224

Biochimica et Biophysica Acta, 571 (1979) 2 2 4 - - 2 3 2 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 68859

CHITIN SYNTHASE ACTIVITY FROM THE SLIME V A R I A N T OF NEUROSPORA CRASSA

C L A U D E P. S E L I T R E N N I K O F F *

Department of Basic Microbiology, Merck Institute for Therapeutic Research, Rahway, NJ 07065 (U.S.A.) (Received March 13th, 1979)

Key words: Chitin synthase activity; Slime variant; (N. crassa)

Summary Chitin synthase (UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-/3-acetamidodeoxy-D-glucosyltransferase, EC 2.4.1.16) activity from the wall-less variant of Neurospora crassa (slime) was partially characterized. The slime enzyme activity was found to be similar to that reported for slime-like and wild-type chitin synthase activities with respect to the following: specific activity, particulate cell-fraction localization, activation by N-acetylglucosamine, apparent Km with respect to substrate, pH optimum and ion requirement. It appears that the phenotype of slime cannot be solely accounted for by the absence of chitin synthase enzyme activity.

Introduction Interest in this laboratory centers around cell-wall assembly in the filamentous Ascomycete Neurospora crassa. Wild-type N. crassa under routine laboratory conditions grows as a mycelium whose hyphal cell walls contain 25--35% fl(1 -+ 3) glucan, 2--8% galactosamine polymer, 12--15% proteins and peptides, 15--20% undefined glucans and 10--15% chitin [1--8]. Chitin synthase (UDP-2-acetamide-2-deoxy-D-glucose :chitin 4-fl-acetamidodeoxyD-glucosyltransferase, EC 2.4.1.16) catalyses the reaction: uridine-5'-diphospho-N-acetylglucosamine (UDPGlcNAc) + acceptor -+ acceptor-GlcNAc + UDP and has been implicated as an essential activity for normal cell-wall biosynthesis n o t only in Neurospora [9--11] but also in a number of taxonomically diverse

* Present address: D e p a r t m e n t of Anatomy, University of C o l o r a d o Medical Center, D e n v e r , CO 8 0 2 6 2 , U.S.A.

225 fungi [12--17]. The general properties of the enzyme appear to be very similar amongst all fungi examined; e.g., Mg 2÷ requirement, activation by N-acetylglucosamine (GlcNAc), particulate fraction localization and inhibition by UDP and polyoxin [11--17]. Isolates of the slime variant Neurospora grow without the presence of detectable surface-bound cell-wall material, i.e., as populations of protoplasts [18--22]. Inquiries into the molecular basis of the slime p h e n o t y p e have led to an examination of cell-wall-polymer biosynthetic enzymes. This study reports the partial characterization of chitin synthase activity from the slime variant and suggests that the wall-less nature of slime cannot be simply accounted for by the absence of chitin synthase activity. Materials and Methods Chemicals Radioactive UDP[1-14C-GlcN]GlcNAc was purchased from New England Nuclear and ICN. Diacetylchitobiose and tetraacetylchitotetraose were generous gifts of Dr. D.R. Sonneborn (University of Wisconsin, Madison). Biochemicals were purchased from Sigma Chemical Company; other chemicals were of reagent grade and distilled water was used throughout. Growth o f N. crassa wild-type and slime strains The slime variant [fz (no number); sg {no number), arg-1 (B369), cr-1 {B123), aur (34508), os-1 (B135), A -- Fungal Genetics Stock Center No. 326] was obtained from Dr. R.L. Metzenberg (University of Wisconsin, Madison). This strain of slime grew as a homogeneous suspension of protoplasts. The wildtype used was 74-OR8-1a (FGSC 988). Cultures of the slime variant were grown using Nelson's Medium B in liquid shaken culture essentially as described by Nelson et al. [18]. Exponentially growing cultures were harvested by centrifugation (500 X g; 5 min) and washed twice with ice-cold fresh medium. Cell-pellets were quick-frozen in liquid N2 and stored at --70°C until needed. No detectable change in enzyme specific activity was noted for storage periods up to one month. Cultures of wild-type were initiated by conidial suspensions (final concentration 1 . 1 0 s conidia/ml) into 2-liter flasks containing 300 ml of Nelson's Medium B [18]. Flasks were shaken {160 rev./min) on a New Brunswick Shaker Table for 24 h at 28°C. Cells were harvested by centrifugation, washed twice with ice-cold distilled water, frozen in liquid N2 and stored at --70°C. Preparation o f particulate cell-fractions Cells of slime were quantitatively disrupted by thawing cell pellets in ice-cold 50 mM Tris-HC1 (pH 8.0), 1 mM MgC12. Crude lysates were centrifuged at 1000 X g for 5 min at 2°C. Supernatants were Centrifuged for 60 rain at 100 000 X g (average g) at 2°C; resulting pellets were resuspended in buffer by a brief {approx. 2--3 s) sonication treatment. Wild-type cells were disrupted by sonication {Ultrasonics Sonifier ® equipped with a microtip; 10 X 15 s bursts at 50 W with 90-s cooling periods between bursts) at 4°C with 25 ~m glass beads. Disruption was judged to be ~>95% (phase contrast microscopy). High-speed pellets were prepared as described above.

