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.     

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.     

Molecular control of phenoloxidase-induced melanin synthesis in an insect

The melanization reaction induced by activated phenoloxidase in arthropods must be tightly controlled because of excessive formation of quinones and excessive systemic melanization damage to the hosts. However, the molecular mechanism by which phenoloxidase-induced melanin synthesis is regulated in vivo is largely unknown. It is known that the Sp?tzle-processing enzyme is a key enzyme in the production of cleaved Sp?tzle from pro-Sp?tzle in the Drosophila Toll pathway. Here, we provide biochemical evidence that the Tenebrio molitor Sp?tzle-processing enzyme converts both the 79-kDa Tenebrio prophenoloxidase and Tenebrio clip-domain SPH1 zymogen to an active melanization complex. This complex, consisting of the 76-kDa Tenebrio phenoloxidase and an active form of Tenebrio clip-domain SPH1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Interestingly, we found the phenoloxidase-induced melanization reaction to be tightly regulated by Tenebrio prophenoloxidase, which functions as a competitive inhibitor of melanization complex formation. These results demonstrate that the Tenebrio Toll pathway and the melanization reaction share a common serine protease for the regulation of these two major innate immune responses.     

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 novel 43-kDa protein as a negative regulatory component of phenoloxidase-induced melanin synthesis

The melanization reaction induced by activated phenoloxidase in arthropods is important in the multiple host defense innate immune reactions, leading to the sequestration and killing of invading microorganisms. This reaction ought to be tightly controlled because excessive formation of quinones and systemic hypermelanization are deleterious to the hosts, suggesting that a negative regulator(s) of melanin synthesis may exist in hemolymph. Here, we report the purification and cloning of a cDNA of a novel 43-kDa protein, from the meal-worm Tenebrio molitor, which functions as a melanization-inhibiting protein (MIP). The deduced amino acid sequence of 352 residues has no homology to known sequences in protein data bases. When the concentration of the 43-kDa protein was examined by Western blot analysis in a melanin-induced hemolymph prepared by injection of Candida albicans into T. molitor larvae, the 43-kDa protein specifically decreased in the melanin-induced hemolymph compared with control hemolymph. Recombinant MIP expressed in a baculovirus system had an inhibitory effect on melanin synthesis in vitro. RNA interference using a synthetic 445-mer double-stranded RNA of MIP injected into Tenebrio larvae showed that melanin synthesis was markedly induced. These results suggest that this 43-kDa MIP inhibits the formation of melanin and thus is a modulator of the melanization reaction to prevent the insect from excessive melanin synthesis in places where it should be inappropriate.     

Evidence for proteins involved in prophenoloxidase cascade Eisenia fetida earthworms

The prophenoloxidase cascade represents one of the most important defense mechanisms in many invertebrates. Following the recognition of microbial saccharides by pattern recognition molecules, proteinases cleave inactive prophenoloxidase to its active form, phenoloxidase. Phenoloxidase is a key enzyme responsible for the catalysis of the melanization reaction. Final product melanin is involved in wound healing and immune responses. Prophenoloxidase cascade has been widely described in arthropods; data in other invertebrate groups are less frequent. Here we show detectable phenoloxidase activity in 90-kDa fraction of the coelomic fluid of earthworms Eisenia fetida. Amino acid sequencing of peptides from the active fraction revealed a partial homology with invertebrate phenoloxidases and hemocyanins. Moreover, the level of phenoloxidase activity is lower and the activation slower as compared to other invertebrates.     

A lipopolysaccharide- and beta-1,3-glucan-binding protein from hemocytes of the freshwater crayfish Pacifastacus leniusculus. Purification, characterization, and cDNA cloning

