21 has trypsin-like specificity. After active HP21 was added to the plasma, there was a major increase in PO activity. HP21 cleaved proPO activating proteinase-2 precursor (proPAP-2) after Lys(153) and generated an amidase activity, which activated proPO in the presence of serine proteinase homolog-1 and 2. In summary, we have discovered and reconstituted a branch of the proPO activation cascade in vitro: beta-1,3-glucan recognition--proHP14 autoactivation--proHP21 cleavage--PAP-2 generation--proPO activation--melanin formation.     

Interaction of beta-1,3-glucan with its recognition protein activates hemolymph proteinase 14, an initiation enzyme of the prophenoloxidase activation system in Manduca sexta

A serine proteinase pathway in insect hemolymph leads to prophenoloxidase activation, an innate immune response against pathogen infection. In the tobacco hornworm Manduca sexta, recombinant hemolymph proteinase 14 precursor (pro-HP14) interacts with peptidoglycan, autoactivates, and initiates the proteinase cascade (Ji, C., Wang, Y., Guo, X., Hartson, S., and Jiang, H. (2004) J. Biol. Chem. 279, 34101-34106). Here, we report the purification and characterization of pro-HP14 from the hemolymph of bacteria-injected M. sexta larvae. The zymogen, consisting of a single polypeptide with a molecular mass of 68.5 kDa, is truncated at the amino terminus. It is converted to a two-chain active form in the presence of beta-1,3-glucan (a fungal cell wall component) and beta-1,3-glucan recognition protein-2. The 45-kDa heavy chain contains four low-density lipoprotein receptor A repeats, one Sushi domain, and one unique cysteine-rich region, whereas the 30-kDa light chain contains a serine proteinase domain, which was labeled by [(3)H]diisopropyl fluorophosphate. Pro-HP14 in the plasma strongly binds curdlan, zymosan, and yeast and interacts with peptidoglycan and Micrococcus luteus. Addition of autoactivated HP14 elevated phenoloxidase activity level in the larval plasma. Recombinant M. sexta serpin-1I reduced prophenoloxidase activation by inhibiting HP14. These data are consistent with the current model on initiation and regulation of the prophenoloxidase activation cascade upon recognition of pathogen-associated molecular patterns by specific pattern recognition proteins.     

Isolation, purification and characterization of beta-1,3-glucan binding protein from the plasma of marine mussel Perna viridis

A beta-1,3-glucan binding protein (betaGBP) specific for laminarin (a beta-1,3-glucan) was detected for the first time in a mollusc, Perna viridis. betaGBP was isolated and purified from the plasma using laminarin precipitation and affinity chromatography on laminarin-Sepharose 6B, respectively. It agglutinated bakers yeast, bacteria, and erythrocytes and enhanced prophenoloxidase (proPO) activity of the plasma in a dose-dependent manner. The purified betaGBP appeared as a single band in native-PAGE and the purity was conformed by HPLC. The protein has a molecular weight estimate of 510kDa as determined by SDS-PAGE and in isoelectric focusing the purified betaGBP was focused as a single band at pI 5.3. betaGBP was found to possess inherent serine protease activity but lacked beta-1,3-glucanase activity and all these results suggest that plasma betaGBP of P. viridis functions as a recognition molecule for beta-1,3-glucan on the surface of microbial cell walls. This recognition and binding lead to the activation of the prophenoloxidase cascade mediated by the inherent serine protease activity of betaGBP. Presence of agglutinating activity and serine protease activity shows that betaGBP is a bifunctional protein. The findings are discussed in light of the importance of this protein in the innate immune response of P. viridis, and they implicate evolutionary link with similar proteins found in other invertebrates.     

