Chitin biosynthesis by a fungal membrane preparation Evidence for a transient non-crystalline state of chitin

Transcription

1 Eur. J. Biohem. 158, (1986) 0 FEBS 1986 Chitin biosynthesis by a fungal membrane preparation Evidene for a transient non-rystalline state of hitin Cornelis A. VERMEULEN and Joseph G. H. WESSELS Department of Plant Physiology, Biologial Centre, University of Groningen, Haren (Reeived February 19/April 14, 1986) - EJB Chitin synthase ativity of membrane preparations from hyphae of Shizophyllum ommune was strongly inhibited by added hitinase beause hitin immediately after its synthesis was highly suseptible to hitinase. In the absene of synthesis, hitin beame more resistant to hitinase with time. Chitin synthesized in the presene of the optial brightener Calofluor White M2R was extremely suseptible to degradation by hitinase and this suseptibility was maintained for a long time. X-ray diffration analysis of hitin synthesized in the presene of Calofluor revealed the absene of rystallinity as long as the material was kept in wet onditions. After drying, disrete defletions harateristi for a-hitin appeared onomitant with a derease in the suseptibility for hitinase. These results strongly suggest the existene of a gap between polymerization and rystallization of hitin hains. In a previous report [l] we have shown that hitin in growing apies of Shizophyllum ommune has properties different from those of hitin in lateral walls. The newly synthesized hitin is extremely suseptible to a hitinase from Serratia marexens and is largely soluble in dilute mineral aid. The ensuing gradual loss of these properties was asribed to ovalent linkage of water-soluble (1 + 3)-b-gluan hains to hitin, resulting in an alkali-resistant hitin - P-gluan omplex and to rystallization of unsubstituted hitin hains. If polymerization and rystallization of hitin were simultaneous proesses, the proposed transitions at the apex of growing hyphae would be diffiult to explain. However, inreasing evidene is available favouring the view that a gap exists between polymerization and rystallization of mirofibrillar wall omponents. Stains like Calofluor White and Congo Red, whih bind to ellulose, hitin and other P-linked polysaharides [2-41 have been espeially used to inhibit ordered assembly of ellulose [5-101 and hitin hains [ll, 121. In growing hyphal tips whih bind Calofluor White [13] the effets may be omplex, beause of the presene of other ell-wall polymers that interat with hitin [14, 151. To avoid this ompliation the effets of hitinase and Calofluor were studied in an in vitro system derived from S. ommune, whih allows for the sythesis of rystalline mirofibrillar hitin [16]. MATERIALS AND METHODS Culture onditions Basidiospores from S. ommune inoulated at a onentration of 7 x 10' ml- ' in minimal medium were grown for 36 h at 25 "C as desribed before [16, 171. Correspondene to C. A. Vermeulen, Vakgroep Plantenfysiologie, Biologish Centrum, Rijksuniversiteit Groningen, 30 Kerklaan, NL NN Haren, The Netherlands Abbreviations. GlNA, N-aetyl-D-gluosamine ; UDP-GlNA, UDP-N-aetylgluosamine; (GICNAC)~, aetylhitobiose. Enzymes. Chitin synthase or Uridine-5'-diphosphate-2-aetamido - 2-deoxy - D - gluose : hitin p - aetamidodeoxygluosyl- transferase (EC ); hitinase or poly[l,4-(2-aetamido-2- deoxy)-b-d-gluoside]glyanohydrolase (EC ). Preparation of a mixed membrane fration Myelium, harvested by entrifugation, was washed with 0.01 M Tris/HCl, ph 7.5 ontaining 2 mm EDTA and 0.6 mm phenylmethylsulfonyl fluoride (isolation buffer) and broken in a X-press (HP 25, BIOX, Stokholm, Sweden). After thawing and low-speed entrifugation (350 x g for 15 min) the supernatant fration was entrifuged at x g for 1 h at 4 C. The pellet was resuspended in 0.01 M Tris/ HCl ph 7.5 ontaining 10 mm MgC12 (inubation buffer) and again entrifuged at x g for 1 h at 4 C on a ushion of inubation buffer ontaining 65% surose and 25% glyerol. The material aumulating on and partly in the surose ushion was withdrawn with a syringe leaving behind residual ell-wall fragments on the bottom of the tube. The isolated mixed membrane fration was diluted approximately ten times with inubation buffer and stored at -80 C in 2-ml samples. Chitin synthase assay Before initiation of the hitin synthase assay thawed samples of mixed membrane fration were inubated with 0.3% digitonin for 15 min (unless indiated otherwise). The hitin synthase system ontained 2 vol. mixed membrane fration and 1 vol. substrate (6 mm UDP-GlNA) plus 60 mm GlNA in inubation buffer supplemented with UDP-[U- ''C]GlNA (10.4 GBq mmol-') to a final speifi ativity of 3-6 Bq nmol-'. In assays set up to determine the breakdown by hitinase of briefly labelled hitin, the hitin synthase ativity was first bloked by addition of 1 vol. inubation buffer ontaining 200 pg ml- ' Polyoxin D and 0.5% Nonidet P40 before adding the hitinase. Inubations were at 26 "C and the reations were terminated by addition of aeti aid to 0.6 M. The preipitated material was olleted on Whatman GF/A glass-fiber diss and washed with a mixture (80: 20) of 1 M aeti aid and 96% ethanol and finally with 96% ethanol. The filters were dried and the radioativity was determined in 5 ml toluene ontaining 0.4% (w/v) diphenyloxazole (PPO).

