Daizo Koga

Insect endochitinases: Glycoproteins from moulting fluid, integument and pupal haemolymph of Manduca sexta L.☆

Three endochitinases have been isolated from the tobacco hornworm, Manduca sexta, by ammonium sulphate fractionation and chromatographic procedures. The purified enzymes show single bands on sodium dodecyl sulphate acrylamide gel electrophoresis and have apparent mol. wts of 7.5 × 104 (I), 6.2 × 104 (II) and 5.0 × 104 (III). They are present in prepharate pupal integument, larval integument prior to apolysis, moulting fluid and pupal haemolymph. I and III are glycoproteins that contain glucosamine and several neutral hexoses. II was not examined for carbohydrate. The endochitinases cleave glycol chitin and chitin oligosaccharides ultimately to disaccharide and trisaccharide products. The longer substrates are preferred and no activity is expressed toward p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucopyranoside, the β (1 → 4) linked dimer of N-acetylglucosamine or Micrococcus lysodeikticus cell walls. Km and kcat values for glycol chitin are 0.2 mg/ml and 2 sec−1, respectively. The endochitinases are quite stable over a wide range of pH and exhibit maximum activity around pH 6 with the oligosaccharide substrates. Antibody raised against III cross reacts with II but not with I. The endochitinases in integument and moulting fluid probably digest chitin in old cuticle prior to moulting.

HPLC Analysis of Anomeric Formation and Cleavage Pattern by Chitinolytic Enzyme.

The reactions of N-acetylchitooligosaccharides with chitinolytic enzyme were analyzed by HPLC using a Tosoh TSK-Gel amide-80 column with 70% acetonitrile as an eluent. We separated α and β anomeric forms of N-acetylchitooligosaccharides, and obtain the following advantages of this HPLC method. 1. We can easily identify the reaction mechanism of chitinolytic enzymes by this method, distinguishing the inverting mechanism showing α anomer formation from the retaining mechanism showing β anomer formation. 2. We can also estimate the cleavage patterns of N-acetylchitooligosaccharides by chitinolytic enzymes by using natural substrates.

Plant chitinase as a possible biocontrol agent for use instead of chemical fungicides.

We investigated whether a plant chitinase can be used as a biocontrol agent instead of chemical fungicides by spraying chitinase E (family 19; class IV) from a yam (Dioscorea opposita Thunb) alone or together with β-1,3-glucanase directly onto the surface of a powdery mildew infecting strawberry berries and leaves. Results were observed by eye and with a scanning electron microscope. The powdery mildew infecting the strawberries was degraded, mainly by the chitinase, and the disease did not appear again for more than 2 weeks. These results indicated that this kind of plant chitinase might be safe and biodegradable biocontrol agent for use instead of conventional fungicides.

Purification and characterization of Bombyx mori chitinases.

Two isozymes of chitinase (EC 3.2.1.14) were purified from the fifth-instar larvae of Bombyx mori by chromatography on DEAE-Cellulofine A-500, hydroxylapatite, Butyl-Toyopearl 650M, and Fractogel EMD DEAE 650 (M). These two isozymes were glycoproteins with different apparent molecular masses of 65 and 88 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pHs of the 65 and 88 kDa chitinases were 5.5 and 6.5, respectively, towards a short substrate, N-acetylchitopentaose (GlcNAc5), whereas their high activities were observed in a wide pH range between 4 and 10 towards a longer substrate, glycolchitin. Steady-state kinetic analysis of these chitinases was performed using a series of N-acetylchitooligosaccharides (GlcNAcn, n = 2-6) and glycolchitin as the substrates. Kinetic parameters for both chitinases could be obtained in the hydrolysis of glycolchitin, but not in that of N-acetylchitooligosaccharides because of strong substrate inhibition. Both chitinases similarly hydrolysed N-acetylchitooligosaccharides except for GlcNAc2 as follows: GlcNAc3 to GlcNAc plus GlcNAc2, GlcNAc4 to two molecules of GlcNAc2, GlcNAc5 to GlcNAc2 plus GlcNAc3, and GlcNAc6 to GlcNAc2 plus GlcNAc4 as well as two molecules of GlcNAc3. These results suggest that these chitinases are endo-type hydrolases, and preferred the longer-chain N-acetylchitooligosaccharides. With respect to activity, the 65 kDa chitinase was 1.7-fold more active than the 88 kDa chitinase with regard to the initial velocity in the reaction of 0.1 mM N-acetylchitooligosaccharides (GlcNAcn, n = 3-6), whereas in the overall reaction of glycolchitin (kcat/K(m)), the 88 kDa chitinase was four times more active than the 65 kDa chitinase. Regarding the affinity (1/K(m)) to glycolchitin, the affinity of the 88 kDa chitinase was 5.8-fold higher than that of the 65 kDa chitinase. The protein amino acid and gene nucleotide sequences were partly determined. Both N-terminal amino acid sequences of the 65 and 88 kDa chitinases were identical as ADSRARIVXYFSNWAVYRPG. The partial amino acid (113 amino acids) and nucleotide sequences (278 nucleotides) analysed from a mixture of 65 and 88 kDa chitinases included the two conserved regions of the family of 18 glycosyl hydrolases. All these results suggest that the B. mori chitinases are similar to Manduca sexta chitinase in primary structure and kinetic behaviour, and may be involved in the initial and intermediate stages of chitin degradation.