Purification and characterization of a beta-1,3-glucan binding protein from plasma of the crayfish Pacifastacus leniusculus

The plasma of the crayfish Pacifastacus leniusculus contains a protein which is able to bind to laminarin (a soluble beta-1,3-glucan) and which has been isolated by two independent methods, affinity precipitation with a beta-1,3-glucan or immunoaffinity chromatography. The purified beta-1,3-glucan binding protein was homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a monomeric glycoprotein with a molecular mass of approximately 100,000 Da and an isoelectric point of approximately 5.0. Amino acid analysis showed a very high similarity with the amino acid composition of beta-1,3-glucan binding proteins recently purified from two insects, the cockroach Blaberus craniifer and the silkworm Bombyx mori. The N-terminal amino acid sequence was determined to be: H2N-Asp-Ala-Gly-X-Ala-Ser-Leu-Val-Thr-Asn-Phe-Asn-Ser-Ala-Lys-Leu-X-X-Ly s--- Using monospecific rabbit polyclonal antibodies, the presence of this protein has also been shown within the blood cells. The purified beta-1,3-glucan binding protein did not show any peptidase or phenoloxidase activity but was able to enhance the activation of hemocyte-derived peptidase and prophenoloxidase only in the presence of the beta-1,3-glucan, laminarin, whereas mannan, dextran (alpha-glucan), or cellulose (beta-1,4-glucan) incubated with the beta-1,3-glucan binding protein had no effect on these enzyme activities. The beta-1,3-glucan binding protein could only be affinity-precipitated from crayfish plasma by the beta-1,3-glucans laminarin or curdlan (an insoluble beta-1,3-glucan), while mannan or dextran did not bind to the beta-1,3-glucan binding protein. No hemagglutinating activity of the purified beta-1,3-glucan binding protein could be detected.     

A beta1,3-glucan recognition protein from an insect, Manduca sexta, agglutinates microorganisms and activates the phenoloxidase cascade

Pattern recognition proteins function in innate immune responses by binding to molecules on the surface of invading pathogens and initiating host defense reactions. We report the purification and molecular cloning of a cDNA for a 53-kDa beta1,3-glucan-recognition protein from the tobacco hornworm, Manduca sexta. This protein is constitutively expressed in fat body and secreted into hemolymph. The protein contains a region with sequence similarity to several glucanases, but it lacks glucanase activity. It binds to the surface of and agglutinates yeast, as well as gram-negative and gram-positive bacteria. Beta1,3-glucan-recognition protein in the presence of laminarin, a soluble glucan, stimulated activation of prophenoloxidase in plasma, whereas laminarin alone did not. These results suggest that beta1,3-glucan-recognition protein serves as a pattern recognition molecule for beta1,3-glucan on the surface of fungal cell walls. After binding to beta1,3-glucan, the protein may interact with a serine protease, leading to the activation of the prophenoloxidase cascade, a pathway in insects for defense against microbial infection.     

Distinct carbohydrate recognition domains of an invertebrate defense molecule recognize Gram-negative and Gram-positive bacteria

Coelomic fluid of Eisenia foetida earthworms (Oligochaeta, Annelida) contains a 42-kDa defense molecule named CCF for coelomic cytolytic factor. By binding microbial antigens, namely the O-antigen of lipopolysaccharide (LPS), beta-1,3-glucans, or N,N'-diacetylchitobiose present, respectively, on Gram-negative bacteria or yeast cell walls, CCF triggers the prophenoloxidase activating pathway. We report that CCF recognizes lysozyme-predigested Gram-positive bacteria or the peptidoglycan constituent muramyl dipeptide as well as muramic acid. To identify the pattern recognition domains of CCF, deletion mutants were tested for their ability to reconstitute the prophenoloxidase cascade in E. foetida coelomic fluid depleted of endogenous CCF in the presence of LPS, beta-1,3-glucans, N,N'-diacetylchitobiose, and muramic acid. In addition, affinity chromatography of CCF peptides was performed on immobilized beta-1,3-glucans or N,N'-diacetylchitobiose. We found that the broad specificity of CCF for pathogen-associated molecular patterns results from the presence of two distinct pattern recognition domains. One domain, which shows homology with the polysaccharide and glucanase motifs of beta-1,3-glucanases and invertebrate defense molecules located in the central part of the CCF polypeptide chain, interacts with LPS and beta-1,3-glucans. The C-terminal tryptophan-rich domain mediates interactions of CCF with N,N'-diacetylchitobiose and muramic acid. These data provide evidence for the presence of spatially distinct carbohydrate recognition domains within this invertebrate defense molecule.     