226

Enzyme assay Chitin synthase activity was monitored in 25-pl reaction mixtures containing 50 mM Tris-HC1 (pH 8.0), 1 mM MgC12, 12.5 mM GlcNAc, 3.3 mM UDP[1-14C]GlcNAc (19 000 dpm/mixture) and aliquots of resuspended highspeed pellets. Mixtures were incubated at 30°C and reactions terminated by boiling for 2--3 min. Reaction mixtures were separated by descending chromatography for 16--18 h using Whatman 1 paper and I M ammonium acetate (pH 7.5)/ethanol (3 : 7, v/v) as solvent. Chromatographically immobile radioactivity (chitin) was determined by liquid scintillation counting using Aquasol II (New England Nuclear). Chitin formation was linear with respect to time for 15 min and linear with respect to protein concentration (50--150 tLg/reaction mixture). Less than 10% enzyme activity was present after 24 h incubation at either 4°C or 30°C; enzyme activity remained stable for up to 2 weeks after high-speed pellets were quick-frozen in liquid N2 and stored at --70°C. Chitinase treatments For some experiments chromatographically immobile radioactivity was treated with chitinase (EC 3.2.1.14): dried areas from origins (1 cm X 2 cm) were immersed in water (5 ml) containing 3 units/ml chitinase, 100 ~g/ml chloramphenicol, 100 pg/ml streptomycin, 1000 units/ml Penicillin G. After treatment for 72 h at 28°C with shaking (160 rev./min) in sealed tubes, the samples were washed twice with distilled water and dried in scintillation vials. Solutions were pooled, lyophylized, resuspended in water and spotted on Whatman 1 paper and separated by chromatography as described above. Distribution of radioactivity was determined by liquid scintillation counting of chromatograms cut into 1 cm pieces. Miscellaneous procedures Protein was determined by the method of Lowry et al. [23], using bovine serum albumin (Fraction V) as standard. Enzyme activity is reported in units (nmol GlcNAc incorporated into chitin/min) at 30°C. Results

Chitin synthase activity in centrifugal fractions from wild-type and slime variant cells Exponentially growing cultures of wild-type and the wall-less variant, slime, were harvested, disrupted, and centrifugal fractions assayed for chitin synthetase activity. The results in Table I demonstrate that although enzyme activity was detected in all fractions tested for both slime and wild-type, the majority of the activity was recovered in high-speed particulate fraction (100 000 × g pellets). It is n o t e w o r t h y that a higher percentage of enzyme activity was recovered in 100 000 × g supernatants derived from slime cells (30%) than that recovered in supernatant fractions derived from wild-type cells (9%). Further, it was observed that the specific activities of wild-type and slime crude extracts were not significantly different.

