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Biosynthesis of yeast mannan
573
Biochimica et Biophysica Acta, 4 2 8 ( 1 9 7 6 ) 5 7 3 - - 5 8 2 © Elsevier Scientific Publishing Company,
A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 27886
BIOSYNTHESIS OF YEAST MANNAN DIVERSITY OF MANNOSYLTRANSFERASES IN THE MANNAN-SYNTHESIZING ENZYME SYSTEM FROM YEAST
VLADIMIR
FARKA~,
VLADIMIR
M. V A G A B O V
a and ~TEFAN
BAUER
Institute o f Chemistry, Slovak Academy o f Sciences, 809 33 Bratislava, (Czechoslovakia), and a Insitute o f Biochemistry and Physiology o f Microorganisms, Academy o f Sciences o f the U.S.S.R. Pustchino on the Oka (U.S.S.R.) (Received November 24th, 1975)
Summary 1. A microsomal enzyme preparation from the yeast Saccharomyces cerevisiae catalyzes the transfer of mannosyl units from GDPmannose to mannose and a number of mannose-containing oligosaccharides and glycosides whereby different glycosidic bonds are formed. 2. Of the compounds tested besides mannose, only those containing an s-linked mannosyl unit at the nonreducing position of their molecule were effective as acceptors. Monodeoxyanalogues of mannose as well as ~-mannose phosphates did not serve as acceptors in the above reaction. 3. The structure of the p r o d u c t formed with mannose as acceptor was determined to be O-~-D-mannosyl-(1-+ 2)-mannose; with ~Man(1-~ 6)mannose as the acceptor, the product was ~Man(1-~6)~Man(1-*6)mannose and with ~Man(1-~2)mannose the product was tentatively characterized as a mixture of ~Man(1-~3)~Man(1-~2)mannose and ~Man(1-~2)~Man(1-*2)mannose. 4. The enzymes catalyzing the formation of different types of glycosidic bonds differed in their a¢ceptor specificity, pH-activity curves and rates of heat denaturation. 5. Radioactive disaccharids were unable to enter the mannan protein molecule in the cell-free system while free radioactive mannose did incorporate into polysaccharide to a minor extent under the same conditions. Abbreviations: aMan(l--*2)mannose, O-~-D-mannosyl-(l-~2)-rnannose; czMan(l--~3)mannose, O-~-Drnannosyl-(1--~3)-mannose; 13Man(1--*4)mannose, O-~-D-mannosyl-(l--*4)-mannose; c~Man(1-~6)mannose, O-a-D-mannosyl-(1-->6)mannose; aMan(1--~2)aMan(l"-*2)manr~ose; O ' ~ - D - m a n n o s y l - ( l ~ 2 ) mannosyl-~(1--*2)-mannose; aMan(l--*3)~Man(1--*2)mannose, O-¢~-D-mannosylo(l-~3)mannosyl"a(1--~2)-mannose; aMan(1--*3)c~Man(1--~2)c~Man(1--*2)mannose, O-a-D-mannosyI-(1-*3)rnannosyl-~(1--~2)mannosyl-~(l--*2)-mannose; CH3-
574 Introduction It has been reported by different workers that a particulate enzyme complex from yeast, the so-called "mannan-synthetase", catalyzes mannosyl transfer from GDPmannose to endogenous acceptors mannoprotein [1--4] and lipids [3,5--9] as well as to exogenous, mannose containing acceptors [10]. Since the latter group of substrates, used in the cell-free system is supposed to mimick the functions of the endogenous acceptors, the studies employing well-defined, low-molecular weight c o m p o u n d s as exogenous acceptors could represent a useful approach to investigation the properties of the individual mannosyltransferases involved in the biosynthesis of yeast mannan without need to separate them from the particulate enzyme complex. Using a similar m e t h o d to Schutzbach and Ankel [11,12] were able to demonstrate the existence of at least four mannosyltransferases, each of them catalyzing the formation of a distinct type of glycosidic linkage, involved in the biosynthesis of xylomannan in Cryptococcus laurentii. An analogous approach was used in investigating the substrate specificity of different glycosyltransferases participating in the formation of blood-group substances [13,14]. Yeast mannan exists in cell walls as a glycoprotein whose polysaccharide part consists of an a(1-~6)-linked backbone to which short side chains containing a(1-~2) and a(1-~3)-linked mannosyl units are attached by a(1-~2) bonds. The polysaccharide part is linked via di-N-acetylchitobiose by an N-glycosidic bond to an asparagine residue in the protein moiety of the mannoprotein molecule [15,16]. Besides that, presumably on the same molecule, mono-, di-, tri- and tetrasaccharides of mannose containing a(1--2) and a(1-~3)glycosidic linkages are linked by an O-glycosidic bond to serine and/or threonine residues on the protein [15,16]. Our results, obtained by using low-molecular weight, mannose containing c o m p o u n d s as acceptors of mannosyl units from GDPmannose, demonstrate the existence of at least three different mannosyltransferases catalyzing the synthesis of different glycosidic linkages in the yeast mannan-synthesizing enzyme system. The possible role of the individual mannosyltransferases in the biosynthesis of yeast mannan is discussed. Materials and Methods
Substrates aMan(l-~2)mannose, a mixture of aMan(l-~2)aMan(1-~2)mannose and aMan(l-~3)aMan(l-~2)mannose, and aMan(1-~3)aMan(1-~2)aMan(1-~2)mannose were prepared by acetolysis of baker's yeast mannan [17]. aMan(l-~3)mannose, flMan(-~4)mannose and aMan(1-~6)mannose were p r e p a r e d . b y acid reversion of mannose in 6 M HC1 [18]. The structure of the prepared oligosaccharides was confirmed by mass spectrometry of permethylated alditols [ 19], specific optical rotation, cleavage with sweet almond emulsin a-mannosidase and cochromatography with standards. Monodeoxyanalogues of mannose were prepared by Drs. Kuc~r and Z~mock~ of our Institute. GDPmannose was purchased from Sigma Chemical Co., St. Louis, Mo., U.S.A. and GDP-[U-'4C]mannose, specific activity 74 Ci/mol, was from the Radiochemical Centre,
575 Amersham, England. Methyl- and p-nitrophenyl-mannosides were from KochLight (England).
Enzyme preparation Particulate enzyme preparations was obtained as follows: Logarithmic-phase cells, Saccharomyces cerevisiae strain CCY 21-4-13, were collected by centrifugation from the growth medium [20], washed with cold distilled water and with cold 0.05 M Tris • HC1 buffer (pH 7.2) containing 0.5 M mannitol and 1 mM EDTA. All subsequent operations were carried out at 0--4°C. The cells suspended in the same buffer with ballotini beads (diameter 0.5 mm) were disintegrated in a r o t a t o r y disintegrator cooled from the outside with solid CO2 ethanol mixture. After rupture of cells, the ballotini beads were decanted several times with the above buffer and combined washings were centrifuged at 1500 X g for 15 min. The sediment containing some ballotini beads and cell wall fragments was discarded and the supernatant was spun at 10 000 X g for 15 min. The sediment was discarded and the resulting supernatant was centrifuged at 100 000 X g for 30 min. The pellet was washed once with the buffer containing 0.5 M mannitol and EDTA and once with the same buffer w i t h o u t mannitol and EDTA. Final pellet was resuspended in the 0.05 M Tris-HC1 buffer (pH 7.2) using 0.1 ml of the buffer per enzyme from 1 g of wet yeast. Freshly prepared e n z y m e was used in the experiments. Protein was determined according to Lowry et al. [21].