Nonproteolytic serine proteinase homologs are involved in prophenoloxidase activation in the tobacco hornworm,Manduca sexta

In insects, the prophenoloxidase activation system is a defense mechanism against parasites and pathogens. Recognition of parasites or pathogens by pattern recognition receptors triggers activation of a serine proteinase cascade, leading to activation of prophenoloxidase-activating proteinase (PAP). PAP converts inactive prophenoloxidase (proPO) to active phenoloxidase (PO), which then catalyzes oxidation of phenolic compounds that can polymerize to form melanin. Because quinone intermediates and melanin are toxic to both hosts and pathogens, activation of proPO must be tightly regulated and localized. We report here purification and cDNA cloning of serine proteinase homologs (SPHs) from the tobacco hornworm, Manduca sexta, which interact with PAP-1 in proPO activation. Two SPHs were co-purified from plasma of M. sexta larvae with immulectin-2, a C-type lectin that binds to bacterial lipopolysaccharide. They contain an amino-terminal clip domain connected to a carboxyl-terminal serine proteinase-like domain. PAP-1 alone cannot efficiently activate proPO, but a mixture of SPHs and PAP-1 was much more effective for proPO activation. Immulectin-2, proPO and PAP-1 in hemolymph bound to the immobilized recombinant proteinase-like domain of SPH-1, indicating that a complex containing these proteins may exist in hemolymph. Since immulectin-2 is a pattern recognition receptor that binds to surface carbohydrates on pathogens, such a protein complex may localize activation of proPO on the surface of pathogens. SPH, which binds to immulectin-2, may function as a mediator to recruit proPO and PAP to the site of infection.     

Pattern recognition proteins in Manduca sexta plasma

Recognition of nonself is the first step in mounting immune responses. In the innate immune systems of both vertebrates and arthropods, such recognition, termed pattern recognition, is mediated by a group of proteins, known as pattern recognition proteins or receptors. Different pattern recognition proteins recognize and bind to molecules (molecular patterns) present on the surface of microorganisms but absent from animals. These molecular patterns include microbial cell wall components such as bacterial lipopolysaccharide, lipoteichoic acid and peptidoglycan, and fungal beta-1,3-glucans. Binding of pattern recognition proteins to these molecular patterns triggers responses such as phagocytosis, nodule formation, encapsulation, activation of proteinase cascades, and synthesis of antimicrobial peptides. In this article, we describe four classes of pattern recognition proteins, hemolin, peptidoglycan recognition protein, beta-1,3-glucan recognition proteins, and immulectins (C-type lectins) involved in immune responses of the tobacco hornworm, Manduca sexta.     

Structural insights into recognition of triple-helical beta-glucans by an insect fungal receptor

The innate ability to detect pathogens is achieved by pattern recognition receptors, which recognize non-self-components such as β1,3-glucan. β1,3-Glucans form a triple-helical structure stabilized by interchain hydrogen bonds. β1,3-Glucan recognition protein (βGRP)/gram-negative bacteria-binding protein 3 (GNBP3), one of the pattern recognition receptors, binds to long, structured β1,3-glucan to initiate innate immune response. However, binding details and how specificity is achieved in such receptors remain important unresolved issues. We solved the crystal structures of the N-terminal β1,3-glucan recognition domain of βGRP/GNBP3 (βGRP-N) in complex with the β1,3-linked glucose hexamer, laminarihexaose. In the crystals, three structured laminarihexaoses simultaneously interact through six glucose residues (two from each chain) with one βGRP-N. The spatial arrangement of the laminarihexaoses bound to βGRP-N is almost identical to that of a β1,3-glucan triple-helical structure. Therefore, our crystallographic structures together with site-directed mutagenesis data provide a structural basis for the unique recognition by such receptors of the triple-helical structure of β1,3-glucan.     

Activation of serum prophenoloxidase in arthropod immunity. The specificity of cell wall glucan activation and activation by purified fungal glycoproteins of crayfish phenoloxidase.