227 TABLE I D I S T R I B U T I O N O F C H I T I N S Y N T H A S E A C T I V I T Y IN C E N T R I F U G A L AND WILD-TYPE EXTRACTS

FRACTIONS OF SLIME

C r u d e exi~acts o f slime and w i l d - t y p e cells w e r e p r e p a r e d as d e s c r i b e d . E x t r a c t s w e r e c e n t r i f u g e d at 1 0 0 0 X g f o r 5 rain at 2°C. R e s u l t i n g s u p e r n a t a n t s w e r e c e n t r i f u g e d a t 1 0 0 0 0 0 X g f o r 60 m i n a t 2°C. F r a c t i o n s w e r e a s s a y e d f o r c h i t i n s y n t h a s e a c t i v i t y and e n z y m e units n o r m a l i z e d t o a c c o u n t for t h e diff e r e n c e in p r o t e i n c o n t e n t b e t w e e n w i l d - t y p e and slime e x t r a c t s . Cell-type

Wild-type

Slime

Fraction

Chitin s y n t h a s e a c t i v i t y Total units

% Distribution

supernatant pellet supernatant pellet

159 133 21 10 107

-86 14 9 91

Crude lysate 1 000 X g supernatant 1 0 0 0 X g pellet 100 000 X g supernatant 100 000 X gpeBet

160 126 30 33 77

-80 20 30 70

Crude lysate 1 000 X g 1 000 X g 100 000 X g 100 000 X g

Formation of chitin by chitin synthase activity from high-speed particulate fractions of slime cells Reaction mixtures containing aliquots of resuspended 100 000 × g pellets derived from slime cells and UDP[ 14C] GlcNAc as substrate were incubated for various times and, after inactivation, mixtures were separated by descending chromatography. Three separate peaks of radioactivity were found when the distribution of radioactivity in chromatograms was determined. One peak corresponded to UDP[14C]GlcNAc (unincorporated substrate), another was chromatographically immobile (chitin; see below) while the third was at an R F GlcNAc of approximately 0.9. This peak had less than 10% of the radioactivity found in chitin and was not investigated further *. Only the UDP[ ~4C] GlcNAc peak was present in zero-time controls and in mixtures containing boiled extract. No radioactive GlcNAc was detected in mixtures containing active extracts. That the chromatographically immobile radioactivity was ['4C]GlcNAc incorporated into chitin was determined by treating isolated areas from origins with chitinase. Greater than 98% of the radioactivity was rendered soluble by treatment with chitinase while < 1 0 % was solubilized in treatments lacking chitinase; greater than 97% of the solubilized radioactivity comigrated with authentic diacetylchitobiose.

Properties of slime chitin synthase activity A pH o p t i m u m of pH 8.0--8.2 for slime chitin synthase activity was determined b y lysing cells in buffer adjusted to various pH (ranging from pH 7.2-8.5) and assaying resuspended pellets for chitin synthase activity. Essentially

* This p e a k m o s t l i k e l y r e p r e s e n t s a m i x t u r e o f c h i t o d e x t r i n s [ 1 6 ] .

228

identical results were obtained when cells were lysed in pH 8.0 buffer and 100 000 X g pellets resuspended in buffers adjusted to various pH. Slime chitin synthase activity was found to be active in vitro without added metal divalent cations but maximum activity occurred in the presence of 1 mM MgC12. Neither Mn 2+ nor Ca 2+ could effectively substitute for Mg 2÷. That chitin synthase activity may exist in extracts as an ion-enzyme complex is suggested by complete inhibition of activity by 10 mM EDTA. The effect on enzyme activity of varying substrate (UDPGlcNAc) concentrations was determined at four GlcNAc concentrations and the data plotted by the standard Lineweaver-Burk method (Fig. 1). Note that GlcNAc stimulates enzyme activity and there exists a non-linear relationship between reciprocal velocity and reciprocal substrate concentration at each of GlcNAc concentrations tested; only at a high GlcNAc concentration (12.5 mM) was a linear relationship approximated. By extrapolating the linear portions of the curves (high substrate concentrations) it was found that the apparent Km for UDPGlcNAc was related to GlcNAc concentration (Fig. 2). When the data in Fig. 1 were replotted as reciprocal velocity versus reciprocal GlcNAc concentration, it was found that the x-axis intercepts (--Ka GlcNAc) were related to substrate concentrations.