2 41 2 Thin-layer hromazography Chitinase-degraded 14C-labelled hitin (3 p1) was hromatographed on ellulose thin-layer plates with pyridine/ethyl aetate/aeti aid/water (5 : 5 : 1 : 3 by vol.) and the plates were sanned for radioativity (Berthold Sanner, FRG). X-ray dgiiration analysis For X-ray analysis of the produts of synthesis, reation mixtures were saled up to 7.5 ml and non-radioative UDP- GlNA was used. Synthesis in the presene of Calofluor White M2R was done by adding 50 pg ml- ' Calofluor at the start of synthesis and 20 pg ml-' at 15-min intervals during the ourse of a 2-h period of synthesis. Reations were terminated by adding 1 vol. inubation buffer ontaining Polyoxin D (200 pg ml-') and 0.5% Nonidet P40. Insoluble material was olleted by entrifugation, washed three times with inubation buffer ontaining 1% digitonin to redue the lipid ontent, and finally washed with distilled water. X-ray analysis of wet and dry material was done by Ni-filtered Cu-K irradiation through a pinhole 40 mm long and 0.5 mm wide. In order to keep samples wet during irradiation a small ylinder of multilayered paper was plaed over the samples. After wetting the inside of the ylinder with water, the ylinder was losed with a thin plasti foil. Fig. 1. Effet of hitinose on the synthesis ofhitin. Inubation mixtures (1 50 J) ontaining various onentrations of hitinase were inubated at 26 C for 5 min in the presene (0-0) or absene (0-0) of 0.3% digitonin I UDP-GLNAr I 1 1GLNA)z n :I Chemials Chitinase from S. maresens was purhased from Serva (Heidelberg, FRG), UDP-[U-'4C]GlNA (10.4 GBq mmol- ') from the Amersham lnternational (UK) and Calofluor White M2R from Polysienes In. (Warrington, PA, USA). Polyoxin D was a gift from the Kaken Co. (Tokyo, Japan). Crustaean hitin from Fluka (Buhs, Switzerland) was purified aording to Skujins et al. [18]. C ) v/ - RESULTS Sensitivity of hitin for hitinase Conformational hanges ourring in hitin during and after synthesis, for instane rystallization of individual hitin hains, are likely to interfere with the proess of degradation of hitin by hitinase. Fig. 1 shows that the amount of hitin synthesized an be redued to almost zero by the presene of high onentrations of hitinase, provided that digitonin is present. In the absene of digitonin approximately 25% of the hitin was apparently inaessible to hitinase, possibly beause of the synthesis of hitin within losed membrane vesiles. The lowered amount of hitin is most probably due to degradation of hitin in the hitin assay mixture beause diaetylhitobiose is produed both when hitinase is present during synthesis and when preformed hitin is inubated with hitinase (Fig. 2). If the redution in net synthesis of radioative hitin by added hitinase ourred by degradation of not yet rystallized hitin hains, the addition of non-radioative amorphous hitin but not of rystalline hitin should ounterat the effet of hitinase. To avoid the diffiulty of preparing non-rystalline hitin, this predition was tested in the following experiment. Chitin was synthesized with non-radioative UDP-GlNA as a substrate. At various times during the period of synthesis, whih lasted for 90 min, a sample was provided with radioative UDP-GlNA and the rate of synthesis was determined both in the absene and presene of a fixed onentration of hitinase. The inhibition by hitinase of the appearane of radioative hitin should then depend on the amount of non-rystalline hitin present at the time of measurement whereas the aumulated rystalline hitin should have little effet. Fig. 3 shows that the hitin whih effetively ompetes for hitinase during synthesis of radioative hitin aumulates during the first 5-10 min of synthesis, then remains at a onstant level for some time, although the total amount of hitin inreases, and delines when the rate of synthesis falls after 40 min. Apparently, in the first 10 rnin the rate of synthesis exeeds the rate of rystallization so that non-rystalline hitin aumulates. During the period