A lipopolysaccharide- and beta-1,3-glucan-binding protein (LGBP) was isolated and characterized from blood cells (hemocytes) of the freshwater crayfish Pacifastacus leniusculus. The LGBP was purified by chromatography on Blue-Sepharose and phenyl-Sepharose, followed by Sephacryl S-200. The LGBP has a molecular mass of 36 kDa and 40 kDa on 10% SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respectively. The calculated mass of LGBP is 39,492 Da, which corresponds to the native size of LGBP; the estimated pI of the mature LGBP is 5.80. LGBP has binding activity to lipopolysaccharides as well as to beta-1,3-glucans such as laminarin and curdlan, but peptidoglycan could not bind to LGBP. Cloning and sequencing of LGBP showed significant homology with several putative Gram-negative bacteria-binding proteins and beta-1, 3-glucanases. Interestingly, LGBP also has a structure and functions similar to those of the coelomic cytolytic factor-1, a lipopolysaccharide- and glucan-binding protein from the earthworm Eisenia foetida. To evaluate the involvement of LGBP in the prophenoloxidase (proPO) activating system, a polyclonal antibody against LGBP was made and used for the inhibition of phenoloxidase (PO) activity triggered by the beta-1,3-glucan laminarin in the hemocyte lysate of crayfish. The PO activity was blocked completely by the anti-LGBP antibody. Moreover, the PO activity could be recovered by the addition of purified LGBP. These results suggest that the 36-kDa LGBP plays a role in the activation of the proPO activating system in crayfish and thus seems to play an important role in the innate immune system of crayfish.     

The pro-phenoloxidase of coleopteran insect, Tenebrio molitor, larvae was activated during cell clump/cell adhesion of insect cellular defense reactions

To characterize the proteins involved in cell clump/cell adhesion of insect cellular defense reactions, we induced the cell clump/cell adhesion reaction in vitro with the hemolymph of larvae of the coleopteran insect, Tenebrio molitor. The 72 kDa protein was specifically enriched in the residues of cell clump/cell adhesion and was purified to homogeneity. A cDNA clone for the 72 kDa protein was isolated. We found that the 72 kDa protein was an activated phenoloxidase from Tenebrio pro-phenoloxidase. We suggest that activated phenoloxidase is involved in the cell clump/cell adhesion reaction as well as in the synthesis of melanin.     

Phenoloxidase activity in the hemolymph of the spiny lobster Panulirus argus

The prophenoloxidase activating system plays a major role in the defense mechanism of arthropods. In the present study, the phenoloxidase activity and its location in the hemolymph of the spiny lobster Panulirus argus is presented. Phenoloxidase activity was observed in the hemocyte lysate supernatant (HLS) and plasma after their incubation with trypsin. Higher amounts of trypsin were required to activate the HLS prophenoloxidase, due to the presence of a trypsin inhibitor in this fraction. Activation of prophenoloxidase was found when HLS was incubated with calcium, with an optimal pH between 7.5 and 8. This spontaneous activity is due to the prophenoloxidase activating enzyme, a serine proteinase that activates the prophenoloxidase once calcium ions were available. SDS was able to induce phenoloxidase activity in plasma and hemocyte fractions. Prophenoloxidase from HLS occurs as an aggregate of 300kDa. Electrophoretic studies combining SDS-PAGE and native PAGE indicate that different proteins produced the phenoloxidase activity found in HLS and plasma. Thus, as in most crustaceans, Panulirus argus contains a prophenoloxidase activating system in its hemocyte, comprising at least the prophenoloxidase activating enzyme and the prophenoloxidase. Finally, it is suggested that phenoloxidase activity found in plasma is produced by hemocyanin.     

Purification of prophenoloxidase from crayfish blood cells,and its activation by an endogenous serine proteinase

A prophenoloxidase was purified from blood cells of the crayfish Pacifastacus leniusculus. The purified proenzyme was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and had a molecular mass of 76 kDa under both non-reducing and reducing conditions. The crayfish prophenoloxidase was a glycoprotein, with an isoelectric point of about 5.4.A 36 kDa serine proteinase, isolated and purified from crayfish blood cells (Aspán et al., 1990b, Insect Biochem.20, 709–718), could convert the 76 kDa prophenoloxidase to phenoloxidase by an apparent proteolytic cleavage, since the molecular masses of two active enzymes, phenoloxidases, were 60 and 62 kDa. A commercial serine proteinase, trypsin, activated prophenoloxidase to phenoloxidase, and as a result a 60 kDa protein was produced.In the blood cells of crayfish four serine proteinases or ³H-DFP binding proteins are present, with masses of 36, 38, 50 and 67 kDa. However, ³H-DFP labelling of proteins in blood cells lysate, prepared in its inactive form, only yielded labelled bands of 50 and 67 kDa, whereas addition of an elicitor to prophenoloxidase system activation, a β-1,3-glucan, resulted in the appearance of four ³H-DFP labelled proteins, with molecular masses of 67, 50, 38 and 36 kDa, respectively. Thus, the 36 kDa endogenous serine proteinase, the prophenoloxidase activating enzyme, ppA, may be present as an inactive precursor in crayfish blood cells. The 38 and 36 kDa proteinases could both cleave the chromogenic peptide S-2337 [Bz-Ile-Glu-(γ-O-Piperidyl)-Gly-Arg-p-nitroaniline], and specifically bind prophenoloxidase.These results show that crayfish prophenoloxidase, the terminal enzyme of the prophenoloxidase activating cascade, a proposed defence pathway in arthropod blood, can be converted to active enzyme by an apparent proteolytic cleavage, not only by a commercial proteinase, but also by an endogenous serine type proteinase.     

