Isolation and purification of a cell adhesion factor from crayfish blood cells

Isolated granular haemocytes (blood cells) from the crayfish Pacifastacus leniusculus attached and spread in vitro on coverslips coated with a lysate of crayfish haemocytes. No cell adhesion activity was detected in crayfish plasma. The cell adhesion activity was only present in haemocyte lysates in which the prophenoloxidase (proPO) activating system (Soderhall and Smith, 1986a, b) had been activated; either by lipopolysaccharide (LPS), the beta-1,3-glucan laminarin, or by preparing the lysate in 5 mM Ca2+. Both lysates of granular or of semigranular haemocytes could mediate adhesion. After A23187-induced exocytosis of the granular cells, cell adhesion activity could be generated in the secreted material if it was incubated with laminarin. The factor responsible for cell adhesion was isolated from an active haemocyte lysate and purified by ammonium sulfate precipitation, cation exchange chromatography and Con A-Sepharose; it had a molecular mass of approximately 76 kD on an SDS-polyacrylamide gel. An antibody to this 76-kD band inhibited cell adhesion. Ca2+ was necessary in the medium for the cells to adhere to the adhesion factor. With cyanide or azide, the cells attached but failed to spread. It is suggested that in vivo the cell adhesion factor is stored in the secretory granules of the semigranular and the granular cells in a putative inactive pro-form, which can be released during exocytosis and, in the presence of beta- 1,3-glucans or LPS, be activated outside the cells to mediate cell attachment and spreading, processes of essential importance in arthropod host defense.     

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.     

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.     

A cell adhesion factor from crayfish haemocytes has degranulating activity towards crayfish granular cells

A factor able to mediate cell adhesion of semigranular and granular haemocytes of the crayfish Pacifastacus leniusculus was recently purified from crayfish haemocyte lysate (Johansson and Söderhäll, J. Cell Biol.106, 1795–1803, 1988). It is a protein with a mass of 76 kDa, and its activity seems to be generated concomitantly with the activation of the prophenoloxidase (proPO) activating system. In this paper, we present evidence that this same protein is also responsible for the previously reported degranulating activity of a crayfish haemocyte lysate, in which the proPO system has been activated. First, the 76 kDa band in SDS-polyacrylamide electrophoresis seems to be a single protein, since in isoelectric focusing the purified cell adhesion factor fraction migrated as one band with an isoelectric point of 7.2. Second, this fraction was also able to degranulate crayfish granular cells in vitro, and third, antibodies to this 76 kDa protein, which are known to block cell adhesion, could also inhibit degranulation in vitro.     

褐飞虱和桃蚜粗提液中酚氧化酶原激活系统的激活特点

采用研磨法制备褐飞虱Nilaparvata lugens (Stål)和桃蚜Myzus persicae (Sulzer)粗提液,用于研究几种化学因子对粗提液中酚氧化酶原激活系统(proPO-AS)的激活特点。结果表明, 在0.1～100 mmol/L的Ca²⁺浓度范围内,虱液和蚜液的酚氧化酶(PO)活性随Ca²⁺浓度升高而增强,并在30 mmol/L处PO活性达到最高,在此限之上PO活性反而下降。当昆布多糖浓度从10^-4 mg/mL升至10 mg/mL时,虱液和蚜液的PO活性也随之升高,但再提高昆布多糖浓度却未见PO活性的峰顶出现。在6种葡聚糖对PO的激活作用中,昆布多糖和酵母聚糖能显著增强PO活性,但curdlan对虱液和蚜液的PO无明显激活作用,而甘露聚糖、右旋糖苷和纤维素则降低PO活性。这些结果显示β-1,3-葡聚糖能有效激活proPO-AS。Ca²⁺和丝氨酸蛋白酶抑制剂PMSF加样顺序的不同也会影响PO活性。     

Purification and partial characterization of a beta-1,3-glucan-binding-protein membrane receptor from blood cells of the crayfish Pacifastacus leniusculus.

