Molecular cloning, expression, and functional analysis of the chitin synthase 1 gene and its two alternative splicing variants in the white-backed planthopper, Sogatella furcifera (Hemiptera: Delphacidae).

Chitin synthase is responsible for chitin synthesis in the cuticles and cuticular linings of other tissues in insects. We cloned two alternative splicing variants of the chitin synthase 1 gene (SfCHS1) from the white-backed planthopper, Sogatella furcifera. The full-length cDNA of the two variants (SfCHS1a and SfCHS1b) consists of 6408 bp, contains a 4719-bp open reading frame encoding 1572 amino acids, and has 5' and 3' non-coding regions of 283 and 1406 bp, respectively. The two splicing variants occur at the same position in the cDNA sequence between base pairs 4115 and 4291, and consist of 177 nucleotides that encode 59 amino acids but show 74.6% identity at the amino acid level. Analysis in different developmental stages showed that expression of SfCHS1 and SfCHS1a were highest just after molting, whereas SfCHS1b reached its highest expression level 2 days after molting. Further, SfCHS1 and SfCHS1a were mainly expressed in the integument, whereas SfCHS1b was predominately expressed in the gut and fat body. RNAi-based gene silencing inhibited transcript levels of the corresponding mRNAs in S. furcifera nymphs injected with double-stranded RNA of SfCHS1, SfCHS1a, and SfCHS1b, resulted in malformed phenotypes, and killed most of the treated nymphs. Our results indicate that SfCHS1 may be a potential target gene for RNAi-based S. furcifera control.

Sorting signals that mediate traffic of chitin synthase III between the TGN/endosomes and to the plasma membrane in yeast.

Traffic of the integral yeast membrane protein chitin synthase III (Chs3p) from the trans-Golgi network (TGN) to the cell surface and to and from the early endosomes (EE) requires active protein sorting decoded by a number of protein coats. Here we define overlapping signals on Chs3p responsible for sorting in both exocytic and intracellular pathways by the coats exomer and AP-1, respectively. Residues 19DEESLL24, near the N-terminal cytoplasmically-exposed domain, comprise both an exocytic di-acidic signal and an intracellular di-leucine signal. Additionally we show that the AP-3 complex is required for the intracellular retention of Chs3p. Finally, residues R374 and W391, comprise another signal responsible for an exomer-independent alternative pathway that conveys Chs3p to the cell surface. These results establish a role for active protein sorting at the trans-Golgi en route to the plasma membrane (PM) and suggest a possible mechanism to regulate protein trafficking.

Purification of an active, oligomeric chitin synthase complex from the midgut of the tobacco hornworm.

Chitin formation depends on the activity of a family II glycosyltransferase known as chitin synthase, whose biochemical and structural properties are largely unknown. Previously, we have demonstrated that the chitin portion of the peritrophic matrix in the midgut of the tobacco hornworm, Manduca sexta, is produced by chitin synthase 2 (CHS-2), one of two isoenzymes encoded by the Chs-1 and Chs-2 genes (also named Chs-A and Chs-B), and that CHS-2 is located at the apical tips of the brush border microvilli. Here we report the purification of the chitin synthase from the Manduca midgut as monitored by its activity and immuno-reactivity with antibodies to the chitin synthase. After gel permeation chromatography, the final step of the developed purification protocol, the active enzyme eluted in a fraction corresponding to a molecular mass between 440 and 670 kDa. Native PAGE revealed a single, immuno-reactive band of about 520 kDa, thrice the molecular mass of the chitin synthase monomer. SDS-PAGE and immunoblotting indicated finally that an active, oligomeric complex of the chitin synthase was purified. In summary, the chitin synthase from the midgut of Manduca may prove to be a good model for investigating the enzymes' mode of action.

Immunoaffinity purification of the class V chitin synthase of Wangiella (Exophiala) dermatitidis.

The class V chitin synthase is unique because it has a myosin motor-like domain fused to its catalytic domain. The biochemical properties of this enzyme and its function remain undefined beyond the knowledge that it is the only single chitin synthase required for sustained cell growth at elevated temperatures and, consequently, virulence. This report describes our successful efforts to isolate and purify an active and soluble form of the enzyme from the cell membranes of Wangiella by using a specific polyclonal antibody. To our knowledge, this is the first purification of a single chitin synthase of a filamentous fungus.

Elucidating the biosynthesis of chitin filaments and their configuration with specific proteins and electron microscopy.