Assay procedures A typical incubation mixture contained 4 pmol of Tris • HC1 buffer (pH 7.2), 0.5 pmol of MnC12, 50 nmol of GDP-[U-14C] mannose (specific activity 0.2 Ci/mol), 1 mmol of the respective exogenous acceptor when used, and 0.15-0.30 mg (as protein) of the particulate enzyme preparation in 50 pl total volume; The incubation was carried out at 30°C for 30 min. To measure the transfer of mannose from GDP-[U-~4C]mannose to exogenous acceptors the reaction was stopped by addition of 0.1 ml ethanol and after standing for 10 min in the ice the tubes were centrifuged for 5 min in a clinical centrifuge. 0.1 ml of the supernatant from each tube was then taken by a micropipette and applied on a chromatographic paper and chromatographed in solvent system A. In cases when incorporation to endogenous glycoprotein was assayed the reaction was terminated by addition of 1 ml ethanol I M a m m o n i u m acetate mixture (2 : 1, by vol.) and the tubes were kept in the ice for 10 min. They were then centrifuged in a clinical centrifuge for 5 min and sediments were twice washed with the same mixture. For determination of radioactivity in precipitates, the procedure of Neame and H o m e w o o d [22] was adopted. To the washed sediments in the glass or plastic incubation tubes (size 0.8 × 5.0 cm) 0.2 ml of water was added and the sediments were suspended by thorough mixing in a vortex mixer. Then 0.5 ml of Instagel emulsifier (Packard Instrument Co.) was added to each sample and the tubes were heated at 40°C for 10 min in a water bath. The contents of the tubes were then mixed with the emulsifier and after cooling the tubes in the ice they were capped with Parafilm and placed into e m p t y scintillation vials and counted at 5--7°C in the scintillation spectromerter (Packard Tri-Carb, Modell 3330).
576
The described procedure is time- and material saving and gives fairly reproducible results by avoiding the losses of radioactive material during its transfer from incubation tubes into the counting vials.
Paper chromatography Descending chromatography was carried out on Whatman no. 1 papers using the following solvent systems: A, ethyl acetate/pyridine/water (5 : 3 : 2, by vol.); B, n-propanol/ethylacetate/water (7 : 1 : 2, by vol); C, acetone~n-butanol~ water (7 : 2 : 1, by vol.). Results
Mannosyl transfer from GDPmannose to exogenous acceptors The microsomal enzyme preparations from the yeast Saccharomyces cerevisiae catalyzes the transfer of mannose from GDP-[U-14C]mannose to free mannose and a number of low-molecular-weight, mannose containing exogenous acceptors. The transfer seemed to be successive since in many cases formation of a series of higher homologues was observed. Comparison of the acceptor activity of various exogenous acceptors (see Table I) shows that a-configuration of mannosyl unit located at the nonreducing end of the acceptor molecule is essential for its acceptor capability. Nevertheless, the oligosaccharides containing an a(1-*3)-linked mannosyl unit in the terminal position were relatively poor acceptors in comparison to the other a-linked oligosaccharides. None of m o n o d e o x y a n a l o g u e s of mannose were able TABLE
I
ACCEPTOR ACCEPTORS
SPECIFICITY
OF
MANNOSYL
TRANSFER
FROM
GDP-MANNOSE
TO
EXOGENOUS
C o n c e n t r a t i o n o f t h e r e s p e c t i v e a c c e p t o r s in t h e i n c u b a t i o n m i x t u r e w a s 2 0 r a M . B e s i d e s t h e s e a c c e p t o r s listed, D-glucose, N-acetyl-D-glucosamine, mannitol, inositol, 3-deoxy-D-mannose, 4-deoxy-D-mannose a n d 6 - d e o x y - D - m a n n o s e w e r e t e s t e d . All t h e s e c o m p o u n d s w e r e f o u n d t o b e i n e f f e c t i v e as a c c e p t o r s . Acceptor
nmol of the reaction p r o d u c t f o r m e d in 3 0 rain per mg of enzyme protein *
Nature of the newly formed bond
D-mannose
38.5 107.2 13.0 0
a ( 1 ~ 2) a ( l ~ 3 ) , a ( 1 --~ 2 ) n.d.
90.0 101.9
a ( 1 --~ 6 ) n.d.
6.6 143.4 237.5 0 1.4 1.0 0
n.d. n.d. n.d.
c~ M a n ( 1 --~ 2 ) m a n n o s e c~ M a n (1 ~ 3 ) m a n n o s e fl M a n (1 --* 4 ) m a n n o s e a M a n (1 --* 6 ) m a n n o s e Mannotriose * * a M a n (1 --~ 3 ) a M a n ( 1 --~ 2 ) a M a n ( 1 -~ 2 )
mannose CH3-a-mannose
Nph-~-mannose Nph-fl-mannose Mannose 1-phosphate Mannose 6-phosphate 2-deoxy-D-glucose
n.d. n.d.
* T h e s u m o f r a d i o a c t i v i t y f o u n d in all l o w - m o l e c u l a r w e i g h t p r o d u c t s f o r m e d w i t h t h e g i v e n a c c e p tot. ** A m i x t u r e o f c ~ M a n ( 1 - - * 2 ) ~ M a n ( 1 - - + 2 ) m a n n o s e a n d ~Man(1--*3)c~Man(1--~2)mannose o b t a i n e d b y acetolysis of yeast mannan [17].
577 to serve as acceptors. The small amounts of radioactive disaccharides found in the incubation mixtures when a-mannose phosphates were tested as acceptors can be ascribed to the presence of traces of mannose in the samples of mannose phosphates used rather than to their acceptor ability. The highest acceptor affinity was found with a-glycosides of mannose; methyl-a-mannose and Nph-a-mannose.