A receptor for the 100 kDa beta-1,3-glucan-binding protein [Duvic, B. and S?derh?ll, K. (1990) J. Biol. Chem. 265, 9327-9332] has been purified from hemocyte membranes of the crayfish Pacifastacus leniusculus. The purification was achieved by DEAE-cellulose chromatography of detergent-solubilized membranes. The receptor had an apparent molecular mass of 350 kDa when subjected to native polyacrylamide-gel electrophoresis and was composed of two non-covalently associated subunits of about 230 kDa and 90 kDa, as judged by SDS/polyacrylamide-gel electrophoresis or two-dimensional electrophoresis. The receptor could only bind the beta-1,3-glucan-binding protein if this protein had previously reacted with a beta-1,3-glucan, laminarin, and the binding site was located on the 230 kDa subunit. The binding of laminarin-treated beta-1,3-glucan-binding protein to its receptor was a saturable process and binding data indicated a single high-affinity-binding site with a Kd of 0.35 +/- 0.15 microM as determined by Scatchard analysis. The receptor had a requirement for divalent cations and a pH optimum of 6.5 for binding the laminarin-treated beta-1,3-glucan-binding protein. Laminarin, as well as oligosaccharides such as D-glucose, sialic acid, N-acetyl glucosamine or methyl-alpha-D-mannoside, could not affect the binding of the beta-1,3-glucan-binding protein to its receptor.     

Purification and characterization of an exo-beta-1,3-glucanase produced by Trichoderma asperellum

Trichoderma asperellum produces at least two extracellular beta-1,3-glucanases upon induction with cell walls from Rhizoctonia solani. A beta-1,3-glucanase was purified by gel filtration and ion exchange chromatography. A typical procedure provided 35.7-fold purification with 9.5% yield. The molecular mass of the purified exo-beta-1,3-glucanases was 83.1 kDa as estimated using a 12% (w/v) SDS-electrophoresis slab gel. The enzyme was only active toward glucans containing beta-1,3-linkages and hydrolyzed laminarin in an exo-like fashion to form glucose. The K(m) and V(max) values for exo-beta-1,3-glucanase, using laminarin as substrate, were 0.087 mg ml(-1) and 0.246 U min(-1), respectively. The pH optimum for the enzyme was pH 5.1 and maximum activity was obtained at 55 degrees C. Hg(2+) strongly inhibited the purified enzyme.     

Purification and characterization of a prophenoloxidase activating enzyme from crayfish blood cells

A proteinase was purified from crayfish haemocytes by affinity chromatography on heparin-sepharose and phenyl-sepharose, followed by DEAE-cellulose ion-exchange chromatography. This proteinase could mediate the conversion of prophenoloxidase (proPO) to its active form, phenoloxidase (PO), and its was therefore designated a prophenoloxidase activating enzyme, ppA.The purified ppA had a molecular mass of about 36,000 Da. Since ppA was a proteinase able to cleave chromogenic peptide substrates of trypsin, and serine proteinase inhibitors were strongly inhibitory towards ppA activity, the enzyme appeared to be a serine type proteinase. It exhibited maximal enzyme activity at neutral and slightly alkaline pH, and was sensitive to heat inactivation at 58°C.     

Regulation of 36-kDa beta-1,3-glucanase synthesis in Trichoderma harzianum

The effect of carbon sources on the level of beta-1,3-glucanases in the culture filtrates of Trichoderma harzianum (Tc) was investigated. Enzyme activity was detected in all carbon sources, but highest levels were found when laminarin and purified cell walls were used. Three isoforms of beta-1,3-glucanase were produced during growth of the fungus on purified cell walls. Two isoforms were produced on chitin, chitosan, N-acetylglucosamine and laminarin, while only one was detected when the fungus was grown on cellulose and glucose. A 36-kDa beta-1,3-glucanase (GLU36) was secreted from T. harzianum (Tc) grown on all carbon sources tested as demonstrated by Western blot analysis. We found that a significant increase in the level of GLU36 in the culture filtrate follows glucose exhaustion, suggesting that this enzyme is controlled by carbon catabolite repression.     

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.     

Isolation and partial characterization of an 87-kilodalton beta-1,3-glucanase from Bacillus circulans IAM1165.

Bacillus circulans IAM1165 produces at least two extracellular beta-1,3-glucanases that lyse fungal cell walls. One of these extracellular enzymes was purified to homogeneity. The molecular mass was 87 kDa, and the pI was 4.3. The optimum temperature of the enzyme reaction was 70 degrees C when laminarin (a soluble beta-1,3-glucan) was used as the substrate. The pH range of the enzyme was broad (pH 4.5 to 9.0), and the optimum pH was 6.5. The enzyme is an endo beta-1,3-glucanase and has a random cleavage pattern.     