To deepen the knowledge of chitin synthesis, a yeast mutant has been used as a model. Purified chitin synthase I-containing vesicles (chitosomes) with a diameter of 85 to 120 nm are identified by electron microscopy to eject tiny fibers upon addition of UDP-N-acetylglucosamine. The filigree of extruded filaments fused gradually into a large three-dimensional network, which is degradable by a chitinase. The network is targeted and restructured by the Streptomyces chitin-binding protein CHB1, which has a very high affinity only for alpha-chitin. Within the chitosomes, filaments are found to be highly condensed within consecutive oval fibroids, which are specifically targeted by the alpha-chitin-binding protein. The presented data give new insights to the generation of chitin filaments with an antiparallel (alpha) configuration. [image: see text]

Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III activity and chitin chain length.

Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of Saccharomyces cerevisiae and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p prenylation. We provide evidence that Chs4p is farnesylated by showing that purified Chs4p is recognized by anti-farnesyl antibody and is a substrate for farnesyl transferase (FTase) in vitro and that inactivation of FTase increases the amount of unmodified Chs4p in yeast cells. We demonstrate that abolition of Chs4p prenylation causes a approximately 60% decrease in CSIII activity, which is correlated with a approximately 30% decrease in chitin content and with increased resistance to the chitin binding compound calcofluor white. Furthermore, we show that lack of Chs4p prenylation decreases the average chain length of the chitin polymer. Prenylation of Chs4p, however, is not a factor that mediates plasma membrane association of the protein. Our results provide evidence that the prenyl moiety attached to Chs4p is a factor modulating the activity of CSIII both in vivo and in vitro.

Isolation of the CHS4 gene of Paracoccidioides brasiliensis and its accommodation in a new class of chitin synthases.

The nucleotide sequence of a chitin synthase gene (PbrCHS4) of the dimorphic fungal human pathogen Paracoccidioides brasiliensis has been determined. A homology search with the deduced amino acid sequence of PbrChs4 (1744 aa) reveals the presence of two distinct domains, an N-terminal domain showing up to 30% homology to myosin motor-like domains and a C-terminal domain with up to 68% homology to chitin synthases, as has been reported for some class V chitin synthases. However, unlike class V chitin synthases with myosin motor-like domains, PbrChs4 does not present characteristic signatures of myosin motor-like domains. Also, although the Chs domain presents the closest homology to other fungal class V enzymes, it is low enough to consider PbrChs4 as belonging to a new class, which we propose as class VII.

The yeast Chs4 protein stimulates the trypsin-sensitive activity of chitin synthase 3 through an apparent protein-protein interaction.

Inducible overexpression of the CHS4 gene under the control of the GAL1 promoter increased Chs3p (chitin synthase 3) activity in Saccharomyces cerevisiae several fold. Approximately half of the Chs3p activity in the membranes of cells overexpressing Chs4p was extracted using CHAPS and cholesteryl hemisuccinate. The detergent-extractable Chs3p activity appeared to be non-zymogenic because incubation with trypsin decreased enzyme activity in both the presence and absence of the substrate, UDP-N-acetylglucosamine. Western blotting confirmed that Chs3p was extracted from membranes by CHAPS and cholesteryl hemisuccinate and revealed that Chs4p was also solubilized using these detergents. Yeast two-hybrid analysis with truncated Chs4p demonstrated that the region of Chs4p between amino acids 269 and 563 is indispensable not only for eliciting the non-zymogenic activity of Chs3p but also for binding of Chs4p to Chs3p. Neither the EF-hand motif nor a possible prenylation site in Chs4p was required for these activities. Thus, it was demonstrated that stimulation of non-zymogenic Chs3p activity by Chs4p requires the amino acid region from 269 to 563 of Chs4p, and it seems that Chs4p activates Chs3p through protein-protein interaction.

Characterization of chitin synthase 2 of Saccharomyces cerevisiae. II: Both full size and processed enzymes are active for chitin synthesis.