Structure o f the products Sodium borohydride reduction of the oligosaccharide products followed by acid hydrolysis resulted in a complete liberation of radioactive mannose indicating that the newly attached radioactive mannosyl moieties were present at non-reducing termini of the reaction products. Upon incubation with sweet almond emulsin a-mannosidase the products completely liberated radioactive mannose indicating exclusive formation of a-mannosidic bonds. For the purpose of structure determination the mannobiose was prepared in scaled up incubation mixture using D-[1-14C]mannose as the acceptor and nonradioactive GDPmannose as the mannosyl donor. The formed disaccharide was isolated and purified by means of preparative paper chromatography. Upon sodium borohydride reduction followed by acid hydrolysis the disaccharide yielded D-[1-14C]mannitol as the only radioactive product. The disaccharide cochromatographed well with authentic aMan(1-*2)mannose in solvent system B. Its structure was further confirmed by periodate oxidation. The disaccharide (40 000 cpm) was reduced with NaBH4 and then oxidized in 1 ml of 0.1 M acetate buffer (pH 5) containing 0.02 M sodium periodate. After 72 h at 4°C excess NaBH4 was added to reduce remaining periodate and the formed aldehydes. After overnight standing at room temperature the mixture was desalted over a mixed-bed resin and evaporated to dryness under reduced pressure. The residue was dissolved in 0.5 ml of 2 M HC1 and the hydrolysis was carried out at 100°C for 3 h in a sealed tube. After removal of HC1 by repeated evaporation with methanol the mixture was spotted on the chromatographic paper and the chromatography was carried out in the solvent system C for 8 h. Measurement of the radioactivity on the paper chromatogram revealed the presence of [14C] glycerol as the only radioactive product of the periodate oxidation of the disaccharide. When the radioactive disaccharide was mixed with authentic aMan(1-~2)mannose and aMan(1-*6)mannose and heated with phenylhydrazine hydrochloride under conditions described by Kuhn and Baer [23] practically all radioactivity remained in the supernatant over the precipitated phenylosazone of aMan(1-~6)mannose. These results give rather strong evidence that the h y d r o x y l group at C-2 of reducing mannosyl unit in the investigated disaccharide is blocked by a glycosidic b o n d and, consequently, that the structure of disaccharide formed from mannose and GDPmannose is O-a-D-mannosyl-(1-~2)-mannose. The trisaccharides for structure determination were prepared from GDP[U34C] mannose as mannosyl donor and aMan(1-~2)mannose and aMan(1-~6)mannose as the respective acceptors. The nature of the newly formed mannosidic bonds was deduced indirectly, on the basis of the relative stability of formed trisaccharides towards acetolysis. The kinetic data obtained by Rosen-
578 feld and Ballou [24] with different oligosaccharides show that, under conditions of acetolysis, the a(1-~6)-glycosidic linkage in mannooligosaccharides is split about 280 times faster than a(1-*2)-bond and, at the same time, about 21 times faster than a (1-~ 3)-bond. The radioactive trisaccharides were acetylated [24] and subjected to acetolysis in the mixture of acetic anhydride/acetic acid/H2SO4 (10 : 10 : 1, by vol.). After 16 h of acetolysis at 40°C the products were deacetylated and resolved b y paper chromatography in solvent system A. Over 97% of the trisaccharide prepared from aMan(1-~6)mannose was hydrolysed in the course of described procedure releasing radioactive mannose and traces of radioactive mannobiose. On the other hand, about 41% of the trisaccharide prepared from aMan(1-~2)mannose remained unhydrolysed under the same conditions the remaining 59% decomposed to radioactive mannose (50%) and radioactive mannobiose (9%). These results indicated that in case of trisaccharide formed from aMan(1-~ 6)mannose the nature of the newly formed glycosidic bond is a(1-*6) and hence the structuTe of the trisaccharide is aMan(1-*6)aMan(1-*6)mannose which is reflected in its high lability towards acetolysis. With the trisaccharide from aMan(1-~2)mannose the situation seems to be more complicated. If the newly formed bond in the trisaccharide were exclusively a(1-~2), over 99% of it should remain intact after 16 h acetolysis. However if it were exclusively a(1-*3) bond that was formed in the course of mannosyl transfer to aMan(1-*2)mannose, there should remain theoretically only about 38% of unhydrolysed mannotriose and practically no mannobiose after 16 h acetolysis (calculated on the basis of the rate constants given in ref. 24). We supposed therefore that in the latter case it is reasonable to assume that both a(1-~ 3 ) a n d a(1-~ 2) bonds were formed with a aMan(1-* 2)mannose as acceptor, the a(1-~3) bonds being more preferred. Whether this is due to unspecificity of corresponding mannosyltransferases towards acceptor or to heterogeneity of the sample of mannobiose used as the acceptor is n o t known.
Effect o f pH and metal ion requirement The observation that different kinds of linkages were formed with different acceptors promted us to investigate in more detail the properties of the individual mannosyltransferasees. As can be seen from the Fig. 1, the enzymes catalyzing the mannosyl transfer from GDPmannose to mannose, aMan(1-~6) mannose and to aMan(1-~2)mannose, respectively, differ in the shape of their pH-activity curves. In all investigated reactions Mn 2+was the best activator. In the reactions with aMan(1-~2)mannose and methyl-a-mannose Mg 2÷ activated to about 15% of the maximal activity with Mn 2÷ while with aMan(1-*3)mannose the Mg 2÷ activated the reaction to 30% of the value obtained with Mn 2÷. Thermal inactivation studies The diversity of the enzymes catalyzing the mannosyl transfer to different acceptors can be well observed also in the kinetics of thermal denaturation of the corresponding enzymes. The particulate enzyme preparation suspended in 0.05 M Tris. HC1 buffer (pH 7.2) was heated at 45°C and at different time intervals the samples were withdrawn and the activity of the enzyme towards
579
I
I
I
I
I
I
I
A 3
e~
2 o m
~=
1
m =IE
0 6 E
4 2 m
0
I -O"-~l
I
t
I
i
i
6
7
8
9
10
8 x
6
=
4
E ==-
5
Fig. 1. E f f e c t o f p H o n t h e rate o f m a n n o s y l t r a n s f e r t o A , m a n n o s e ; B, ~ M a n ( 1 - * 6 ) m a n n o s e ; C, ~ M a n ( 1 - ~ 2 ) m a n n o s e . ( o ) s u c c i n a t e ; ( e ) i m i d a z o l e / H C l ; ( ~ ) Tris - HC1; (A) g l y c i n e / N a O H b u f f e r s . T h e c o m p o s i t i o n o f t h e r e a c t i o n m i x t u r e s w a s t h e s a m e as in t h e s t a n d a r d assay e x c e p t t h a t t h e c o n c e n t r a t i o n o f t h e r e s p e c t i v e b u f f e r w a s 80 r a M .
different acceptors was determined in a standard incubation mixture. As can be seen in the Fig. 2, the rate of mannosylation of the endogenous acceptors dropped rapidly upon the heat treatment of the enzyme. The observed decrease in the,mannoprotein synthesis was almost paralleled by the inactivation of the enzyme catalyzing the transfer of mannosyl units to aMan(1-~6)mannose. The activity of the enzyme functioning with aMan(1-*2)mannose as acceptor was much less decreased while the enzyme catalyzing the monnosyl transfer to mannose was practically unaffected by the heat treatment. The latter two reactions were even slightly stimulated by short-term heat treatment of the particulate enzyme preparation.
Inability of free mannooligosaccharides to enter the tnannoprotein molecule Free radioactive mannose when incubated with the enzyme in the presence of nonradioactive GDPmannose was observed to enter into the endogenous mannoprotein. The observed incorporation was, however less than 2% of the
580
1.0 ,8
.4
.]
I
I
I
0
5
10
I
15 mitt it 45"C
Fig. 2. E f f e c t of h e a t t r e a t m e n t of t h e p a r t i c u l a t e e n z y m e p r e p a r a t i o n
o n t h e r a t e of m a n n o s y l t r a n s f e r
f r o m G D P - [ U - 1 4 c ] m a n n o s e to: (-~.) m a n n o s e ; (A) ~ M a n ( l ~ 2 ) m a n n o s e ; endogenous mannoprotein.