This study shows that the activation of crayfish serum prophenoloxidase by carbohydrates was specific for beta-1,3-glucans. Fractionation of the beta-1,3-glucan laminaran into laminaran M and laminaran G showed that both activated the proenzyme, but the G-chain had somewhat higher affinity for the proenzyme. Methylation analysis of these two fractions revealed that there were no 1,6-linkages present. Laminaripentaose, a linear pentasaccharide composed of (1 leads to 3)-linked beta-D-glucopyranosyl residues was also active but had a lower affinity for the proenzyme than laminaran G. Laminaran completely inhibited the activation of prophenoloxidase by the pentaose. In the concentrations tested, laminaran was not inhibitory to the phenoloxidase activity. Purified extracellular glycoproteins of the parasitic fungus Aphanomyces astaci also strongly activated crayfish serum prophenoloxidase. Only high molecular weight glycoproteins were effective. Exo-beta-1,3-glucanase treatment decreased the activating capacity, suggesting that at least part of the glycoproteins consisted of beta-1,3-glucans. The significance of these results in the defence against parasitic fungi in crayfish is discussed.     

Gram-negative bacteria-binding protein, a pattern recognition receptor for lipopolysaccharide and beta-1,3-glucan that mediates the signaling for the induction of innate immune genes in Drosophila melanogaster cells

Pattern recognition receptors, non-clonal immune proteins recognizing common microbial components, are critical for non-self recognition and the subsequent induction of Rel/NF-kappaB-controlled innate immune genes. However, the molecular identities of such receptors are still obscure. Here, we present data showing that Drosophila possesses at least three cDNAs encoding members of the Gram-negative bacteria-binding protein (DGNBP) family, one of which, DGNBP-1, has been characterized. Western blot, flow cytometric, and confocal laser microscopic analyses demonstrate that DGNBP-1 exists in both a soluble and a glycosylphosphatidylinositol-anchored membrane form in culture medium supernatant and on Drosophila immunocompetent cells, respectively. DGNBP-1 has a high affinity to microbial immune elicitors such as lipopolysaccharide (LPS) and beta-1,3-glucan whereas no binding affinity is detected with peptidoglycan, beta-1,4-glucan, or chitin. Importantly, the overexpression of DGNBP-1 in Drosophila immunocompetent cells enhances LPS- and beta-1,3-glucan-induced innate immune gene (NF-kappaB-dependent antimicrobial peptide gene) expression, which can be specifically blocked by pretreatment with anti-DGNBP-1 antibody. These results suggest that DGNBP-1 functions as a pattern recognition receptor for LPS from Gram-negative bacteria and beta-1, 3-glucan from fungi and plays an important role in non-self recognition and the subsequent immune signal transmission for the induction of antimicrobial peptide genes in the Drosophila innate immune system.     

Phagocytosis by human neutrophils is stimulated by a unique fungal cell wall component

Innate immunity depends upon recognition of surface features common to broad groups of pathogens. The glucose polymer beta-glucan has been implicated in fungal immune recognition. Fungal walls have two kinds of beta-glucan: beta-1,3-glucan and beta-1,6-glucan. Predominance of beta-1,3-glucan has led to the presumption that it is the key immunological determinant for neutrophils. Examining various beta-glucans for their ability to stimulate human neutrophils, we find that the minor cell wall component beta-1,6-glucan mediates neutrophil activity more efficiently than beta-1,3-glucan, as measured by engulfment, production of reactive oxygen species, and expression of heat shock proteins. Neutrophils rapidly ingest beads coated with beta-1,6-glucan while ignoring those coated with beta-1,3-glucan. Complement factors C3b/C3d are deposited on beta-1,6-glucan more readily than on beta-1,3-glucan. Beta-1,6-glucan is also important for efficient engulfment of the human pathogen Candida albicans. These unique stimulatory effects offer potential for directed stimulation of neutrophils in a therapeutic context.     