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.     

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.     

Innate immunity in a pyralid moth: functional evaluation of domains from a beta-1,3-glucan recognition protein

Invertebrates, like vertebrates, utilize pattern recognition proteins for detection of microbes and subsequent activation of innate immune responses. We report structural and functional properties of two domains from a beta-1,3-glucan recognition protein present in the hemolymph of a pyralid moth, Plodia interpunctella. A recombinant protein corresponding to the first 181 amino-terminal residues bound to beta-1,3-glucan, lipopolysaccharide, and lipoteichoic acid, polysaccharides found on cell surfaces of microorganisms, and also activated the prophenoloxidase-activating system, an immune response pathway in insects. The amino-terminal domain consists primarily of an alpha-helical secondary structure with a minor beta-structure. This domain was thermally stable and resisted proteolytic degradation. The 290 residue carboxyl-terminal domain, which is similar in sequence to glucanases, had less affinity for the polysaccharides, did not activate the prophenoloxidase cascade, had a more complicated CD spectrum, and was heat-labile and susceptible to proteinase digestion. The carboxyl-terminal domain bound to laminarin, a beta-1,3-glucan with beta-1,6 branches, but not to curdlan, a beta-1,3-glucan that lacks branching. These results indicate that the two domains of Plodia beta-1,3-glucan recognition protein, separated by a putative linker region, bind microbial polysaccharides with differing specificities and that the amino-terminal domain, which is unique to this class of pattern recognition receptors from invertebrates, is responsible for stimulating prophenoloxidase activation.     

A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca2+ in coleopteran and Tenebrio molitor larvae.

To elucidate the biochemical activation mechanism of the insect pro-phenoloxidase (pro-PO) system, we purified a 45-kDa protein to homogeneity from the hemolymph of Tenebrio molitor (mealworm) larvae, and cloned its cDNA. The overall structure of the 45-kDa protein is similar to Drosophila masquerade serine proteinase homologue, which is an essential component in Drosophila muscle development. This Tenebrio masquerade-like serine proteinase homologue (Tm-mas) contains a trypsin-like serine proteinase domain in the C-terminal region, except for the substitution of Ser to Gly at the active site triad, and a disulfide-knotted domain at the amino-terminal region. When the purified 45-kDa Tm-mas was incubated with CM-Toyopearl eluate solution containing pro-PO and other pro-PO activating factors, the resulting phenoloxidase (PO) activity was shown to be independent of Ca2+. This suggests that the purified 45-kDa Tm-mas is an activated form of pro-PO activating factor. The55-kDa zymogen form of Tm-mas was detected in the hemolymph when PO activity was not evident. However, when Tenebrio hemolymph was incubated with Ca2+, a 79-kDa Tenebrio pro-PO and the 55-kDa zymogen Tm-mas converted to 76-kDa PO and 45-kDa Tm-mas, respectively, with detectable PO activity. Furthermore, when Tenebrio hemolymph was incubated with Ca2+ and beta-1,3-glucan, the conversion of pro-PO to PO and the 55-kDa zymogen Tm-mas to the 45-kDa protein, was faster than in the presence of Ca2+ only. These results suggest that the cleavage of the 55-kDa zymogen of Tm-mas by a limited proteolysis is necessary for PO activity, and the Tm-mas is a pro-PO activating cofactor.     