When chitin synthase 2 of Saccharomyces cerevisiae was overexpressed in yeast cells using GAL1 promoter, deletion of the N-terminal 193 amino acids significantly increased the level of the protein without affecting its characteristics. We partially purified N-terminally truncated chitin synthase 2 by product entrapment and ion exchange column chromatography, and found that it was active even without trypsin treatment when appropriate divalent cations were present in the reaction mixture. This chitin synthase activity was independent of the N-terminal 193 amino acid truncation, because partially purified full length enzyme also exhibited the activity without trypsin treatment in the presence of appropriate cations. Furthermore, the molecular weights of these two forms of chitin synthase 2 were coincident with those estimated from the deduced amino acid sequence, and most of the chitin synthase 2 in the yeast membrane was present as an unprocessed form, as judged from its molecular weight. Treatment of either full length or truncated enzyme with trypsin, however, further increased the enzyme activity by four to fivefold, and produced a 35 kDa polypeptide that specifically reacted with monoclonal antibody raised against the region containing the putative active site of chitin synthase 2. Thus, it appears that predominant native (unprocessed) chitin synthase 2 is active, but the 35 kDa region encompassing the active site is sufficient for the catalytic activity.

Nikkomycin-resistant mutants of Mucor rouxii: physiological and biochemical properties.

We isolated three nikkomycin-resistant mutants of the dimorphic fungus M. rouxii which were physiologically characterized regarding their response to yeast-phase inducing conditions and their sensitivity to bacilysin. Mutant strains G21 and G23, showed a qualitatively normal, though delayed, dimorphic transition and partial cross-resistance to bacilysin. Mutant strain G27 showed an altered dimorphism, producing a high proportion (50%) of hyphal cells, and a wild-type sensitivity to bacilysin. Cell-free extracts from this mutant exhibited an activity of both basal and protease-activated chitin synthetase which was overexpressed as compared with the parental strain and mutants G21 and G23. Results are discussed in terms of the different genetic background of the mutants.

Effect of digitonin on membrane-bound and chitosomal chitin synthetase activity in protoplasts from yeast cells of Candida albicans.

The effect of digitonin on chitin synthetase present in membrane (MMF) and cytoplasmic fractions (chitosomes) (CF) from C. albicans yeast protoplasts has been determined. The zymogen is preferentially, but not exclusively, solubilized by digitonin from MMF. Centrifugation of distinct solubilized preparations, containing either zymogen, in vivo active enzyme and/or trypsin activated enzyme, on linear sucrose gradients suggests that both zymogen and trypsin activated enzyme sediment slightly slower than the active enzyme, pointing out differences between the activation processes in vivo and in vitro or, alternatively, that both enzyme activities (active in vivo and zymogenic) correspond to different gene products. The detection of a zymogenic activity under certain conditions (0.5 mg ml-1 of digitonin and 64 micrograms ml-1 of trypsin) also suggests the existence of more than one pool of zymogenic enzyme in the MMF. Digitonin sensitizes the chitosomal (CF) proenzyme to trypsin: activation is enhanced by low digitonin concentrations in the presence of 8 micrograms ml-1 of protease, whereas activity strongly decreases in the presence of 64 micrograms ml-1 of trypsin. Digitonin does not produce zymogen activation per se in absence of exogenous protease. Furthermore, chitosome structure is modified into particles with low buoyant densities.

Unexpected destruction of chitosomal chitin synthetase by an endogenous protease during sucrose density gradient purification.

Because of their intrinsic low buoyant density, chitosomes can be separated from crude cell homogenates (1000 g or 35,000 g supernatants) of Mucor rouxii by isopycnic sedimentation in sucrose density gradients. To accelerate and simplify the isolation of chitosomes, we centrifuged the cell-free extracts at ultrahigh speed (in a fixed-angle rotor at forces up to 311,000 g Rav) and found that the duration of centrifugation was critical. Prolonged centrifugation at ultrahigh speed caused severe distortion of the chitin synthetase profile in the gradient as the peak of chitosomal chitin synthetase nearly disappeared. We traced the problem to a soluble protease(s) that moved into the chitosome band during protracted centrifugation and destroyed the chitin synthetase activity. The interfering protease was a soluble protein with a sedimentation coefficient of 4.6 S and a pH optimum of 7-7.5, and it was sensitive to PMSF (phenylmethylsulfonyl fluoride), indicating that it was a serine protease. Unlike other proteases, it destroyed chitin synthetase but did not activate the chitin synthetase zymogen. The interfering protease could be eliminated either by adding PMSF to the cells immediately after breakage or by removing the upper part of the sucrose gradient midway through the centrifugation of the cell-free extract and then completing the sedimentation with the 'decapitated' gradient.

Two chitin synthases in Saccharomyces cerevisiae.