Specificity of activation of slime chitin synthase activity by GlcNAc Various compounds structurally related to GlcNAc were added to reaction mixtures containing resuspended high-speed particulate fractions and mixtures I

I

I

I

I

18

I o o

14. 10 L 6 >

I0

I

I

I

[

I

I

I 2

I 4

1 6

I 8

I I0

I 12

II

8

g

0

I O2:

I 0,4

I 0.6

I 0.8

I/S (ram -I)

I 1.0

I 1.2

6

~ 1.4

GlcNAc

14

(raM)

Fig. 1. E f f e c t o f v a r y i n g s u b s t r a t e ( U D P G l c N A c ) c o n c e n t r a t i o n s a t f i x e d G l c N A c c o n c e n t r a t i o n s o n s l i m e c h i t i n s y n t h a s e a c t i v i t y . R e s u s p e n d e d h i g h - s p e e d p a r t i c u l a t e f r a c t i o n s w e r e i n c u b a t e d in m i x t u r e s c o n t a i n i n g the i n d i c a t e d U D P G l c N A c c o n c e n t r a t i o n s a n d t h e f o l l o w i n g G l c N A c c o n c e n t r a t i o n s : o o, none; e g, 3.1 raM; • A 6,25 raM; A ~, 1 2 . 5 m M . A f t e r i n c u b a t i o n f o r 7.5 a n d 15 r a i n at 3 0 ° C , t h e a m o u n t o f c h i t i n f o r m e d w a s d e t e r m i n e d , v e l o c i t y ( n m o l p r o d u c t / r a i n ) c a l c u l a t e d a n d the data r e p l o t t e d in the d o u b l e r e c i p r o c a l f o r m . Each p o i n t r e p r e s e n t s an i n d i v i d u a l d e t e r m i n a t i o n . T h e x-axis i n t e r c e p t s (--Kin) were d e t e r m i n e d by linear regression analysis of the linear p o r t i o n s of the curves. F i g . 2. D e p e n d e n c e o f a p p a r e n t K m o f s l i m e c h i t i n s y n t h a s e a c t i v i t y o n G l c N A c c o n c e n t r a t i o n . T h e x - a x i s i n t e r c e p t s ( - - K i n ) in Fig. 1 w e r e p l o t t e d as f u n c t i o n o f G l c N A c c o n c e n t r a t i o n .

229 T A B L E II EFFECT OF VARIOUS SUGARS ON SLIME CHITIN SYNTHASE ACTIVITY R e a c t i o n m i x t u r e s c o n t a i n i n g a l i q u o t s o f r e s u s p e n d e d slime h i g h - s p e e d pellets, 3.3 m M U D P [ 14 C] G l c N A e and the indicated additions were incubated for 15 min and the radioactivity incorporated into chitin determined. Additions to reaction mixture

Radioactivity incorporated (cpm)

None 12.5 mM N-acetylmannosamine 12.5 mM N-acetylgalactosamine 12.5 mM glucosamine 12.5 mM galactosamine 12.5 raM glucose 12.5 mM galactose 12.5 mM GlcNAc 10 mM diacetylchitobiose 10 mM tetraacetylchitotetraose 10 mM diacetylchitobiose, 10 mM tetraacetylchitotetraose 12.5 mM GlcNAc, 10 mM diacetylchitobiose 12.5 mM GIcNAc, 10 mM tetraacetylchitotetraose 12.5 mM GlcNAc, 10 mM diacetylchitobiose, 10 mM tetraacetylehitotetraose