(©) c~Man(1-~6)m~nnose; a n d (e)
rate measured with the same concentration of GDP-[U-14C]mannose. Radioactive aMan(1-*2)mannose isolated from large-scale incubation mixtures, as well as radioactive aMan(1-~6)mannose prepared by acid reversion of [14C]mannose [18] did not incorporate into the mannoprotein under the same conditions. Discussion
The observation made by Lehle and Tanner [10] and confirmed by us, that mannose and mannose-containing low-molecular weight acceptors are able to serve as mannosyl acceptors from GDPmannose in reactions catalyzed by yeast mannan synthetase, opens the possibility to investigate the properties of the individual mannosyltransferase participating in the biosynthesis of yeast mannan. Our experiments have confirmed that a-configuration of the acceptor mannosyl unit is necessary for the enzyme-catalyzed mannosyl transfer. The observation that oligosaccharides containing an a(1-~3) linked nonreducing terminal mannosyl unit are relatively poor acceptors may explain w h y a(1-*3)-linked mannosyl units are located predominantly at the nonreducing ends of ~-elimin-
581
able oligosaccharides and side chains of the polymannose part in the yeast mannan protein . The observed relatively high activity of mannosyltransferases towards a-glycosides of mannose, namely methyl* -mannose and Nphe -mannose could be explained as due to lower polarity of these compounds as compared with that of mannose and mannooligosaccharides. It can be anticipated that compounds with decreased polarity penetrate easier than neutral sugars into the membrane-bound enzyme complex. Also important is the observation that none of the monodeoxyanalogues of mannose was able to serve as acceptor in the investigated enzyme system. Biely et al. [25] have found that one of these compounds, namely 2-deoxy-Dglucose enters into the yeast mannoprotein in vivo. They observed, however, that 2-deoxy-D-glucose was incorporated almost exclusively into the polymannose part of mannoprotein but not into the oligosaccharides attached to serine and/or theonine, The 2-deoxy-D-glucose incorporated into the polysaccharide moiety of yeast manno protein was located mostly in the side chains and in many cases it was further glycosylated by mannose. It can be therefore assumed that the enzymes active with D-mannose but inactive with its 2-deoxyanalogue as acceptor are most probably involved in the biosynthesis of P-eliminable carbohydrate moiety rather than in the formation of polysaccharide part of mannoprotein. Hence, the enzyme catalyzing the formation of ar(l+2)-bond with D-mannose as acceptor may be considered as being involved in the biosynthesis of P-eliminable saccharides attached to serine and/or threonine. The second investigated enzyme, namely that catalyzing the formation of e( l-+6)-linkage with exogenous aMan(l+6)mannose as acceptor is most probably participating in the biosynthesis of cr(l-+6)-linked main chain in the polysaccharide part of mannoprotein. This assumption comes from the fact that cr(l+6)-glycosidic bonds were not found in the oligosaccharides attached to serine and threonine [26,27]. The observation that the kinetics of thermal inactivation of this enzyme is almost parallel with decrease in the rate of mannoprotein synthesis caused by heat treatment of the enzyme gives another support to the above assumption. The formation of aMan( l+ 3)cuMan(1+2)mannose and aMan( l- 2)cyMan (1-+2)mannose from cYMan(l+2)mannose can be considered as a result of simultaneous action of two respective mannosyl transferases on the same acceptor. On the basis of the present information it is difficult to assign a precise role to these two enzymes in the biosynthesis of yeast mannoprotein. Experiments with cross-inhibition (Farkas, V., unpublished results) have shown that the enzyme catalyzing the formation of a(l+2)-glycosidic bonds with uMan(l+B)mannose as acceptor is not identical with the a(1+2)mannosyltransferase acting with D-mannose as acceptor. Perhaps the most interesting results were obtained with thermal inactivation studies. All three investigated reactions with exogenous acceptors exhibited different kinetics of thermal inactivation. This fact documents well the diversity of mannosyltransfereases participating in the biosynthesis of yeast mannan. It is noteworthy that although the rate of overall mannan biosynthesis was considerably reduced by heat treatment of the enzyme, almost no change in the
582
amount of radioactivity incorporated into fl-eliminable saccharidic moiety was observed [ 2 8 ] . Our failure to demonstrate'the incorporation of labelled aMan(1-~2)mannose and aMan(1-+6)mannose into mannoprotein in the complete incubation mixture rules out the probability that these oligosaccharides in the free state might be the precursors or intermediates in the biosynthesis of yeast mannan. The observed slight incorporation of free mannose into mannoprotein observed under identical conditions can be explained as due to the mannosyl exchange at the level of dolichol monophosphate mannose which plays a role of intermediate in the biosynthesis of mannoprotein [ 3,5--9]. 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
Behrens, N.H. a n d Cabib, E. ( 1 9 6 8 ) J. Biol. Chem. 2 4 3 , 5 0 2 - - 5 0 9 K o z a k , L.P. a n d B r e t t h a u e r , R.K. ( 1 9 7 1 ) B i o c h e m i s t r y 9+ 1 1 1 5 - - 1 1 2 2 S e n t a n d r e u , R. a n d L a m p e n , J . O . ( 1 9 7 1 ) FEBS L e t t . 14, 1 0 9 - - 1 1 3 B r e t t h a u o r , R . K . a n d Chen Tsay, G. ( 1 9 7 4 ) A r c h . B i o c h e m . B i o p h y s . 1 6 4 , 1 1 8 - - 1 2 6 T a n n e r , W. ( 1 9 6 9 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 35, 1 4 4 - - 1 5 0 T a n n e r , W., J u n g , P. a n d Behrens, N.H. ( 1 9 7 1 ) FEBS Lett. 1 6 , 2 4 5 - - 2 4 8 J u n g , P. a n d T a n n e r , W. ( 1 9 7 3 ) Eur. J. B i o c h e m . 37, 1--6 B r e t t h a u e r , R.K.+ Wu, S. a n d Irwin, W.E. ( 1 9 7 3 ) Biochim. B i o p h y s . A c t a 304, 7 3 6 - - 7 4 7 S h a r m a , C.B., B a b c z i n s k i , P., Lehle, L. a n d T a n n e r , W. ( 1 9 7 4 ) Eur. J. B i o c h e m . 46, 35---41 Lehle, L. a n d T a n n e r , W. ( 1 9 7 4 ) Biochim. B i o p h y s . A c t a 3 5 0 , 2 2 5 - - 2 3 5 S c h u t z b a c h , J.S. a n d Ankel+ H. ( 1 9 7 1 ) J. Biol. Chem. 2 4 6 , 2 1 8 7 - - 2 1 9 4 S c h u t z b a c h , J.S. a n d A n k e l , H. ( 1 9 7 2 ) J. Biol. Chem. 2 4 7 , 6 5 7 4 - - 6 5 8 0 H e a r n , V.M., S m i t h , Z.G. a n d Watkins, W.M. ( 1 9 6 8 ) B i o c h e m . J. 1 0 9 , 3 1 5 - - 3 1 7 K o b a t a , A., G r o l l m a n , E.F. a n d G i n s b u r g , V. ( 1 9 6 8 ) A r c h . B i o c h e m . Biophys. 1 2 4 , 6 0 9 - - 6 1 2 Nakajima+ T. a n d Ballou, C.E. ( 1 9 7 4 ) J. Biol. Chem. 2 4 9 , 7 6 7 9 - - 7 6 8 4 N a k a j i m a , T. a n d Ballou, C.E. ( 1 9 7 4 ) J. Biol. Chem. 2 4 9 , 7 6 8 5 - - 7 6 9 4 K o c o u r e k , J. a n d Ballou, C.E. ( 1 9 6 9 ) J. Bacteriol. 1 0 0 , 1 1 7 5 - - 1 1 8 1 J o n e s , J . K . N . a n d N i c h o l s o n , W.H. ( 1 9 5 8 ) J. C h e m . Soc. 2 7 - - 3 3 K a r k k ~ i n e n , J. ( 1 9 7 0 ) C a r b o h y d r . Res. 14, 2 7 - - 3 3 Biely, P., Kr~itky, Z.+ Kova~ik, J. a n d Bauer, S. ( 1 9 7 1 ) J. Bacteriol. 107, 1 2 1 - - 1 2 9 L o w r y , O.H., R o s e b r o u g h , N.J., F a r r , A.L. a n d R a n d a l l , R.J. ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 Neame, K.D. a n d H o m e w o o d , C.A. ( 1 9 7 4 ) Anal. B i o c h e m . 5 7 , 6 2 3 - - 6 2 7 K u h n , R. a n d Baer, H.H. ( 1 9 5 4 ) C h e m . Ber. 87, 1 5 6 0 - - 1 5 6 7 R o s e n f e l d , L. a n d Ballou, C.E. ( 1 9 7 4 ) C a r b o h y d r . Res. 2 8 7 - - 2 9 8 Biely, P., K r ~ t k y , Z. a n d Bauer, S. ( 1 9 7 4 ) Biochim. Biophys. A c t a 3 5 2 , 2 6 8 - - 2 7 4 R a s c h k e , W.C., K e r n , K . A . , Antalis, C. a n d BaUou, C.E. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 4 6 6 0 - - 4 6 6 6 Ballou, C.E., K e r n , K . A . a n d R a s c h k e , W.C. ( 1 9 7 3 ) J. Biol. C h e m . 248, 4 6 6 7 - - - 4 6 7 3 Farka~, V. Bauer, ~. a n d V a g a b o v , V.M. ( 1 9 7 6 ) B i o c h i m . Biophys. A c t a 4 2 8 , 5 8 3 - - 5 9 0