Purification and characterization of two β-N-acetylhexosaminidases from the tobacco hornworm, Manduca sexta (L.) (Lepidoptera: Sphingidae)☆

Abstract β- N -Acetylhexosaminidases were detected in 10 insects including species of Lepidoptera, Coleoptera, Hemiptera, and Orthoptera. Two enzymes were purified from the tobacco hornworm, Manduca sexta (L.). EI was detected in larval and pharate pupal molting fluid, integument, and pupal hemolymph while EII was found in larval and pupal hemolymphs. They are acidic hydrolases with similar molecular weights (6.1 × 10 4 ), molar extinction coefficients at 280 nm (1.9 × 10 5 liters mol −1 cm −1 ), and pH optima (pH 6). They differ in the number of polypeptide chains per molecule (EI is a single chain and EII consists of two polypeptide chains), amino acid composition, extent of glycosylation (EII is probably a glycoprotein), isoelectric point (p I EI = 5.9 and p I EII ∼- 5.1), tissue distribution, and reactivities toward nitrophenylated N -acetylglucosamine ( k cat,I = 328 s −1 and k cat,II = 103 s −1 ) and N,N′ -diacetylchitobiose ( k cat,I = 307 s −1 and k cat,II = 3 s −1 ). These results suggest that EI is a chitinase and that EII may function as a hexosaminidase in vivo .

Kinetics and mechanism of exochitinase and β-N-acetylhexosaminidase from the tobacco hornworm, Manduca sexta L. (Lepidoptera : Sphingidae)

Abstract Exochitinase (EI) from moulting fluid, integument and pupal haemolymph of Manduca sexta was greater than two orders of magnitude more active in hydrolyzing a series of N- acetylglucosamine oligosaccharides than the β-N- acetylhexosaminidase (EII) from larval and pupal haemolymphs. Product analyses showed that both enzymes hydrolyzed oligosaccharide substrates following an exocleavage pattern beginning at the nonreducing end. No transglycosylation occurred. Analyses of kinetic parameters obtained with EI in hydrolyzing βGlcNAc2 or βGlcNAc6 suggested that two ionizable groups take part in the catalysis, one of pK 3.7–3.8 affecting the K m and the other of pK 8.1 involved with the kcat. EI exhibited either linear or parabolic competitive inhibition patterns depending on the type of inhibitor used and was also inhibited by high concentrations of substrate. EII displayed only linear competitive inhibition patterns and was not inhibited by substrate. The most potent inhibitors were δ-lactone derivatives of N- acetylglocosamine and N- acetylgalactosamine with K i ⋍ 10 and 1 μM for EI and EII, respectively. These results suggested that the active site of EI consists of subsites which bind larger substrates than the active site of EII, which enables EI to hydrolyze chitin oligosaccharides more rapidly.

Immunoblot analysis of chitinolytic enzymes in integument and molting fluid of the silkworm, Bombyx mori, and the tobacco hornworm, Manduca sexta

Abstract The occurrence of proteins related to chitinolytic enzymes in integument of the silkworm, Bombyx mori, and the tobacco hornworm, Manduca sexta, during the larval-pupal transformation was determined by immunoblot analysis using rabbit polyclonal antibodies for B. mori chitinase (EC 3.2.1.14) and β-N-acetylglucosaminidase (EC 3.2.1.30) as probes. Similar temporal patterns of appearance and immunoreactivities of chitinase-like and β-N-acetylglucosaminidase-like proteins were observed in the two species. Several chitinase-like proteins (MWapp 50–200 kDa) were resolved by denaturing electrophoresis. During the latter part of the fifth larval stadium, the major immunoreactive protein in B. mori integument and molting fluid had an apparent molecular mass of 88 kDa, which was previously observed (Koga et al., 1989). In M. sexta integument on days 6–8 of the fifth larval stadium and in molting fluid, the major immunoreactive protein had an apparent molecular mass of 97 kDa, which was 22 kDa larger than the 75 kDa chitinase detected in molting fluid (Koga et al., 1983). In integument on days 3–7 of the fifth larval stadium, another immunoreactive protein with an apparent molecular mass of 119 kDa was present. In contrast to multiple immunoreactive chitinase-like proteins, only a single major β-N-acetylglucosaminidase-immunoreactive protein was detected in integument and molting fluid from either species. The immunoreactive β-N-acetylglucosaminidase-like proteins had an apparent molecular mass of 67.5 kDa and a pI of 5.0, which are identical values to those of β-N-acetylglucosaminidases determined previously. The β-N-acetylglucosaminidases cleaved N-acetylchitooligosaccharides from the non-reducing end in an exo-fashion. The results of this study suggest that chitinases are synthesized in B. mori and M. sexta integuments as zymogens, which are activated by limited proteolysis whereas β-N-acetylglucosaminidases are not.

