FEMS Microbiology Letters 120 (1994) 169-176 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier

169

FEMSLE 06049

Effect of tunicamycin on a-galactosidase secretion by Aspergillus nidulans and the importance of N-glycosylation

S a n t i a g o R ios , I n m a c u l a d a F e r n f i n d e z - M o n i s t r o l a n d F e r n a n d o L a b o r d a *

Departamento de Microbiologfa y Parasitologfa, Facultad de Farmacia, Universidad de Alcald de Henares, Campus Universitario, 28871 Alcald de Henares, Madrid Spain

(Received 3 March 1994; revision received 12 April 1994; accepted 25 April 1994)

Abstract: Aspergillus nidulans released a-galactosidase into the culture medium during the exponential growth on either lactose or galactose as the only carbon source. This enzyme is a glycoprotein. Its treatment with endoglycosidases produces a reduction in its molecular mass but the resulting enzyme conserved some of their carbohydrate components in addition to its enzymatic activity. Mycelia of A. nidulans growing in the presence of tunicamycin synthesized an underglycosylated a-galactosidase which was not released into the culture media but remained bound to the cell-wall. Tunicamycin did not prevent the synthesis and secretion of a-galactosidase by protoplasts. N-linked oligosaccharide chains seem not to be essential for the synthesis and secretion of a-galactosidase of A. nidulans, but they could be necessary for proper targeting at the extracellular level.

Key words: u-galactosidase; Aspergillus nidulans; Secretion

Introduction

a-Galac tos idase (a-D-galactoside galactohy- drolase; E.C. 3.2.1.22) is an enzyme widely dis- t r ibuted among micro-organisms that is able to hydrolyze a variety of simple a-galactosides as well as more complex polysaccharides [1]. a - Galactosidase f rom Aspergillus nidulans has been chosen as a model of extracellular hydrolases to study prote in secret ion in f i lamentous fungi [2]. Extracellular enzymes (as are many o ther se-

* Corresponding author. Tel: 34 (01) 885 46 21; Fax: (01) 885 46 60.

cre ted proteins) are glycosylated molecules. N- glycosylation is one of the major post-transla- t ional modificat ions which occurs when the pro- tein molecules are t ranspor ted th rough the sub- cellular compar tments : endoplasmic ret iculum and Golgi apparatus. The addit ion of the glycan moiety is of key impor tance to the propert ies of glycoproteins, ensuring their stability and correct processing th rough the secretory pathway [3]. However , prote in glycosylation in f i lamentous fungi is still poorly defined and little is known about its influence on prote in secretion.

Tunicamycin (TM) is a nucleot ide antibiotic which inhibits the t ransfer of N-acetyl glu- cosamine phospha te to dolichol phosphate , the

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first step in the synthesis of N-linked oligosaccha- rides [4]. When both animal cells and eukaryotic microorganisms are treated with TM in vivo, they produce under-, or non-glycosylated proteins [5]. The in vitro treatment of glycoproteins with the Endo H enzyme (endo-/3-N-acetylglucosamini- dase H; E.C. 3.2.1.96)-which hydrolyses the bond between the two GIuNAc residues of the oligosaccharide core-also yields under- or non- glycosylated proteins. [6].

In this report, the glycoprotein nature of a- galactosidase purified from A. nidulans was stud- ied. Moreover, mycelial cells and protoplasts were treated with TM in order to obtain information about the physiological significance of the N-lin- ked carbohydrate chains on a-galactosidase se- cretion.

Materials and Methods

Organism and growth conditions A. nidulans 2.3, kindly donated by Prof. J.F.

Peberdy (Department of Life Sciences, University of Nottingham, UK) was grown in Aspergillus complete medium (ACM) [7] supplemented with either glucose or galactose at 20 g 1-1. A conidial suspension was inoculated into 100 ml Erlen- meyer flasks containing 20 ml of ACM to give a final concentration of 2 X 106 conidia m1-1. Cul- tures were incubated on a rotary shaker (200 rpm) at 28°C.

Fractionation of A. nidulans cultures Mycelia grown for 24 h in ACM medium were

recovered by filtration through nylon cloth. Cul- ture filtrates were used as an extracellular en- zyme solution. Mycelia were washed with distilled water, lyophilized and weighted. Then, they were homogenized in a mortar and resuspended, at a concentration of 50 mg m1-1, in 25 mM Tris/phosphate buffer (pH 6.7) plus 0.1 mM dithiothreitol, 1 mM EDTA and 1 mM phenyl- methyl sulphonylfluoride (PMSF). After eentrifu- gation (10,000 × g, 30 min, 4°C), the collected supernatant was considered as the soluble myeelial extract. The pellet was washed with lysis buffer until no malate dehydrogenase activity (taken as control for cytoplasmic contamination)

was detected. Finally it was resuspended in the same volume of lysis buffer and used as the cell wall fraction (insoluble mycelial extract).