Biochimica et Biophysica Acta, 4 2 8 ( 1 9 7 6 ) 5 7 3 - - 5 8 2 © Elsevier Scientific Publishing Company,
A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 27886
BIOSYNTHESIS OF YEAST MANNAN DIVERSITY OF MANNOSYLTRANSFERASES IN THE MANNAN-SYNTHESIZING ENZYME SYSTEM FROM YEAST
VLADIMIR
FARKA~,
VLADIMIR
M. V A G A B O V
a and ~TEFAN
BAUER
Institute o f Chemistry, Slovak Academy o f Sciences, 809 33 Bratislava, (Czechoslovakia), and a Insitute o f Biochemistry and Physiology o f Microorganisms, Academy o f Sciences o f the U.S.S.R. Pustchino on the Oka (U.S.S.R.) (Received November 24th, 1975)
Summary 1. A microsomal enzyme preparation from the yeast Saccharomyces cerevisiae catalyzes the transfer of mannosyl units from GDPmannose to mannose and a number of mannose-containing oligosaccharides and glycosides whereby different glycosidic bonds are formed. 2. Of the compounds tested besides mannose, only those containing an s-linked mannosyl unit at the nonreducing position of their molecule were effective as acceptors. Monodeoxyanalogues of mannose as well as ~-mannose phosphates did not serve as acceptors in the above reaction. 3. The structure of the p r o d u c t formed with mannose as acceptor was determined to be O-~-D-mannosyl-(1-+ 2)-mannose; with ~Man(1-~ 6)mannose as the acceptor, the product was ~Man(1-~6)~Man(1-*6)mannose and with ~Man(1-~2)mannose the product was tentatively characterized as a mixture of ~Man(1-~3)~Man(1-~2)mannose and ~Man(1-~2)~Man(1-*2)mannose. 4. The enzymes catalyzing the formation of different types of glycosidic bonds differed in their a¢ceptor specificity, pH-activity curves and rates of heat denaturation. 5. Radioactive disaccharids were unable to enter the mannan protein molecule in the cell-free system while free radioactive mannose did incorporate into polysaccharide to a minor extent under the same conditions. Abbreviations: aMan(l--*2)mannose, O-~-D-mannosyl-(l-~2)-rnannose; czMan(l--~3)mannose, O-~-Drnannosyl-(1--~3)-mannose; 13Man(1--*4)mannose, O-~-D-mannosyl-(l--*4)-mannose; c~Man(1-~6)mannose, O-a-D-mannosyl-(1-->6)mannose; aMan(1--~2)aMan(l"-*2)manr~ose; O ' ~ - D - m a n n o s y l - ( l ~ 2 ) mannosyl-~(1--*2)-mannose; aMan(l--*3)~Man(1--*2)mannose, O-¢~-D-mannosylo(l-~3)mannosyl"a(1--~2)-mannose; aMan(1--*3)c~Man(1--~2)c~Man(1--*2)mannose, O-a-D-mannosyI-(1-*3)rnannosyl-~(1--~2)mannosyl-~(l--*2)-mannose; CH3-
574 Introduction It has been reported by different workers that a particulate enzyme complex from yeast, the so-called "mannan-synthetase", catalyzes mannosyl transfer from GDPmannose to endogenous acceptors mannoprotein [1--4] and lipids [3,5--9] as well as to exogenous, mannose containing acceptors [10]. Since the latter group of substrates, used in the cell-free system is supposed to mimick the functions of the endogenous acceptors, the studies employing well-defined, low-molecular weight c o m p o u n d s as exogenous acceptors could represent a useful approach to investigation the properties of the individual mannosyltransferases involved in the biosynthesis of yeast mannan without need to separate them from the particulate enzyme complex. Using a similar m e t h o d to Schutzbach and Ankel [11,12] were able to demonstrate the existence of at least four mannosyltransferases, each of them catalyzing the formation of a distinct type of glycosidic linkage, involved in the biosynthesis of xylomannan in Cryptococcus laurentii. An analogous approach was used in investigating the substrate specificity of different glycosyltransferases participating in the formation of blood-group substances [13,14]. Yeast mannan exists in cell walls as a glycoprotein whose polysaccharide part consists of an a(1-~6)-linked backbone to which short side chains containing a(1-~2) and a(1-~3)-linked mannosyl units are attached by a(1-~2) bonds. The polysaccharide part is linked via di-N-acetylchitobiose by an N-glycosidic bond to an asparagine residue in the protein moiety of the mannoprotein molecule [15,16]. Besides that, presumably on the same molecule, mono-, di-, tri- and tetrasaccharides of mannose containing a(1--2) and a(1-~3)glycosidic linkages are linked by an O-glycosidic bond to serine and/or threonine residues on the protein [15,16]. Our results, obtained by using low-molecular weight, mannose containing c o m p o u n d s as acceptors of mannosyl units from GDPmannose, demonstrate the existence of at least three different mannosyltransferases catalyzing the synthesis of different glycosidic linkages in the yeast mannan-synthesizing enzyme system. The possible role of the individual mannosyltransferases in the biosynthesis of yeast mannan is discussed. Materials and Methods
Substrates aMan(l-~2)mannose, a mixture of aMan(l-~2)aMan(1-~2)mannose and aMan(l-~3)aMan(l-~2)mannose, and aMan(1-~3)aMan(1-~2)aMan(1-~2)mannose were prepared by acetolysis of baker's yeast mannan [17]. aMan(l-~3)mannose, flMan(-~4)mannose and aMan(1-~6)mannose were p r e p a r e d . b y acid reversion of mannose in 6 M HC1 [18]. The structure of the prepared oligosaccharides was confirmed by mass spectrometry of permethylated alditols [ 19], specific optical rotation, cleavage with sweet almond emulsin a-mannosidase and cochromatography with standards. Monodeoxyanalogues of mannose were prepared by Drs. Kuc~r and Z~mock~ of our Institute. GDPmannose was purchased from Sigma Chemical Co., St. Louis, Mo., U.S.A. and GDP-[U-'4C]mannose, specific activity 74 Ci/mol, was from the Radiochemical Centre,
575 Amersham, England. Methyl- and p-nitrophenyl-mannosides were from KochLight (England).
Enzyme preparation Particulate enzyme preparations was obtained as follows: Logarithmic-phase cells, Saccharomyces cerevisiae strain CCY 21-4-13, were collected by centrifugation from the growth medium [20], washed with cold distilled water and with cold 0.05 M Tris • HC1 buffer (pH 7.2) containing 0.5 M mannitol and 1 mM EDTA. All subsequent operations were carried out at 0--4°C. The cells suspended in the same buffer with ballotini beads (diameter 0.5 mm) were disintegrated in a r o t a t o r y disintegrator cooled from the outside with solid CO2 ethanol mixture. After rupture of cells, the ballotini beads were decanted several times with the above buffer and combined washings were centrifuged at 1500 X g for 15 min. The sediment containing some ballotini beads and cell wall fragments was discarded and the supernatant was spun at 10 000 X g for 15 min. The sediment was discarded and the resulting supernatant was centrifuged at 100 000 X g for 30 min. The pellet was washed once with the buffer containing 0.5 M mannitol and EDTA and once with the same buffer w i t h o u t mannitol and EDTA. Final pellet was resuspended in the 0.05 M Tris-HC1 buffer (pH 7.2) using 0.1 ml of the buffer per enzyme from 1 g of wet yeast. Freshly prepared e n z y m e was used in the experiments. Protein was determined according to Lowry et al. [21].