Purification and characterization of a 54 kDa chitinase from Bombyx mori

The 54 kDa protein that was suggested to be processed from the 65 kDa and 88 kDa chitinases of Bombyx mori [Koga et al., Insect Biochem. Mol. Biol. 27, 757‐767 (1997)] was purified and proved to be a third chitinase (EC 3.2.1.14). This chitinase was purified from the fifth larval instar ofB. mori by chromatography on DEAE-Cellulofine A‐500, hydroxylapatite, Butyl-Toyopearl 650M, and Fractogel EMD DEAE 650(M) columns. The apparent molecular mass was confirmed to be 54 kDa by SDS‐PAGE. Its optimum pH was 6.0 toward a short substrate, N-acetylchitopentaose (GlcNAc5), while in its reaction with a longer substrate, glycolchitin, the enzyme showed a wide pH-range between 4.0 and 10. Kinetic parameters for the chitinase could be obtained in the hydrolysis of glycolchitin but not in that of N-acetylchitooligosaccharides (GlcNAc n, n=2‐6) because of substrate inhibition. The chitinase hydrolyzed N-acetylchitooligosaccharides except for dimer as follows: trimer to monomer plus dimer, tetramer to two molecules of dimer, pentamer to dimer plus trimer, and hexamer to dimer plus tetramer as well as two molecules of trimer. These results suggest that the 54 kDa chitinase is an endo-type hydrolase and preferred the longer-chain N-acetylchitooligosaccharides. Moreover, the anomeric forms of N-acetylchitooligosaccharides were analyzed in the reaction with the 54-kDa chitinase. It was revealed that this enzyme cleaves the substrate to produce the b anomeric product. With respect to inhibition of the 54 kDa chitinase, it was specifically inhibited by allosamidin in a competitive way with Ki values depending on the pH of the reaction mixture (Ki=0.01320.746 μM). Comparing the properties and kinetic behavior of this chitinase with those of the 88 and 65 kDa chitinases from B. mori, regarding the specific activity of the three enzymes, the 65-kDa chitinase was 2.15 and 2.8 times more active than the 88 and 54-kDa chitinases, respectively. However, in the overall reaction of glycolchitin ( kcat/Km), the 88-kDa enzyme was 4 and 40 times more active than the 65-kDa and the 54-kDa enzymes, respectively. Concerning the affinity (1/Km) to glycolchitin, the 88 kDa chitinase affinity (at pH 6.5) was 5.8 times higher than that of the 65 kDa chitinase (at pH 5.5) and 4.0 times higher than that of the 54 kDa chitinase (at pH 6.0). These kinetic results suggest that B. mori chitinases are processed during ecdysis from the larger chitinase to smaller ones that leads to changes in their kinetic properties such as Km, kcat and kcat/Km successively.

Appearance of chitinolytic enzymes in integument of Bombyx mori during the larval-pupal transformation. Evidence for zymogenic forms

Abstract The appearance of chitinolytic enzymes, chitinase and β-N-acetylglucosaminidase, involved in ecdysis of the silkworm, Bombyx mori, was investigated using integuments prepared from fifth instar larvae during and after spinning behavior just before the larval-pupal transformation. β-N-Acetylglucosaminidase activity appeared a day after the beginning of spinning (SP1) and gradually increased for 2 more days (SP3), while chitinase activity appeared later at the SP3 stage (1 day before the ecdysis). It was shown by immunoblotting that the changes in activity were due to increases in the amounts of enzymes present. A probable zymogenic form of chitinase, whose molecular weight was about 215 kDa, was detected during spinning period by immunoblotting using anti-65-kDa chitinase antibody. The zymogen was observed 2 days before the appearance of enzyme activity. High molecular proteins (120–190 kDa) related to β-N-acetylglucosaminidase were also observed throughout the spinning period by immunoblotting, but this appearance pattern was different from that of chitinase. The results support, at least in the case of chitinase the hypothesis, that insect chitinolytic enzymes are synthesized as inactive precursors which are activated by limited proteolysis.

A genomic clone for a chitinase gene from the silkworm, Bombyx mori: structural organization identifies functional motifs.

A genomic clone containing the chitinase gene was isolated and characterized from the silkworm, Bombyx mori, using the polymerase chain reaction technique directed by primers designed from the chitinase cDNA of the insect previously cloned [Insect Biochem. Molec. Biol. 28 (1998) 163]. Results from nucleotide sequence analysis of the gene and its PCR amplification suggest that the B. mori genome probably has only one copy of chitinase gene. The gene is organized into 10 coding regions, exons, interrupted by noncoding regions, introns. The motifs encoded and exon organization of the gene are almost identical to the related gene from Manduca sexta. The results suggest that the domain organization of chitinase genes may be conserved among insect cuticular chitinase genes and that they are more complex than their counterparts in plants. The possibilities of intron splicing from the primary transcripts are also discussed.