Preparation and fractionation of protoplasts Protoplasts were obtained from mycelium of

A. nidulans grown for 18 h in ACM with 2 g ! -1 glucose according to Hamlyn et al. [8]. After 2 h of incubation in the lytic enzyme solution (5 mg m1-1 Novozym 234, 50 mM K2H2PO4, pH 6.5 and 0.7 M KCI) protoplasts were harvested, washed and transferred into a minimum medium [7] containing 2 g 1-1 galactose and 0.7 M KCI. After 6 h of incubation in this medium, they were recovered by centrifugation (2,000 x g for 10 min) and the supernatant was used as a solution of proteins secreted by protoplasts. Protoplasts were washed twice in 0.7 M KC1 and disrupted, using vigorous mixing by vortex, in distilled water. Lysed protoplasts were centrifuged at 18,000 × g for 60 min to separate the membrane fraction (pellet) from the cytosol (supernatant).

Tunicamycin treatment When A. nidulans mycelium was incubated in

the presence of TM, the antibiotic was added to the culture media, dissolved as a stock solution in 25 mM NaOH, to give 5 and 10 ~g m1-1 final concentration. Equivalent volumes of 25 mM NaOH were added to control flasks without TM. Cultures of A. nidulans grown for 24 h in the presence and absence of TM were filtered through nylon cloth. Culture filtrates were used as an extracellular enzyme solution. Mycelia were washed, homogenized, and divided in two frac- tions-the soluble mycelial extract and the cell wall fraction-as indicated above.

In other independent experiment, protoplasts from A. nidulans were incubated for 6 h in Aspergillus minimal medium [7], which contained galactose, in the presence and absence of 5 and 10/xg ml-1 TM. After incubation, samples were divided in protein secreted solution, membranes and cytosol as detailed above.

Thermostability study of a-galactosidase Enzyme thermostability was determined by in-

cubating the soluble mycelial extract prepared

from A. nidulans-which was grown both in the presence and absence of TM-at 55°C for differ- ent times. Then, residual a-galactosidase activity was measured in the enzymatic solution.

Endoglycosidase treatment of purified a-galac- tosidase

Purified a-galactosidase [2] was treated with Endo H. The native enzyme (0.4 mU per mg of protein) suspended in 50 mM sodium citrate buffer (pH 5.5) plus 1 mM PMSF was incubated with 20 mU Endo H ml-1 for different times at 37°C. In parallel assays, a-galactosidase was de- natured before the enzymatic treatment by boil- ing for 5 min in the same buffer added with 0.4 mg ml-1 SDS. The glycosidic nature of a-galac- tosidase obtained after the different treatments was examined by its ability to bind concanavalin A Sepharose gels (Con A) [2].

Analytical procedures Protein concentration of the different enzy-

matic samples was determined by the method of Lowry [9]. a-Galactosidase activity was assayed as previously described [2]. Malate dehydrogenase activity (E.C. 11.1.37) was measured spectropho- tometrically [10]; one unit was 1 Izmol of malate reduced per minute under the assay conditions.

Electrophoretic techniques Proteins were analysed by SDS-PAGE (12%

w/v acrylamide) [11] and by native-PAGE (7% w/v acrylamide). Gels were stained for proteins with 0.2 g 1-1 Coomassie brilliant blue R-350. a-Galactosidase on native gels was localized staining for enzymatic activity [12]. In some cases, electrophoretic transfer of proteins from SDS- PAGE to Immobilom P membranes (Millipore) were carried out and then proteins were devel- oped with anti a-galactosidase sera as previously described [2].

Materials Endo H and TM were from Sigma. Con A

Sepharose was from Pharmacia. Reagents for electrophoresis, Western blotting and immuno- staining were obtained from Bio-Rad. All other reagents used were of analytical grade.

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Results and Discussion

Endo [3-N-acetylglucosaminidase H treatment A. nidulans a-galactosidase purified from both

the culture filtrate and the mycelial extract are the same enzyme, as was previously determined [2]. This is a glycoprotein, because of its ability to bind Con A. Endo H digestion of the native enzyme produced a decrease of the molecular mass of the enzyme monomer, from 87 to 81 kDa, as observed by SDS-PAGE (Fig. 1A), indicating that some N-carbohydrate chains were released

A

1 2 3 4

- - - 94 - - 67

- - 4 3

B

1 2

Fig. 1. (A) SDS-PAGE of Endo H digested a-galactosidase purified from A. nidulans. The gel was stained for proteins. (B) Native electrophoresis of Endo H digested a-galactosi- dase. The gel was stained for enzymatic activity as described in Materials and Methods (Lane 1) Native a-galactosidase. (Lane 2) Native u-galaetosidase treated with Endo H. (Lane 3) Denatured a-galactosidase treated with Endo H. (Lane 4)

Molecular weight markers.