185 245 130 270 215 215 170 1140 325 530 500 1270 1290 1320

assayed for chitin synthase activity. The results presented in Table II demonstrate that none of the compounds tested was as effective as GlcNAc in stimulating slime chitin synthase activity. However, diacetylchitobiose and tetraacetylchitotetraose stimulated enzyme activity by 1.7 and 3-fold, respectively. The presence of 10 mM tetracetylchitotetraose in reaction mixtures did not alter the effects of varying substrate concentrations at different GlcNAc concentrations on enzyme activity, i.e., non-Michealis-Menten enzyme kinetics observed in Fig. 1; however, the presence of tetraacetylchitotetraose in reaction mixtUres altered the effect on enzyme activity of varying GlcNAc concentrations at fixed substrate (UDPGlcNAc) concentration. The Ka GlcNAc was found to be independent of substrate concentration and was estimated to be 2.6 mM.

T A B L E III EFFECT OF NUCLEOSIDE PHOSPHATES ON SLIME CHITIN SYNTHASE ACTIVITY R e a c t i o n m i x t U r e s c o n t a i n i n g 1 2 . 5 m M G l c N A c , 3 . 3 m M U D p [ 1 4 C ] G l c N A c , r e s u s p e n d e d slime h i g h speed pellets, and the indicated concentrations of each of the listed compounds were incubated for 15 rain and the radioactivity incorporated into chitin determined. Addition

Radioactivity incorporated (cpm)

None 2 mM 5 mM 5 mM 2 mM 5 mM 2 mM 5 mM

1115 690 115 460 850 440 910 225

UTP UTP UDP UMP UMP ATP ATP

230 J

I

40

/

28 24 I/V

20 16

K i ~2,2raM

0

I I

I

I

./

56 32

I

I 2

J ,5

I 4

I 5

UDP (mM)

Fig. 3. C o m p e t i t i v e i n h i b i t i o n o f c h i t i n s y n t h a s e a c t i v i t y b y U D P . R e a c t i o n m i x t u r e s c o n t a i n i n g t h e i n d i c a t e d U D P c o n c e n t r a t i o n s and t h e f o l l o w i n g U D P G l c N A c c o n c e n t r a t i o n s : • e, 3.3 raM; o o, 2 . 5 raM; A A 0 . 8 m M ; w e r e i n c u b a t e d f o r 7 . 5 a n d 15 m i n . T n e a m o u n t o f c h i t i n f o r m e d was d e t e r m i n e d and the results were plotted by the m e t h o d of D i x o n [25]. Each point represents an individual determination.

Effect o f various phospholipids and nucleoside phosphates on slime chitin synthase activity Phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine and phosphatidylinositol (25 ~g/reaction mixture) were each tested for their ability to stimulate chitin synthetase activity (see ref. 15). Phosphatidylinositol stimulated enzyme activity by 4.5 fold while phosphatidylserine and phosphatidylethanolamine increased activity by 2.8 and 1.7 fold, respectively; phosphatidylcholine was without significant effect. Whether stimulation of activity by phospholipids (and especially phosphatidylinositol) is due to stabilization of enzyme activity, to solubilization of enzyme activity rendering it more available to substrate or to the provision of a limiting lipid acceptor (intermediate?) is not known. The indicated concentration of the nucleoside phosphates listed in Table III was added to reaction mixtures and mixtures assayed for chitin formation. All compounds tested inhibited enzyme activity. Since UDP is a product of chitin synthetase activity, inhibition by UDP was investigated further. The data in Fig. 3 show that UDP behaved as a competitive inhibitor (nomenclature of ref. 24) with respect to the substrate, UDPGlcNAc. An inhibition constant (Ki) of 2.2 mM was graphically determined (average Ki from 3 experiments was 2.4 + 0.2 mM). Discussion

Cell wall construction in Neurospora crassa and other filamentous fungi occurs by deposition of carbohydrate polymers and other materials at the