3 41 3 B nl -... *, I n TIME lminl Fig. 3. Effet of preformed hitin on the rat of hitin synthesis in the presene of hitinase. Two bathes of mixed membrane fration were simultaneously inubated with non-radioative substrate ontaining GlNA. One bath (1.2 ml) was supplemented with 1 pm UDP-[U- ''C]GlNA at t = 0 min to determine the aumulation of hitin over a period of 90 rnin (A). From the other bath (3 ml) samples (150 pl) were periodially taken and inubated with radioative substrate for 5 min in the presene or absene of 20 pg ml-' hitinase. The residual substrate onentration at sampling times was alulated, permitting estimation of the rate of synthesis during the 5-min measuring periods (B) and the redution by hitinase of the amount of hitin synthesized (C) I s-1 $-l-o Fig. 4. Kinetis o j hitin degradation. Radioative hitin was synthesized for 2.5 min in 150-p1 samples and the reation was stopped by adding 1 vol. inubation buffer ontaining Polyoxin D and Nonidet P40. Immediately or 90 min later, samples were diluted with the same buffer to obtain various onentrations of hitin and inubated for 5 min with 125 p1 ml-' hitinase. The rate of hitin degradation (V equals the differene between initial and residual hitin ontent) was determined at the various substrate onentrations (S). (0-0) Degradation immediately after synthesis; (0-0) degradation 90 rnin after termination of synthesis up to 40 rnin the rates of synthesis and rystallization are equal so that the amount of non-rystalline hitin remains at a onstant level. It then falls off when the rate of synthesis dereases but rystallization ontinues at the same rate. That hitin after its synthesis beomes gradually more resistant to hitinase an also be shown by inubating the hitin with hitinase immediately or 90 rnin after synthesis. The kinetis of suh degradations by hitinase are presented as Lineweaver-Burk plots in Fig. 4. Immediately after synthesis the veloity of breakdown by hitinase is approximately two times higher than 90 min later, indiating that in the absene of synthesis the hitin beomes less suseptible to hitinase as time proeeds. Chitin olleted after synthesis for 90 min appeared to have about the same low suseptibility to hitinase as hitin synthesized for 2.5 min and then left for 90 min (results not shown). Effet of Calofuor White on the synthesis and enzymati degradation of hitin On the basis of the foregoing results it is reasonable to suggest that hitin after being synthesized exists for a short period in a onfiguration of non-rystallinity and high suseptibility to hitinase. If this is orret one might expet that in the presene of Calofluor White the initial high sensitivity of hitin for degradation by hitinase is maintained for at least a longer period of time. As has been noted by Calofluor (,og ml-1) Fig. 5. Exfet of Calofluor White on hitin synthesis. A mixed-membrane fation was inubated for 5 min with substrate and various onentrations of Calofluor White in a total volume of 150 MI others [19, 201 Calofluor inhibits the synthesis of hitin in vitro (Fig. 9, although the degree of inhibition was somewhat variable depending on the ativity of the membrane preparation. To measure the effet of the presene of Calofluor during synthesis of hitin, onditions were hosen suh that synthesis was only slightly inhibited (18%). Fig. 6 shows that hitin synthesized in the absene of Calofluor is rather suseptible to hitinase immediately after synthesis but this suseptibility dereases with time. Chitin synthesized in the presene of Calofluor is even more sensitive to hitinase as it is almost ompletely degraded by hitinase. In addition, the high suseptibility to hitinase does not derease with time. Calofluor added after termination of the synthesis of hitin has no influene on the suseptibility towards hitinase.