An immune-responsive Serpin regulates the melanization cascade in Drosophila

In arthropods, the melanization reaction is associated with multiple host defense mechanisms leading to the sequestration and killing of invading microorganisms. Arthropod melanization is controlled by a cascade of serine proteases that ultimately activates the enzyme prophenoloxidase (PPO), which, in turn, catalyzes the synthesis of melanin. Here we report the biochemical and genetic characterization of a Drosophila serine protease inhibitor protein, Serpin-27A, which regulates the melanization cascade through the specific inhibition of the terminal protease prophenoloxidase-activating enzyme. Our data demonstrate that Serpin-27A is required to restrict the phenoloxidase activity to the site of injury or infection, preventing the insect from excessive melanization.     

Cyclic beta-(1,2)-glucan synthesis in Rhizobiaceae: roles of the 319-kilodalton protein intermediate.

Cyclic beta-(1,2)-glucans are synthesized by members of the Rhizobiaceae family through protein-linked oligosaccharides as intermediates. The protein moiety is a large inner membrane molecule of about 319 kDa. In Agrobacterium tumefaciens and in Rhizobium meliloti the protein is termed ChvB and NdvB, respectively. Inner membranes of R. meliloti 102F34 and A. tumefaciens A348 were first incubated with UDP-[14C]Glc and then solubilized with Triton X-100 and analyzed by polyacrylamide gel electrophoresis under native conditions. A radioactive band corresponding to the 319-kDa protein was detected in both bacteria. Triton-solubilized inner membranes of A. tumefaciens were submitted to native electrophoresis and then assayed for oligosaccharide-protein intermediate formation in situ by incubating the gel with UDP-[14C]Glc. A [14C]glucose-labeled protein with an electrophoretic mobility identical to that corresponding to the 319-kDa [14C]glucan protein intermediate was detected. In addition, protein-linked radioactivity was partially chased when the gel was incubated with unlabeled UDP-Glc. A heterogeneous family of cyclic beta-(1,2)-glucans was formed upon incubation of the gel portion containing the 319-kDa protein intermediate with UDP-[14C]Glc. A protein with an electrophoretic behavior similar to the 319-kDa protein intermediate was "in gel" labeled by using Triton-solubilized inner membranes of an A. tumefaciens exoC mutant, which contains a protein intermediate without nascent glucan. These results indicate that initiation (protein glucosylation), elongation, and cyclization were catalyzed in situ. Therefore, the three enzymatic activities detected in situ reside in a unique protein component (i.e., cyclic beta-(1,2)-glucan synthase). It is suggested that the protein component is the 319-kDa protein intermediate, which might catalyze the overall cyclic beta-(1,2)-glucan synthesis.     

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.     

Properties of Leuconostoc mesenteroides B-512FMC constitutive dextransucrase

Leuconostoc mesenteroides B-512FMC, a constitutive mutant for dextransucrase, was grown on glucose, fructose, or sucrose. The amount of cell-associated dextransucrase was about the same for the three sugars at different concentrations (0.6% and 3%). Enzyme produced in glucose medium was adsorbed on Sephadex G-100 and G-200, but much less enzyme was adsorbed when it was produced in sucrose medium. Sephadex adsorption decreased when the glucose-produced enzyme was preincubated with dextrans of molecular size greater than 10 kDa. The release of dextransucrase activity from Sephadex by buffer (20 mM acetate, pH 5.2) was the highest at 28°–30°C. The addition of dextran to the enzyme stimulated dextran synthesis but had very little effect on the temperature or pH stability. Dextransucrase purified by ammonium sulfate precipitation, hydroxyapatite chromatography, and Sephadex G-200 adsorption did not contain any carbohydrate, and it synthesized dextran, showing that primers are not necessary to initiate dextran synthesis. The purified enzyme had a molecular size of 184 kDa on SDS-PAGE. On standing at 4°C for 30 days, the native enzyme was dissociated into three inactive proteins of 65, 62, and 57 kDa. However, two protein bands of 63 and 59 kDa were obtained on SDS-PAGE after heat denaturation of the 184-kDa active enzyme at 100°C. The amount of 63-kDa protein was about twice that of 59-kDa protein. The native enzyme is believed to be a trimer of two 63-kDa and one 59-kDa monomers.     

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.