Assay procedures A typical incubation mixture contained 4 pmol of Tris • HC1 buffer (pH 7.2), 0.5 pmol of MnC12, 50 nmol of GDP-[U-14C] mannose (specific activity 0.2 Ci/mol), 1 mmol of the respective exogenous acceptor when used, and 0.15-0.30 mg (as protein) of the particulate enzyme preparation in 50 pl total volume; The incubation was carried out at 30°C for 30 min. To measure the transfer of mannose from GDP-[U-~4C]mannose to exogenous acceptors the reaction was stopped by addition of 0.1 ml ethanol and after standing for 10 min in the ice the tubes were centrifuged for 5 min in a clinical centrifuge. 0.1 ml of the supernatant from each tube was then taken by a micropipette and applied on a chromatographic paper and chromatographed in solvent system A. In cases when incorporation to endogenous glycoprotein was assayed the reaction was terminated by addition of 1 ml ethanol I M a m m o n i u m acetate mixture (2 : 1, by vol.) and the tubes were kept in the ice for 10 min. They were then centrifuged in a clinical centrifuge for 5 min and sediments were twice washed with the same mixture. For determination of radioactivity in precipitates, the procedure of Neame and H o m e w o o d [22] was adopted. To the washed sediments in the glass or plastic incubation tubes (size 0.8 × 5.0 cm) 0.2 ml of water was added and the sediments were suspended by thorough mixing in a vortex mixer. Then 0.5 ml of Instagel emulsifier (Packard Instrument Co.) was added to each sample and the tubes were heated at 40°C for 10 min in a water bath. The contents of the tubes were then mixed with the emulsifier and after cooling the tubes in the ice they were capped with Parafilm and placed into e m p t y scintillation vials and counted at 5--7°C in the scintillation spectromerter (Packard Tri-Carb, Modell 3330).
576
The described procedure is time- and material saving and gives fairly reproducible results by avoiding the losses of radioactive material during its transfer from incubation tubes into the counting vials.
Paper chromatography Descending chromatography was carried out on Whatman no. 1 papers using the following solvent systems: A, ethyl acetate/pyridine/water (5 : 3 : 2, by vol.); B, n-propanol/ethylacetate/water (7 : 1 : 2, by vol); C, acetone~n-butanol~ water (7 : 2 : 1, by vol.). Results
Mannosyl transfer from GDPmannose to exogenous acceptors The microsomal enzyme preparations from the yeast Saccharomyces cerevisiae catalyzes the transfer of mannose from GDP-[U-14C]mannose to free mannose and a number of low-molecular-weight, mannose containing exogenous acceptors. The transfer seemed to be successive since in many cases formation of a series of higher homologues was observed. Comparison of the acceptor activity of various exogenous acceptors (see Table I) shows that a-configuration of mannosyl unit located at the nonreducing end of the acceptor molecule is essential for its acceptor capability. Nevertheless, the oligosaccharides containing an a(1-*3)-linked mannosyl unit in the terminal position were relatively poor acceptors in comparison to the other a-linked oligosaccharides. None of m o n o d e o x y a n a l o g u e s of mannose were able TABLE
I
ACCEPTOR ACCEPTORS
SPECIFICITY
OF
MANNOSYL
TRANSFER
FROM
GDP-MANNOSE
TO
EXOGENOUS
C o n c e n t r a t i o n o f t h e r e s p e c t i v e a c c e p t o r s in t h e i n c u b a t i o n m i x t u r e w a s 2 0 r a M . B e s i d e s t h e s e a c c e p t o r s listed, D-glucose, N-acetyl-D-glucosamine, mannitol, inositol, 3-deoxy-D-mannose, 4-deoxy-D-mannose a n d 6 - d e o x y - D - m a n n o s e w e r e t e s t e d . All t h e s e c o m p o u n d s w e r e f o u n d t o b e i n e f f e c t i v e as a c c e p t o r s . Acceptor
nmol of the reaction p r o d u c t f o r m e d in 3 0 rain per mg of enzyme protein *
Nature of the newly formed bond
D-mannose
38.5 107.2 13.0 0
a ( 1 ~ 2) a ( l ~ 3 ) , a ( 1 --~ 2 ) n.d.
90.0 101.9
a ( 1 --~ 6 ) n.d.
6.6 143.4 237.5 0 1.4 1.0 0
n.d. n.d. n.d.
c~ M a n ( 1 --~ 2 ) m a n n o s e c~ M a n (1 ~ 3 ) m a n n o s e fl M a n (1 --* 4 ) m a n n o s e a M a n (1 --* 6 ) m a n n o s e Mannotriose * * a M a n (1 --~ 3 ) a M a n ( 1 --~ 2 ) a M a n ( 1 -~ 2 )
mannose CH3-a-mannose
Nph-~-mannose Nph-fl-mannose Mannose 1-phosphate Mannose 6-phosphate 2-deoxy-D-glucose
n.d. n.d.
* T h e s u m o f r a d i o a c t i v i t y f o u n d in all l o w - m o l e c u l a r w e i g h t p r o d u c t s f o r m e d w i t h t h e g i v e n a c c e p tot. ** A m i x t u r e o f c ~ M a n ( 1 - - * 2 ) ~ M a n ( 1 - - + 2 ) m a n n o s e a n d ~Man(1--*3)c~Man(1--~2)mannose o b t a i n e d b y acetolysis of yeast mannan [17].
577 to serve as acceptors. The small amounts of radioactive disaccharides found in the incubation mixtures when a-mannose phosphates were tested as acceptors can be ascribed to the presence of traces of mannose in the samples of mannose phosphates used rather than to their acceptor ability. The highest acceptor affinity was found with a-glycosides of mannose; methyl-a-mannose and Nph-a-mannose.