Disruption of the yeast CHS1 gene, which encodes trypsin-activable chitin synthase I, yielded strains that apparently lacked chitin synthase activity in vitro, yet contained normal levels of chitin (Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L., and Robbins, P. W. (1986) Cell 46, 213-225). It is shown here that disrupted (chs1 :: URA3) strains have a particulate chitin synthetic activity, chitin synthase II, and that wild type strains, in addition to chitin synthase I, have this second activity. Chitin synthase II is measured in wild type strains without preincubation with trypsin, the condition under which highest chitin synthase II activities are obtained in extracts from the chs1 :: URA3 strain. Chitin synthase II, like chitin synthase I, uses UDP-GlcNAc as substrate and synthesizes alkali-insoluble chitin (with a chain length of about 170 residues). The enzymes are equally sensitive to the competitive inhibitor Polyoxin D. The two chitin synthases are distinct in their pH and temperature optima, and in their responses to trypsin, digitonin, N-acetyl-D-glucosamine, and Co2+. In contrast to the report by Sburlati and Cabib (Sburlati, A., and Cabib, E. (1986) Fed. Proc. 45, 1909), chitin synthase II activity in vitro is usually lowered on treatment with trypsin, indicating that chitin synthase II is not activated by proteolysis. Chitin synthase II shows highest specific activities in extracts from logarithmically growing cultures, whereas chitin synthase I, whether from growing or stationary phase cultures, is only measurable after trypsin treatment, and levels of the zymogen do not change. Chitin synthase I is not required for alpha-mating pheromone-induced chitin synthesis in MATa cells, yet levels of chitin synthase I zymogen double in alpha factor-treated cultures. Specific chitin synthase II activities do not change in pheromone-treated cultures. It is proposed that of yeast's two chitin synthases, chitin synthase II is responsible for chitin synthesis in vivo, whereas nonessential chitin synthase I, detectable in vitro only after trypsin treatment, may not normally be active in vivo.

Stabilization of chitin synthetase and purification of chitosomes from several mycelial Mucorales.

Stability of chitin synthetase in cell-free extracts from mycelial fungi was markedly improved by the presence of sucrose in the homogenization media. Breakage of mycelium in sucrose-containing buffer yielded enzyme preparations from which chitosomal chitin synthetase could be purified by a procedure involving ammonium sulfate precipitation, gel filtration and centrifugation in sucrose density gradients. Purified chitosomes catalyzed the synthesis of chitin microfibrils in vitro upon incubation with substrate and activators. Chitosomal chitin synthetase from the filamentous form of M. rouxii was similar to the enzyme from yeast cells, except for the poorer stability and diminished sensitivity to GlcNAc activation of the former.

Fotoactivación in vitro de la quitina sintetasa de los esporangioforos de Phycomyces blakesleeanus / In vitro photoactivation of chitin synthetase from Phycomyces blakesleeanus sporangiophores

Se estudió el mecanismo de la foto-activación de la quitina sintetasa presente en los esporangióforos de Phycomyces en estado IV. La enzima se estabilizó parcialmente cuando los extractos se obtuvieron en presencia de sacarosa; se obtuvo la máxima actividad a pH 7.8 y se estimuló por adición de proteasas exógenas. La iluminación de los extractos con luz blanca produjo una estimulación promdeido en la actividad del 75% en ausencia de tripsina y del 21% en presencia de la proteasa. En la obscuridad, la adición de Ca 2+ produjo una débil estimulación a bajas concentraciones y una inhibición a concentraciones mas altas; su adicion dio lugar a una alta activación por la luz. La iodo-acetamida inhibio la activación de la quitina sintetasa por la luz y por la tripsina. Con estos resultados se propone un modelo para explicar el mecanismo de activación de la quitina sintetasa por la luz

Chitin synthetase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae.

Strains containing a disrupted structural gene for chitin synthetase (chs1::URA3) are defective in chitin synthetase 1 (Chs1) activity but contain normal amounts of chitin (Bulawa, C.E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, L., and Robbins, P. W. (1986) Cell 46, 213-225). We have now detected in such strains a new chitin synthetase activity (Chs2), at levels about 5% of those of Chs1 in wild-type cells. Thus, Chs2 is presumably the physiological agent for chitin deposition in strains with a disrupted CHS1 gene and probably also in wild-type strains. Chs1 and Chs2 share certain properties, such as stimulation by N-acetylglucosamine and by partial proteolysis. They differ sharply, however, in divalent cation specificity and in pH optimum. Chs2 also shows less sensitivity than Chs1 to inhibition by polyoxin D or sodium chloride, a property that was used to demonstrate the presence of Chs2 in wild-type extracts. As in the case of Chs1, most of the Chs2 activity was found to be associated with the plasma membranes. This finding, together with the apparent zymogenic nature of Chs2, is consistent with the hypothesis, previously put forward for Chs1, that localized deposition of chitin is attained by activation of the zymogen form at a specific time and place. Function and significance of the two chitin synthetases are discussed in connection with fungal morphogenesis and evolution.