4 Time after synthesis (rnin) Fig. 6. Ejyet qf Calofluor White on the suseptibility ujhitin towards hitinase. Mixed membrane frations ontaining 0.15% digitonin were inubated for 2.5 rnin with radioative UDP-GlNA in the presene or absene of Calofluor (100 Fg m1-l). Chitin synthesis was then inhibited by dilution with 1 vol. inubation buffer ontaining Polyoxin D and Nonidet P40. After various time intervals hitinase (125 pg rn1-l) was added and the inubation was ontinued for 5 min in the absene or presene of Calofluor (100 pg ml-'). (.----a) No Calofluor present; (0-0) Calofluor only present after synthesis; (A-A) Calofluor present during and after synthesis amounts of suseptible hitin ould not be synthesized by simply inreasing the reation time and adding large amounts of Calofluor at the start of the reation. High onentrations of Calofluor inhibited hitin synthesis while the addition of lower onentrations led to a rapid deline in the onentration of free Calofluor as a result of binding to hitin. Therefore, the synthesis of hitin was allowed to proeed for 2 h with Calofluor added periodially as desribed in Materials and Methods. The hitin synthesized in the presene of Calofluor appeared to be extremely sensitive to hitinase provided it was kept in a wet state (75% degradation by 125 pg ml- ' hitinase in 5 min). After drying the suseptibility to hitinase was strongly redued and beame similar to that of hitin synthesized in the absene of Calofluor (25% degradation by 125 pg ml-' hitinase in 5 min). In the latter ase drying had no effet on the suseptibility towards hitinase. The X-ray diffration pattern of hitin made in the absene of Calofluor shows disrete lines of rystalline a-hitin both in the wet state (Fig. 7D) and after drying (Fig. 7E). This pattern differs somewhat from that of rustaean hitin (Fig. 7A), but we have previously [16] shown that treatment of hitin made in vitro with 1 M KOH for 20min at 60 C leads to a pattern idential to that of rustaean hitin. No suh refletions are present in the ase of hitin synthesized in the presene of Calofluor as long as the material is kept in a wet state (Fig. 7 B). After drying, however, this hitin also shows the refletions typial for rystalline hitin (Fig. 7C). > am Crystal spaings (nm) Fig. I. X-ray diffration analysis of hitin. Inubation mixtures ontaining 0.1 5% digitonin were inubated with non-radioative substrate in the presene or absene of Calofluor White as desribed in Material and Methods. (A) Crustaean hitin; (B) wet material synthesized in the presene of Calofluor; (C) same material as in (B) after drying; (D) wet material synthesized in the absene of Calofluor; (E) same material as in (D) after drying X-ray diffration of hitin synthesized in the presene of Calofluor White The ability of Calofluor White to maintain hitin in a state of high suseptibility to hitinase opened the possibility of synthesizing enough material for a X-ray diffration analysis. In preliminary experiments it was found that large DISCUSSION The present study strongly suggests that hitin after its synthesis by membraneous preparations is initially present in a non-rystalline state and that rystallization is a seondary proess. The results show that immediately after synthesis the hitin is very suseptible to hitinase degradation, whih has been interpreted as indiating its non-rystalline nature [ This is followed by rystallization of the hains as shown by a derease in the suseptibility towards hitinase and the appearane of X-ray defletions. In the presene of Calofluor the non-rystalline state an be maintained if the preparation is not dried down. The experimental data do not permit firm onlusions with respet to the length of the gap between polymerization and rystallization. The results of the ompetition experiment (Fig. 3) suggest that 10 rnin after the start of synthesis the amount of non-rystalline hitin reahes a steady-state level set by the rates of synthesis and rystallization of hitin. Chitin synthesized in the presene of Calofluor for 2.5 min is signifiantly more suseptible to