Structure o f the products Sodium borohydride reduction of the oligosaccharide products followed by acid hydrolysis resulted in a complete liberation of radioactive mannose indicating that the newly attached radioactive mannosyl moieties were present at non-reducing termini of the reaction products. Upon incubation with sweet almond emulsin a-mannosidase the products completely liberated radioactive mannose indicating exclusive formation of a-mannosidic bonds. For the purpose of structure determination the mannobiose was prepared in scaled up incubation mixture using D-[1-14C]mannose as the acceptor and nonradioactive GDPmannose as the mannosyl donor. The formed disaccharide was isolated and purified by means of preparative paper chromatography. Upon sodium borohydride reduction followed by acid hydrolysis the disaccharide yielded D-[1-14C]mannitol as the only radioactive product. The disaccharide cochromatographed well with authentic aMan(1-*2)mannose in solvent system B. Its structure was further confirmed by periodate oxidation. The disaccharide (40 000 cpm) was reduced with NaBH4 and then oxidized in 1 ml of 0.1 M acetate buffer (pH 5) containing 0.02 M sodium periodate. After 72 h at 4°C excess NaBH4 was added to reduce remaining periodate and the formed aldehydes. After overnight standing at room temperature the mixture was desalted over a mixed-bed resin and evaporated to dryness under reduced pressure. The residue was dissolved in 0.5 ml of 2 M HC1 and the hydrolysis was carried out at 100°C for 3 h in a sealed tube. After removal of HC1 by repeated evaporation with methanol the mixture was spotted on the chromatographic paper and the chromatography was carried out in the solvent system C for 8 h. Measurement of the radioactivity on the paper chromatogram revealed the presence of [14C] glycerol as the only radioactive product of the periodate oxidation of the disaccharide. When the radioactive disaccharide was mixed with authentic aMan(1-~2)mannose and aMan(1-*6)mannose and heated with phenylhydrazine hydrochloride under conditions described by Kuhn and Baer [23] practically all radioactivity remained in the supernatant over the precipitated phenylosazone of aMan(1-~6)mannose. These results give rather strong evidence that the h y d r o x y l group at C-2 of reducing mannosyl unit in the investigated disaccharide is blocked by a glycosidic b o n d and, consequently, that the structure of disaccharide formed from mannose and GDPmannose is O-a-D-mannosyl-(1-~2)-mannose. The trisaccharides for structure determination were prepared from GDP[U34C] mannose as mannosyl donor and aMan(1-~2)mannose and aMan(1-~6)mannose as the respective acceptors. The nature of the newly formed mannosidic bonds was deduced indirectly, on the basis of the relative stability of formed trisaccharides towards acetolysis. The kinetic data obtained by Rosen-
578 feld and Ballou [24] with different oligosaccharides show that, under conditions of acetolysis, the a(1-~6)-glycosidic linkage in mannooligosaccharides is split about 280 times faster than a(1-*2)-bond and, at the same time, about 21 times faster than a (1-~ 3)-bond. The radioactive trisaccharides were acetylated [24] and subjected to acetolysis in the mixture of acetic anhydride/acetic acid/H2SO4 (10 : 10 : 1, by vol.). After 16 h of acetolysis at 40°C the products were deacetylated and resolved b y paper chromatography in solvent system A. Over 97% of the trisaccharide prepared from aMan(1-~6)mannose was hydrolysed in the course of described procedure releasing radioactive mannose and traces of radioactive mannobiose. On the other hand, about 41% of the trisaccharide prepared from aMan(1-~2)mannose remained unhydrolysed under the same conditions the remaining 59% decomposed to radioactive mannose (50%) and radioactive mannobiose (9%). These results indicated that in case of trisaccharide formed from aMan(1-~ 6)mannose the nature of the newly formed glycosidic bond is a(1-*6) and hence the structuTe of the trisaccharide is aMan(1-*6)aMan(1-*6)mannose which is reflected in its high lability towards acetolysis. With the trisaccharide from aMan(1-~2)mannose the situation seems to be more complicated. If the newly formed bond in the trisaccharide were exclusively a(1-~2), over 99% of it should remain intact after 16 h acetolysis. However if it were exclusively a(1-*3) bond that was formed in the course of mannosyl transfer to aMan(1-*2)mannose, there should remain theoretically only about 38% of unhydrolysed mannotriose and practically no mannobiose after 16 h acetolysis (calculated on the basis of the rate constants given in ref. 24). We supposed therefore that in the latter case it is reasonable to assume that both a(1-~ 3 ) a n d a(1-~ 2) bonds were formed with a aMan(1-* 2)mannose as acceptor, the a(1-~3) bonds being more preferred. Whether this is due to unspecificity of corresponding mannosyltransferases towards acceptor or to heterogeneity of the sample of mannobiose used as the acceptor is n o t known.
Effect o f pH and metal ion requirement The observation that different kinds of linkages were formed with different acceptors promted us to investigate in more detail the properties of the individual mannosyltransferasees. As can be seen from the Fig. 1, the enzymes catalyzing the mannosyl transfer from GDPmannose to mannose, aMan(1-~6) mannose and to aMan(1-~2)mannose, respectively, differ in the shape of their pH-activity curves. In all investigated reactions Mn 2+was the best activator. In the reactions with aMan(1-~2)mannose and methyl-a-mannose Mg 2÷ activated to about 15% of the maximal activity with Mn 2÷ while with aMan(1-*3)mannose the Mg 2÷ activated the reaction to 30% of the value obtained with Mn 2÷. Thermal inactivation studies The diversity of the enzymes catalyzing the mannosyl transfer to different acceptors can be well observed also in the kinetics of thermal denaturation of the corresponding enzymes. The particulate enzyme preparation suspended in 0.05 M Tris. HC1 buffer (pH 7.2) was heated at 45°C and at different time intervals the samples were withdrawn and the activity of the enzyme towards
579
I
I
I
I
I
I
I
A 3
e~
2 o m
~=
1
m =IE
0 6 E
4 2 m
0
I -O"-~l
I
t
I
i
i
6
7
8
9
10
8 x
6
=
4
E ==-
5
Fig. 1. E f f e c t o f p H o n t h e rate o f m a n n o s y l t r a n s f e r t o A , m a n n o s e ; B, ~ M a n ( 1 - * 6 ) m a n n o s e ; C, ~ M a n ( 1 - ~ 2 ) m a n n o s e . ( o ) s u c c i n a t e ; ( e ) i m i d a z o l e / H C l ; ( ~ ) Tris - HC1; (A) g l y c i n e / N a O H b u f f e r s . T h e c o m p o s i t i o n o f t h e r e a c t i o n m i x t u r e s w a s t h e s a m e as in t h e s t a n d a r d assay e x c e p t t h a t t h e c o n c e n t r a t i o n o f t h e r e s p e c t i v e b u f f e r w a s 80 r a M .
different acceptors was determined in a standard incubation mixture. As can be seen in the Fig. 2, the rate of mannosylation of the endogenous acceptors dropped rapidly upon the heat treatment of the enzyme. The observed decrease in the,mannoprotein synthesis was almost paralleled by the inactivation of the enzyme catalyzing the transfer of mannosyl units to aMan(1-~6)mannose. The activity of the enzyme functioning with aMan(1-*2)mannose as acceptor was much less decreased while the enzyme catalyzing the monnosyl transfer to mannose was practically unaffected by the heat treatment. The latter two reactions were even slightly stimulated by short-term heat treatment of the particulate enzyme preparation.
Inability of free mannooligosaccharides to enter the tnannoprotein molecule Free radioactive mannose when incubated with the enzyme in the presence of nonradioactive GDPmannose was observed to enter into the endogenous mannoprotein. The observed incorporation was, however less than 2% of the
580
1.0 ,8
.4
.]
I
I
I
0
5
10
I
15 mitt it 45"C
Fig. 2. E f f e c t of h e a t t r e a t m e n t of t h e p a r t i c u l a t e e n z y m e p r e p a r a t i o n
o n t h e r a t e of m a n n o s y l t r a n s f e r
f r o m G D P - [ U - 1 4 c ] m a n n o s e to: (-~.) m a n n o s e ; (A) ~ M a n ( l ~ 2 ) m a n n o s e ; endogenous mannoprotein.