Phenoloxidase activity in larval and juvenile homogenates and adult plasma and haemocytes of bivalve molluscs

Phenoloxidase (PO) activity was studied in larval and juvenile homogenates and in the plasma and haemocytes of adult Crassostrea gigas, Argopecten ventricosus, Nodipecten subnodosus, and Atrina maura. Samples were tested for the presence of PO activity by incubation with the substrate L-3, 4-dihydroxyphenylalanine using trypsin, alpha-chymotrypsin, laminarin, lipopolysaccharides (LPS), and sodium dodecyl sulphate (SDS) to elicit activation of prophenoloxidase (proPO) system. PO activity was not detected in larval homogenate. In juvenile homogenate, PO activity was found only in C. gigas and N. subnodosus. PO activity was present in adult samples and was enhanced by elicitors in the plasma of all species tested, but in haemocyte lysate supernatant (HLS) of only N. subnodosus. Activation of proPO by laminarin was suppressed by a protease inhibitor cocktail (P-2714) in plasma and HLS of all species tested.     

Purification and characterization of laminaran hydrolases from Trichoderma viride

At least three extracellular laminaran hydrolases which hydrolyzed laminaran (beta-1,3:1,6-glucan) from Eisenia bicyclis were secreted in wheat bran solid medium by Trichoderma viride U-1. These three enzymes, lam AI, AII, and B, were purified to electrophoretic homogeneity. Their molecular masses were estimated to be 70.1, 70.4, and 45.0 kDa for lam AI, AII, and B, respectively, by SDS-PAGE. Whereas both lam AI and AII could hydrolyze laminarin from Laminaria digitata, lam AII showed higher activity against Laminaria laminarin rather than Eisenia laminaran. On the other hand, lam B preferentially hydrolyzed pustulan, a beta-1,6-glucan. Laminarioligosaccharide was hydrolyzed by lam AI and AII but not B, whereas gentiooligosaccharide was hydrolyzed by only lam B. It showed that lam AI and AII were specific for beta-1,3-linkages, but lam B was specific for beta-1,6-linkages. These results indicated that T. viride U-1 has a multiple glucanolytic enzyme system.     

Molecular cloning, purification and characterization of thermostable beta-1,3-1,4 glucanase from Bacillus subtilis A8-8

A gene encoding a beta-1,3-1,4-glucanase (CelA) belonging to family 5 of glycoside hydrolases was cloned and sequenced from the Bacillus subtilis A8-8. The open-reading-frame of celA comprised 1499 base pairs and the enzyme was composed of 500 amino acids with a molecular mass of 55 kDa. The recombinant beta-1,3-1,4 glucanase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 8.0 and 60 degrees C, respectively. The enzyme was stable within pH 6.0-9.0. It was stable up to 60 degrees C and retained 30% of its original activity at 70 degrees C for 60 min. It hydrolyzed lichenan, CMC, xylan, laminarin, avicel and pNPC, but was inactive towards cellobiose. The enzyme activity was markedly activated by Co2+ and Mn2+, but was strongly inactivated by Fe3+. The truncated gene, devoid of cellulose-binding domain (CBD) showed 60% of activity and bound to avicel.     

A protein with lectin activity in penetration glands of Diplostomum pseudospathaceum cercariae

Homogenates of Diplostomum pseudospathaceum cercariae agglutinated mouse erythrocytes. The haemagglutination could be inhibited by certain glycoconjugates containing beta-1,3- and beta-1,4-glycan chains and also by some simple saccharides. The most potent inhibitors were heparin and some other glycosaminoglycans, bacterial lipopolysaccharides, laminarin (a beta-1,3-glucan) and lactulose. After electrophoresis of cercarial proteins, a dominant double band appeared in the 22-24 kDa region of gels. On blots, this protein bound labelled laminarin and it was also one of the few proteins recognised by mouse antibodies raised against cercarial haemagglutinins. In addition, mouse polyclonal antibodies against the beta-1,3-glucan-binding protein bound exclusively to the 22-24 kDa region on Western blots. Histochemistry revealed strong binding of labelled laminarin to cercarial penetration glands; this reaction was fully blocked by unlabelled laminarin. Other labelled glycoconjugates such as heparin, hyaluronic acid and a bacterial lipopolysaccharide also bound to the glands. Immunohistochemistry confirmed the localisation of the beta-1,3-glucan-binding protein in penetration glands. Reaction of the cercarial protein with immunoglobulins from non-immunised mice was observed on both nitrocellulose membranes and histological sections; this could be blocked by laminarin in incubation buffers. We consider the cercarial haemagglutinin to be a lectin which is identical with the 22-24 kDa beta-1,3-glucan-binding protein. According to the binding specificity and localisation we speculate on a role of this lectin in cercarial penetration into the host, probably as a tissue recognition or antibody rendering factor.