172

from the glycoprotein. When a-galactosidase was denatured and then digested with Endo H, the reduction on the monomer molecular mass was even higher than for the native enzyme (from 87 to 75 kDa) (Fig. 1A; lane 3). Thus, a great num- ber of N-carbohydrate chains appear to be re- leased when the denatured enzyme was digested with Endo H. These observations could signify that some N-oligosaccharide chains in the native molecule are inaccessible to Endo H hydrolysis, but become accessible when the native a-galac- tosidase is denatured [13]. Both, accessible and inaccessible N-oligosaccharide moieties were found at the same proportion, each representing approximately 7% of the total molecular mass.

a-Galactosidase treated with Endo H still con- served its enzymatic activity after native elec- trophoresis (Fig. 1B). If N-linked carbohydrate contributed to the proper folding, the enzyme digested with Endo H would be expected to be partially or completely inactive [14]. Thus, it can be deduced that the N-carbohydrate is not essen- tial for proper folding of this glycoprotein.

a-Galactosidase from A. nidulans digested with Endo H conserved its ability to bind Con A Sepharose gels, indicating that the enzyme was still glycosylated. The remaining carbohydrate is probably formed by O-linked oligosaccharide chains, which are not susceptible to be hydrolysed by Endo H [6].

Effect of tunicamycin on a-galactosidase secretion by the whole mycelium

A. nidulans growing in the presence of TM (5 and 10/zg m1-1) did not secrete a-galactosidase to the culture medium. Nevertheless, enzyme ac- tivity was detected at the cell wall and soluble mycelial extract (Table 1).

SDS-PAGE and electrobloting of A. nidulans proteins obtained after the fungal growth in the presence and absence of TM were revealed by antibodies versus a-galactosidase. Underglycosy- lated enzyme was detected in the soluble mycelial extract and the cell wall fraction and its elec- trophoretical mobility coincided with that of the enzyme digested with Endo H (Fig. 2). Antibod- ies did not detect a-galactosidase in the culture filtrate.

Table 1

Effect of Tunicamycin on a-galactosidase present in the cul- ture filtrate, cell wall fraction and soluble extract of mycelium from Aspergillus nidulans *

Tunicamicyn a-Galactosidase (/.t g ml - x) [mU (mg dry weight) - 1 ]

Culture filtrate Soluble Cell wall mycelial extract ~act ion

0 10 70 24 5 0 80 23

10 0 50 20

Data represents the mean values for three independent exper- iments; *A. nidulans was grown in ACM with increasing amounts of TM.

The lack of N-glycosylation, induced by TM, did not affect either a-galactosidase synthesis or their transport to the cell-wall, although it pre- vented its release to the culture medium.

a-Galactosidase synthesized by A. nidulans growing in the presence of TM was more rapidly inactivated at 55°C than the enzyme formed in the absence of TM (Fig. 3). Similar result was obtained for the enzyme digested with Endo H. Underglycosylated a-galactosidase is more sensi- ble to high temperature than the completely gly- cosylated enzyme, indicating some physiological role of the carbohydrate chains protecting the enzyme against elevated temperature [14].

Effect of tunicamycin on a-galactosidase secretion by protoplasts

The relative distribution of a-galactosidase ac- tivity among proteins secreted by protoplasts, cy- tosol and membranes obtained after lysis of pro- toplasts was studied. When TM was not present in the incubation medium, about 60% of the total a-galactosidase activity was found in the super- natant used as proteins secreted by protoplasts. Regarding the intracellular activity, 15% was de- tected in soluble form in the cytosol and almost 20% in the membranes (Fig. 4). The relative distribution of a-galactosidase activity was not modified when protoplasts were incubated in the presence of TM.