hitinase than hitin synthesized in the absene of Calofluor (Fig. 6). Probably, onformational hanges leading to a redution in suseptibility of hitin for hitinase already start within a 2.5-min period of synthesis. This is supported by the observation that addition of Calofluor immediately after synthesis does not prevent the gradual redution in sensitivity to hitinase as observed when hitin is synthesized in the absene of Calofluor. Therefore, it is tempting to suggest that immediately after synthesis of hitin hains some hydrogen bonding ours, thereby reduing the suseptibility towards hitinase and preventing interation with Calofluor. Only when Calofluor is present during synthesis of the hains may hydrogen bonding between hains be ompletely prevented leading to a more or less permanent state of high suseptibility towards hitinase. Subsequent drying may hange the balane between the interation of hitin hains with Calofluor and the formation

5 41 5 of hydrogen bonds between the hains in favour of the latter, resulting in rystallization. A similar effet of drying has been observed in the ase of ellulose synthesized in the presene of Calofluor White [6]. The in vitro experiments show that synthesis and rystallization of hitin an be dissoiated. This agrees with the reported high suseptibility of hitin towards hitinase at its site of synthesis at the growing hyphal apex [l]. This hitin synthesized at the apex ontinuously moves in subapial diretion to beome part of the mature ell wall while aquiring resistane towards hitinase. The simultaneous extrusion at the apex of large amounts of soluble P-gluans into the wall [26] may further hamper assoiation of hitin hains and thus extend the period of non-rystallinity of hitin. This explains how during this period enzymes in the wall an attah soluble (3 -i 3)-P-gluan hains to the hitin hains resulting in insolubilization of the gluan [15, 271. Combined with partial rystallization of the (substituted) hitin hains and hydrogen bonding between the gluan hains this proess has been suggested to transform a viso-elasti wall at the apex in a rigid ross-linked polymer assemblage as present in the tubular wall [26, 281. We thank Dr J. H. Sietsma for performing the X-ray analysis. REFERENCES 1. Vrmeulen, C. A. & Wessels, J. G. H. (1984) Protoplasma 12, Maeda, H. & Ishida, N. (1967) J. Biohem. (Tokyo) 62, Hughes, J. & M Cully, M. E. (1975) Stain Tehnol. 50, Wood, P. J. (1980) Curbohydr. Res. 85, Benziman, M., Haigler, C. H., Brown, R. M., Jr, White, A. R. & Cooper, K. M. (1980) Pro. Natl Aad. Si. USA 77, Haigler, C. H., Brown, R. M., Jr, & Benziman, M. (1980) Siene (Wash. DC) 210, Quader, H. (1981) Nuturwissenshuften 68, Roberts, E., Seagull, R. W., Haigler, C. H. & Brown, R. M., Jr, (1982) Protoplusma 113, Shnepf, E., Deihgraber, G. & Herth, W. (1982) Protoplusma 110, Colvin, J. R. & Witter, D. E. (1983) Protoplusma 116, Herth, W. (1980) J. Cell. Bid. 87, Elorza, M. V., Rio, H. & Sentandreu, R. (1983) J. Gen. Mirobiol. 129, , 13. Gull, K. & Trini, A. P. J. (1974) Arh. Mirobiol. Y6, Sietsma, J. H. & Wessels, J. G. H. (1977) Biohim. Biophys. Ata 496, Sietsma, J. H. & Wessels, J. G. H. (1979) J. Gen. Mirobiol. 114, Vermeulen, C. A., Raeven, M. B. J. M. & Wessels, J. G. H. (1979) J. Gen. Mirobiol. 114, Vermeulen, C. A. & Wessels, J. G. H. (1983) Curr. Mirobiol. 8, Skujins, J. J., Potgieter, H. J. & Alexander, M. (1965) Arh. Biohem. Biophys. 111, Ronero, C. & Duran, A. (1985) J. Buteriol. 163, Selitrennikoff, C. P. (1984) Exp. Myol. 8, Molano, J., Durin, A. & Cabib, E. (1977) Anal. Biohem. 83, Molano, J., Polahek, I., Duran, A. & Cabib, E. (1979) J. Biol. Chem. 254, Correa, J. U., Elango, N., Polahek, I. & Cabib, E. (1982) J. Biol. Chem. 257, Lopez-Romero, E., Ruiz-Herrera, J. & Bartniki-Garia, S. (1982) Biohim. Biophys. Ata. 702, Zarain-Herzberg, A. & Arroyo-Begovih, A. (1983) J. Gen. Mirobiol. 129, Wessels, J. G. H., Sietsma, J. H. & Sonnenberg, A. S. M. (1983) J. Gen. Mirobiol. 129, Sietsma, J. H. & Wessels, J. G. H. (1981) J. Gen. Mirobiol, 125, Wessels, J. G. )I. (1986) Int. Rev. Cytol. 104,