The S. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo.

The chitin synthase of Saccharomyces is a plasma membrane-bound zymogen. Following proteolytic activation, the enzyme synthesizes insoluble chitin that has chain length and other physical properties similar to chitin found in bud scars. We isolated mutants lacking chitin synthase activity (chs1) and used these to clone CHS1. The gene has an open reading frame of 3400 bases and encodes a protein of 130 kd. The fission yeast S. pombe lacks chitin synthase and chitin. When a plasmid encoding a CHS1-lacZ fusion protein is introduced into S. pombe, both enzymatic activities are expressed in the same ratio as in S. cerevisiae, demonstrating that CHS1 encodes the structural gene of chitin synthase. Three CHS1 gene disruption experiments were performed. In all cases, strains with the disrupted gene have a recognizable phenotype, lack measurable chitin synthase activity in vitro but are viable, contain normal levels of chitin in vivo, and mate and sporulate efficiently.

Isolation of chitin synthetase from Saccharomyces cerevisiae. Purification of an enzyme by entrapment in the reaction product.

Chitin synthetase, in the zymogen form, was extracted with digitonin from a particulate fraction from Saccharomyces cerevisiae and converted into active form by treatment with immobilized trypsin. When the activated enzyme was incubated with UDP-GlcNAc and other components of an assay mixture, a chitin precipitate formed, trapping a large portion of the synthetase. The enzyme was easily extracted frm the chitin gel with a recovery of approximately 50% and an enrichment of approximately 100-fold. Further purification was obtained by repeating the chitin step. After polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the purified synthetase showed a major band corresponding to Mr 63,000, a weaker band at Mr 74,000, and some other minor bands. Under nondenaturing conditions, an Mr of 570,000 was calculated for the enzyme from Stokes radius and sedimentation coefficient determinations. After electrophoresis in a nondenaturing gel and incubation with the components of the standard assay, chitin was formed and precipitated in the gel, yielding an opaque band. Soluble oligosaccharides were not precursors for insoluble chitin, suggesting that synthesis of chitin chains takes place by a processive mechanism. N-Acetylglucosamine stimulated the purified synthetase only slightly and did not participate as a primer in the reaction. The same chain length, somewhat more than 100 units of GlcNAc, was determined in samples of chitin that had been synthesized either in vivo, or with a membrane preparation or with purified synthetase. These results suggest that chitin synthetase itself is capable both of initiating chitin chains without a primer and of determining their length.

Activation of chitin synthetase in permeabilized cells of a Saccharomyces cerevisiae mutant lacking proteinase B.

Digitonin treatment at 30 degrees C of a Saccharomyces cerevisiae mutant lacking proteinase B permeabilized the cells and caused rapid and extensive activation of chitin synthetase in situ. The same result was obtained with a mutant generally defective in vacuolar proteases. By lowering the temperature and using different permeabilization procedures, we showed that increases in permeability and activation are distinct processes. Activation was inhibited by the protease inhibitors antipain and leupeptin, but by pepstatin or chymostatin. Metal chelators were also inhibitory, and their effect was reversed by the addition of Ca2+ but not by Mg2+. Antipain added together with Ca2+ after incubation of the cells in the presence of a chelating agent prevented reversal of inhibition, a result that was interpreted as indicating that antipain acts either on the same step affected by Ca2+ or on a subsequent step. Efforts to obtain activation in cell-free extracts were unsuccessful, but it was possible to extract the synthetase, once activated, by breaking permeabilized cells with glass beads. Treatment of the cell-free extracts with trypsin led not only to increased activity of chitin synthetase, but also to a change in the pH-activity curve and a diminished requirement by the enzyme for free N-acetylglucosamine. These observations suggest that the modification undergone by the synthetase during endogenous activation is different from that brought about by trypsin treatment.