The high proportion of a-galactosidase found as secreted protein could indicate that A. nidu-

1 2 3 4 5 6 7 8 9 10 11 12

- - - - - - - - - - . , - - ~ ~ --6"/

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Fig. 2. Immunoblot analysis of a-galactosidase present in: the soluble mycelium extract (lanes 1, 2, 3); cell wall (lanes 4, 5, 6) and culture filtrate (lanes 9, 10, 11) from A. nidulans grown in the absence (lanes 1, 4, 9) and presence of 5 (lanes 2, 5, 10) and 10 (lanes 3, 6, 11) /~g m1-1 TM. (Lane 7) Denatured a-galactosidase treated with Endo H. (Lane 8) Purified a-galactosidase. (Lane 12)

Molecular weight markers.

lans protoplasts actively secrete this enzyme, but it may have been, also, due to intracellular en- zyme released into the extracellular medium by protoplast lysis [15]. To check this hypothesis the activity of the intracellular enzyme malate dehy- drogenase was measured, but no activity was de- tected in the fraction of proteins secreted by protoplasts. Therefore, it can be concluded that the protoplast preparations were free of lysed cells, and the extracellular a-galactosidase de-

tected must represent actively secreted protein from the protoplast.

The greatest proportion of a-galactosidase re- leased from the protoplasts as compared with the enzyme released by the mycelium to the culture filtrate could be explained if a considerable amount of the secreted enzyme is retained at the cell periphery (periplasm and/or cell wall). It is

10o

100

0 0 10 20

Time (min)

Fig. 3. Temperature stability of a-galactosidase stability syn- thesized by A. nidulans grown in the absence (11 . . . . . . II) and presence of 5 (A . . . . . . A) and 10 (e . . . . . . e ) /~g m1-1 TM (100% of a-galactosidase activity 420 m U / m g proteins).

80

60

.~ 40 N

20

80

.~. 60

40 ~-

2O

A

V B c

Fig. 4. Relative distribution of a-galactosidase activity in cytosol (~]), membrane (t~) and secreted protein (IS]) frac- tions of A. nidulans protoplasts after incubation in the ab- sence (A) and presence of 5 (B) and 10 (C) /~g m1-1 TM (100% of argalactosidase activity 50 m U / 1 0 s protoplasts). Data represent the mean values for three independent experi-

ments.

174

also probably that some of the enzymatic activity determined at the soluble mycelial extract actu- ally was extracellular enzyme present at the cell periphery released during homogenization [16].

Proteins secreted by protoplasts were sub- jected to native-PAGE followed by a-galactosi- dase activity gel staining. Protoplasts incubated in the absence of TM, secreted an a-galactosidase which showed similar electrophoretical mobility than the enzyme purified from A. nidulans [2]. However, when protoplasts were incubated in the presence of TM, a notably modification on the electrophoretical mobility of secreted a-galac- tosidase was observed, which was more pro- nounced when TM concentration was 10/zg ml- 1 (Fig. 5). These results indicated that protoplasts of .4. nidulans, incubated in the presence of TM, secreted underglycosylated active a-galactosi- dase, supporting that N-glycosylation is not nec- essary to the release of this enzyme out of the cell.

There is increasing appreciation of the struc- tural and functional role that carbohydrates may play in the elaboration of biologically active gly- coproteins [3]. However, the results reported by

1 2 3 4 5

m

several authors in lower eukaryotic ceils are con- troversial and variable for each particular protein [5,17,18]. The showed results indicate that, in A. nidulans, N-glycosylation is not strictly necessary to release o~-galactosidase out of the plasmatic membrane, since an active underglycosylated en- zyme was detected in the protoplast extracellular medium in the presence of TM. However, be- cause in the presence of TM .4. nidulans mycelium was unable to release ot-galactosidase to the culture medium, although it was present at the cell wall, changes in glycosylation could result on enzyme mistargeting at the cell periphery. This observations also reflect that the cell wall is not an inert cellular component of the secretory pathway of proteins to the culture medium [16]. It seems that there is some kind of interaction be- tween the glycoprotein and the cell wall and in this interaction the N-carbohydrates are impli- cated. The reason for which underglycosylated a-galactosidase was completely retained at the periplasma space a n d / o r at the cell-wall is not yet known. A possible interpretation is that N- glycan moieties are components of re ta ining/ releasing signals which target the glycoproteins to their final destination (cellular envelope or cul- ture medium) [19]. N-linked carbohydrate deple- tion could alter these signals, preventing the pro- tein release to the culture medium. It is also possible that the lack of N-linked carbohydrate produces alterations on the superficial structure of a-galactosidase, so modifying the normal inter- actions of this protein with the cell-wall polymers, causing its retention at the wall.

Fig. 5. a-Galactosidase activity revealed after native-PAGE of proteins secreted by protoplasts incubated in the absence and presence of TM. Proteins secreted by protoplasts in the absence (lane 1) and presence of 5 (lane 2) and 10 (lane 3)/zg ml-1 TM. Purified a-galactosidase (lane 4). Endo H digested

a-galactosidase (lane 5).

Acknowledgements

This work was partially supported by grant from the Ministerio de Educaci6n y Ciencia to S.R. We thank Mr V. de Sousa for the correction of the English version of this manuscript.

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