231 growing hyphal apex [26--33]. Although many mutants exist which confer aberrant morphology upon the organism as well as a chemically altered cell wall [see Refs. 1, 34, 35], analysis of such mutants has shed little light upon the molecular details of cell-wall assembly. Since the variants slime and its derivative, slime-like [20,36], possess extreme phenotypes, i.e., grow a protoplasts devoid of surface-bound cell-wall material, it was suspected that the molecular block(s) in these strains might be at a key point in cell-wall biosynthesis, perhaps, even at an initial step. From a number of studies using a variety of filamentous fungi it appears that one of the first polymers to be laid down is chitin and that chitin synthase is an essential enzyme activity for continued cell-wall biosynthesis (see Ref. 33). Thus the molecular defect responsible for the slime p h e n o t y p e could have been an absence of chitin synthase activity. However, chitin synthase activity of slime and slime-like resembles that described from a number of fungi and that of wild-type Neurospora crassa (this report, and Refs. 11 and 36). All three enzyme activities were primarily recovered in particulate fractions of cell lysates and the specific activities of crude extracts were not significantly different from those of wild-type [11,36]. That a larger percentage of slime and slime-like enzyme were recovered in 100 000 × g supernatants than that recovered for wild-type {Table I; Ref. 36) may be a direct consequence of the wall-less nature of these mutants. N-Acetylglucosamine acts as a specific allosteric effector of slime variant (Fig. 1 and Table II), slime-like and wild-type enzyme activities [11,36]. All three enzyme activities are stimulated by Mg 2÷ [11,36] and those from wild-type and slime were inhibitable by polyoxin, an analogue of UDPGlcNAc (Refs. 9, 10 and Selitrennikoff, C.P., unpublished results). The pH o p t i m u m of the slime enzyme was pH 8.0--8.2, that for the slime-like enzyme was pH 7.8 [36] and that for wild-type has been variously reported to be pH 7.5 [11] and pH 7.8 [36]; in light of the differences in experimental conditions between these determinations, it is likely that these differences are not significant. The apparent ~Km with respect to UDPGlcNAc for wild-type enzyme activity has been reported to be 1.4 mM [9], 2.1 mM [11], and 4 mM [36]. The apparent Km for the slimelike enzyme was 3.7 mM [36] while that for the slime enzyme was found to be dependent upon GlcNAc concentration (Fig. 2); the Km was 2.2 mM when extrapolated to be the conditions used for wild-type [11]. Further, the slime enzyme shares with other fungal chiti.n synthase the following properties: competitive inhibition by UDP [15,17,28], and stimulation of activity by phospholipids [15]. Taken together these data point to a general similarity of chitin synthase enzyme activity from slime, slime-like and wild-type. The presence of active chitin synthase in extracts from slime and slime-like does not exclude the possibility that in vivo the enzyme is inactive. The lack of enzyme activity could be the result of a block in precursor biosynthesis (hexosamine biosynthetic pathway) or to a defect in the activation of chitin synthase (see Refs. 14, 37--39), for activation of zymogenic chitin synthase activity by intra-cellular proteases). These notions seem excluded b y the following observations: In slime-like the enzymes of the hexosamine biosynthetic pathway were found to be present at essentially the same specific activities as found in wild-type [36] and the pool sizes of hexosamine intermediates and UDPGlcNAc were similar to those found in wild-type [36].

232

Finally, when cultures of slime were grown in the presence of [14C]GlcN, radioactively labeled chitin was formed (Selitrennikoff, C.P., unpublished, and Braymer, D., personal communication). It seems unlikely that the wall-less nature of slime and slime-like can be accounted for by an absence of chitin synthase acitivity.

Acknowledgements I would like to thank Dr. D.R. Sonneborn who introduced me to chitin and chitin synthase and special thanks to Dr. E.L. Dulaney for helpful discussions and critically reading the manuscipt.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 30 31 32 33 34 35 36 37 38 39

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