(©) c~Man(1-~6)m~nnose; a n d (e)
rate measured with the same concentration of GDP-[U-14C]mannose. Radioactive aMan(1-*2)mannose isolated from large-scale incubation mixtures, as well as radioactive aMan(1-~6)mannose prepared by acid reversion of [14C]mannose [18] did not incorporate into the mannoprotein under the same conditions. Discussion
The observation made by Lehle and Tanner [10] and confirmed by us, that mannose and mannose-containing low-molecular weight acceptors are able to serve as mannosyl acceptors from GDPmannose in reactions catalyzed by yeast mannan synthetase, opens the possibility to investigate the properties of the individual mannosyltransferase participating in the biosynthesis of yeast mannan. Our experiments have confirmed that a-configuration of the acceptor mannosyl unit is necessary for the enzyme-catalyzed mannosyl transfer. The observation that oligosaccharides containing an a(1-~3) linked nonreducing terminal mannosyl unit are relatively poor acceptors may explain w h y a(1-*3)-linked mannosyl units are located predominantly at the nonreducing ends of ~-elimin-
581
able oligosaccharides and side chains of the polymannose part in the yeast mannan protein . The observed relatively high activity of mannosyltransferases towards a-glycosides of mannose, namely methyl* -mannose and Nphe -mannose could be explained as due to lower polarity of these compounds as compared with that of mannose and mannooligosaccharides. It can be anticipated that compounds with decreased polarity penetrate easier than neutral sugars into the membrane-bound enzyme complex. Also important is the observation that none of the monodeoxyanalogues of mannose was able to serve as acceptor in the investigated enzyme system. Biely et al. [25] have found that one of these compounds, namely 2-deoxy-Dglucose enters into the yeast mannoprotein in vivo. They observed, however, that 2-deoxy-D-glucose was incorporated almost exclusively into the polymannose part of mannoprotein but not into the oligosaccharides attached to serine and/or theonine, The 2-deoxy-D-glucose incorporated into the polysaccharide moiety of yeast manno protein was located mostly in the side chains and in many cases it was further glycosylated by mannose. It can be therefore assumed that the enzymes active with D-mannose but inactive with its 2-deoxyanalogue as acceptor are most probably involved in the biosynthesis of P-eliminable carbohydrate moiety rather than in the formation of polysaccharide part of mannoprotein. Hence, the enzyme catalyzing the formation of ar(l+2)-bond with D-mannose as acceptor may be considered as being involved in the biosynthesis of P-eliminable saccharides attached to serine and/or threonine. The second investigated enzyme, namely that catalyzing the formation of e( l-+6)-linkage with exogenous aMan(l+6)mannose as acceptor is most probably participating in the biosynthesis of cr(l-+6)-linked main chain in the polysaccharide part of mannoprotein. This assumption comes from the fact that cr(l+6)-glycosidic bonds were not found in the oligosaccharides attached to serine and threonine [26,27]. The observation that the kinetics of thermal inactivation of this enzyme is almost parallel with decrease in the rate of mannoprotein synthesis caused by heat treatment of the enzyme gives another support to the above assumption. The formation of aMan( l+ 3)cuMan(1+2)mannose and aMan( l- 2)cyMan (1-+2)mannose from cYMan(l+2)mannose can be considered as a result of simultaneous action of two respective mannosyl transferases on the same acceptor. On the basis of the present information it is difficult to assign a precise role to these two enzymes in the biosynthesis of yeast mannoprotein. Experiments with cross-inhibition (Farkas, V., unpublished results) have shown that the enzyme catalyzing the formation of a(l+2)-glycosidic bonds with uMan(l+B)mannose as acceptor is not identical with the a(1+2)mannosyltransferase acting with D-mannose as acceptor. Perhaps the most interesting results were obtained with thermal inactivation studies. All three investigated reactions with exogenous acceptors exhibited different kinetics of thermal inactivation. This fact documents well the diversity of mannosyltransfereases participating in the biosynthesis of yeast mannan. It is noteworthy that although the rate of overall mannan biosynthesis was considerably reduced by heat treatment of the enzyme, almost no change in the
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amount of radioactivity incorporated into fl-eliminable saccharidic moiety was observed [ 2 8 ] . Our failure to demonstrate'the incorporation of labelled aMan(1-~2)mannose and aMan(1-+6)mannose into mannoprotein in the complete incubation mixture rules out the probability that these oligosaccharides in the free state might be the precursors or intermediates in the biosynthesis of yeast mannan. The observed slight incorporation of free mannose into mannoprotein observed under identical conditions can be explained as due to the mannosyl exchange at the level of dolichol monophosphate mannose which plays a role of intermediate in the biosynthesis of mannoprotein [ 3,5--9]. 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
Behrens, N.H. a n d Cabib, E. ( 1 9 6 8 ) J. Biol. Chem. 2 4 3 , 5 0 2 - - 5 0 9 K o z a k , L.P. a n d B r e t t h a u e r , R.K. ( 1 9 7 1 ) B i o c h e m i s t r y 9+ 1 1 1 5 - - 1 1 2 2 S e n t a n d r e u , R. a n d L a m p e n , J . O . ( 1 9 7 1 ) FEBS L e t t . 14, 1 0 9 - - 1 1 3 B r e t t h a u o r , R . K . a n d Chen Tsay, G. ( 1 9 7 4 ) A r c h . B i o c h e m . B i o p h y s . 1 6 4 , 1 1 8 - - 1 2 6 T a n n e r , W. ( 1 9 6 9 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 35, 1 4 4 - - 1 5 0 T a n n e r , W., J u n g , P. a n d Behrens, N.H. ( 1 9 7 1 ) FEBS Lett. 1 6 , 2 4 5 - - 2 4 8 J u n g , P. a n d T a n n e r , W. ( 1 9 7 3 ) Eur. J. B i o c h e m . 37, 1--6 B r e t t h a u e r , R.K.+ Wu, S. a n d Irwin, W.E. ( 1 9 7 3 ) Biochim. B i o p h y s . A c t a 304, 7 3 6 - - 7 4 7 S h a r m a , C.B., B a b c z i n s k i , P., Lehle, L. a n d T a n n e r , W. ( 1 9 7 4 ) Eur. J. B i o c h e m . 46, 35---41 Lehle, L. a n d T a n n e r , W. ( 1 9 7 4 ) Biochim. B i o p h y s . A c t a 3 5 0 , 2 2 5 - - 2 3 5 S c h u t z b a c h , J.S. a n d Ankel+ H. ( 1 9 7 1 ) J. Biol. Chem. 2 4 6 , 2 1 8 7 - - 2 1 9 4 S c h u t z b a c h , J.S. a n d A n k e l , H. ( 1 9 7 2 ) J. Biol. Chem. 2 4 7 , 6 5 7 4 - - 6 5 8 0 H e a r n , V.M., S m i t h , Z.G. a n d Watkins, W.M. ( 1 9 6 8 ) B i o c h e m . J. 1 0 9 , 3 1 5 - - 3 1 7 K o b a t a , A., G r o l l m a n , E.F. a n d G i n s b u r g , V. ( 1 9 6 8 ) A r c h . B i o c h e m . Biophys. 1 2 4 , 6 0 9 - - 6 1 2 Nakajima+ T. a n d Ballou, C.E. ( 1 9 7 4 ) J. Biol. Chem. 2 4 9 , 7 6 7 9 - - 7 6 8 4 N a k a j i m a , T. a n d Ballou, C.E. ( 1 9 7 4 ) J. Biol. Chem. 2 4 9 , 7 6 8 5 - - 7 6 9 4 K o c o u r e k , J. a n d Ballou, C.E. ( 1 9 6 9 ) J. Bacteriol. 1 0 0 , 1 1 7 5 - - 1 1 8 1 J o n e s , J . K . N . a n d N i c h o l s o n , W.H. ( 1 9 5 8 ) J. C h e m . Soc. 2 7 - - 3 3 K a r k k ~ i n e n , J. ( 1 9 7 0 ) C a r b o h y d r . Res. 14, 2 7 - - 3 3 Biely, P., Kr~itky, Z.+ Kova~ik, J. a n d Bauer, S. ( 1 9 7 1 ) J. Bacteriol. 107, 1 2 1 - - 1 2 9 L o w r y , O.H., R o s e b r o u g h , N.J., F a r r , A.L. a n d R a n d a l l , R.J. ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 Neame, K.D. a n d H o m e w o o d , C.A. ( 1 9 7 4 ) Anal. B i o c h e m . 5 7 , 6 2 3 - - 6 2 7 K u h n , R. a n d Baer, H.H. ( 1 9 5 4 ) C h e m . Ber. 87, 1 5 6 0 - - 1 5 6 7 R o s e n f e l d , L. a n d Ballou, C.E. ( 1 9 7 4 ) C a r b o h y d r . Res. 2 8 7 - - 2 9 8 Biely, P., K r ~ t k y , Z. a n d Bauer, S. ( 1 9 7 4 ) Biochim. Biophys. A c t a 3 5 2 , 2 6 8 - - 2 7 4 R a s c h k e , W.C., K e r n , K . A . , Antalis, C. a n d BaUou, C.E. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 4 6 6 0 - - 4 6 6 6 Ballou, C.E., K e r n , K . A . a n d R a s c h k e , W.C. ( 1 9 7 3 ) J. Biol. C h e m . 248, 4 6 6 7 - - - 4 6 7 3 Farka~, V. Bauer, ~. a n d V a g a b o v , V.M. ( 1 9 7 6 ) B i o c h i m . Biophys. A c t a 4 2 8 , 5 8 3 - - 5 9 0