A synthetic peptidoglycan fragment as a competitive inhibitor of the melanization cascade

Melanin synthesis is essential for defense and development but must be tightly controlled because systemic hyperactivation of the prophenoloxidase and excessive melanin synthesis are deleterious to the hosts. The melanization cascade of the arthropods can be activated by bacterial lysine-peptidoglycan (PGN), diaminopimelic acid (DAP)-PGN, or fungal beta-1,3-glucan. The molecular mechanism of how DAP- or Lys-PGN induces melanin synthesis and which molecules are involved in distinguishing these PGNs are not known. The identification of PGN derivatives that can work as inhibitors of the melanization cascade and the characterization of PGN recognition molecules will provide important information to clarify how the melanization is regulated and controlled. Here, we report that a novel synthetic Lys-PGN fragment ((GlcNAc-MurNAc-L-Ala-D-isoGln-L-Lys-D-Ala)2, T-4P2) functions as a competitive inhibitor of the natural PGN-induced melanization reaction. By using a T-4P2-coupled column, we purified the Tenebrio molitor PGN recognition protein (Tm-PGRP) without causing activation of the prophenoloxidase. The purified Tm-PGRP recognized both Lys- and DAP-PGN. In vitro reconstitution experiments showed that Tm-PGRP functions as a common recognition molecule of Lys- and DAP-PGN-dependent melanization cascades.     

Characterization and properties of a 1,3-beta-D-glucan pattern recognition protein of Tenebrio molitor larvae that is specifically degraded by serine protease during prophenoloxidase activation

Although many different pattern recognition receptors recognizing peptidoglycan and 1,3-beta-D-glucan have been identified in vertebrates and insects, the molecular mechanism of these molecules in the pattern recognition and subsequent signaling is largely unknown. To gain insights into the action mechanism of 1,3-beta-D-glucan pattern recognition protein in the insect prophenoloxidase (proPO) activation system, we purified a 53-kDa 1,3-beta-D-glucan recognition protein (Tm-GRP) to homogeneity from the hemolymph of the mealworm, Tenebrio molitor, by using a 1,3-beta-d-glucan affinity column. The purified protein specifically bound to 1,3-beta-D-glucan but not to peptidoglycan. Subsequent molecular cloning revealed that Tm-GRP contains a region with close sequence similarity to bacterial glucanases. Strikingly, two catalytically important residues in glucanases are replaced with other nonhomologous amino acids in Tm-GRP. The finding suggests that Tm-GRP has evolved from an ancestral gene of glucanases but retained only the ability to recognize 1,3-beta-D-glucan. A Western blot analysis of the protein level of endogenous Tm-GRP showed that the protein was specifically degraded following the activation of proPO with 1,3-beta-D-glucan and calcium ion. The degradation was significantly retarded by the addition of serine protease inhibitors but not by cysteine or acidic protease inhibitor. These results suggest that 1,3-beta-D-glucan pattern recognition protein is specifically degraded by serine protease(s) during proPO activation, and we propose that this degradation is an important regulatory mechanism of the activation of the proPO system.     

Properties of the prophenoloxidase activating enzyme of the freshwater crayfish, Pacifastacus leniusculus.

The prophenoloxidase activating enzyme (ppA), a serine proteinase catalyzing the conversion of prophenoloxidase to an active phenoloxidase, has a molecular mass of about 36 kDa in its active form. This protein was cloned from a blood cell cDNA library and its corresponding cDNA of 1736 base pairs encodes a zymogenic protein (proppA) of 468 amino acids. An antibody raised against a synthetic peptide derived from a region of the cDNA sequence could efficiently inhibit the beta-1,3-glucan triggered activation of prophenoloxidase in vitro. The C-terminal half of the proppA is composed of a typical serine proteinase domain, with a sequence similar to other invertebrate and vertebrate serine proteinases. The N-terminal half contains a cationic glycine-rich domain, a cationic proline-rich domain and a clip-domain, in which the disulfide-bonding pattern is likely to be identical to those of the horseshoe crab big defensin and mammalian beta-defensins. Antibodies made against both the C- and the N-terminal halves recognize two proppAs under reducing conditions. However, under nonreducing conditions only the anti-C antibody recognized the two proppAs, which suggests that a conformational change takes place upon reduction that allows the anti-N to react with the N-terminal half of proppA. The recombinant clip-domain in crayfish proppA was overexpressed in Escherichia coli and the resulting peptide exhibited antibacterial activity against Gram-positive bacterial strains such as Micrococcus luteus Ml11 and Bacillus megaterium Bm11 with 50% growth inhibitory concentrations of 1.43 microM and 17.9 microM, respectively. These results suggest that the clip-domains in proppAs may function as antibacterial peptides.