146

Bioehimica et Biophysica Acta, 1075(1991) 146-153 © 1991ElsevierSciencePublishersB.V.All rightsreserved0304-4165/91/$03.50 ADONIS 0304416591002370

Characterisation of Oligosaccharides from Drosophila melanogaster Glycoproteins P h i l l i p J. W i l l i a m s l, M a r k R . W o r m a l d t, R a y m o n d A . D w e k t, T h o m a s W . R a d e m a c h e r I, G e o r g e F. P a r k e r 2 a n d D a v i d R . R o b e r t s 2 Glycobiology Unit, Department of Biochemistry, Unh'ersityof Oxford, Oxford (U.h:) and " Department of Genetics, Unirersity of Oxford, Oxford ( U.K.)

(Received7 February 1991) Key words: Oligosaccharide;Characterization;Glycoprotein;(1). melanogaster) An analysis of the released oligosaccharides from a membrane glycoprotein preparation of third instar larvae (3rdlL), and purified larval serum protein 2 (LSP2) from Drosophila melanogaster was performed. Sequential exoglycosidase digestion in combination with high-resolution gel permeation chromatography and partial acetolysis indicated the presence of two series of oligomannosides; one of these series was unusual and characterized by the presence of a core ~ 1 - 6 linked fucose, the other was a typical mammalian oligomannose series containing the following isomers - D r , - D z , - D t z , - D t ~ and - C D t ~ as well as the unprocessed Man9GIcNAc 2 structure. Conventional oligomannose could only be detected in the LSP2 sample. This study opens the way to use powerful molecular and classical genetic techniques to analyse the control and functional significance of glycosylation in higher organisms. Introduction The importance of glycosylation in modifying and controlling discrete biological functions has been strongly implicated in many cases [1]. However, most of these observations come from in vitro studies which show few of the biological features of organisms such as development and behaviour. In an effort to understand more about the role of glycosylation in general we have analysed the oligosaccharides from various preparations of D. melanogast~r. This study has enabled us to characterize the oligosaccharides present and identify the putative glycosyltransferases and glycosidases necessary for their synthesis. The long-term aim is to produce and study mutant strains deficient in specific enzymes involved in protein glycosylation. These mutants should be recessive lethals, but by selecting conditional mutants, e.g., temperature-sensitive

Abbreviations: g.u., glucose units; HRP, horseradish peroxidase; LSP2, larval serum protein 2; GlcNAc-ol, N-acetylglucosaminitol; GIcNAc. N-acetylglucosamine:Man, mannose; Xyl, xylose; Fuc, fucose. Correspondence: P.J. Williams, GlycobiologyUnit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OXI3QU, U.K.

mutants, it will be possible to study the consequence of blocking glycosylation (i) at different developmental stages and (ii) on adult behaviour. Combining conditional mutations and the technique of somatic crossing over will allow us to compare the effect of blocking glycosylation in one structure, while the paired structure in the same organism is normal. Insect glycosylation has received little attention, although the use of insect cell lines and the baculovirus vector system in the production of recombinant glycoproteins has prompted new research in this area. There is strong evidence that a mosquito cell line can produce N-linked oligomannose structures and possibly Nacetylglucosamine O-linked structures [2]. N-Acetylglucosamine has also been observed in O-linkage on as yet undefined D. melanogaster chromosomal proteins [3]. Oligosaccharide-linked dolichol derivatives have been detected, again from mosquito cell lines, indicating that glycoprotein assembly in insects may be analogous to that of higher organisms [4,5]. Complex and hybrid N-linked oligosaccharides have not been observed on mosquito glycoproteins from cell lines, neither have sialyltransferase, galactosyltransferase and N-acetylglycosaminyl transferase activities been detected [5]. Terminal N-acetylglucosamine and fucose residues have been reported on N-linked oligosaccharides from bee venom phospholipase A2 [6]. There is

147 recent evidence that insects can, produce complex Nlinked oligosaccharides [7]. Oligosaccharides were analysed from human plasminogen expressed in a baculovirus-infected lepidopteran cell line, they observed various oligomannose structures, however, 40% of the structures were complex sialylated biantennaries. Antibodies to horseradish peroxidase (anti-HRP) can be used as a neural specific probe in D. melanogaster embryonic neurons [8]. It is now known that anti-HRP can immunoprecipitate at least seventeen different membrane glycoproteins from D. melanogaster embryo central nervous system and that anti-HRP recognizes a neural specific carbohydrate epitope expressed on these glycoproteins [9]. This epitope may be structurally related to M a n a 6 (Man a3XXylfl2)Man/34GIcNAc/34(Fuca3)GIcNAc-ol, which is the major N-linked oligosaccharide of HRP [10]. The/32xylosyl and a3fucosyl residues are thought to be key immunogenic components [11]. Their presence in D. melanogaster has yet to be documented. Carbohydrate analysis of glycoproteins obtained from insect cell lines, which are derived from a single tissue, may not be representative of the pattern of N-linked glycosylation in the whole animal. In this paper we use hydrazine to release oligosaccharides from a D. melanogaster membrane glycoprotein preparation of 3rd instar larvae, and purified larval serum protein, 2 (LSP2), and carry out initial sequence analyses. Material and Methods

Membrane protein preparations D. melanogaster larvae were collected as described previously [12]. The preparation was extensively washed to remove yeast and medium, and then homogem'zed in 50 mM Tris-HCI (pH 8.5) with 0.25 M sucrose and 1 mM EDTA using a teflon glass homogenizer with 0.5 mm clearance. The homogenate was passed through a 750/~m mesh filter and the residue (mainly intact D. melanogaster tissues) was retained by a 100 p.m mesh filter, and homogenised as before using the teflon glass homogenizer. The homogenate was filtered through four layers of cheese cloth and the filtrate was centrifuged at 1000 × g for 10 rain. The supernatant was centrifuged at 1 0 0 0 0 × g for 20 min. The pellet was resuspended in the same buffer and rccentrifuged. The subsequent pellet was suspended in 10 mM Tris-HCI (pH 8.0) as the membran e preparation. The membrane preparation underwent extraction with five volumes of acetone at 4~C (three times), the denatured proteins were resuspended in water and dialysed exhaustively ( M r 12000 cut off) against water at 4°C.

LSP2 was purified as described by Akam et al. [13] with the following modifications: (a) the initial extract

was prepared from LSPI ° larvae which lack the major contaminating protein LSP1 (Roberts et al., unpublished data), and (b) the preparation was not subjected to isoelectric focussing.

Isolation of oligosaccharides Protein samples (1-5 rag) were cryogenically dried prior to release of oligosaccharides using double vacuum distilled anhydrous hydrazine. Released oligosaccharides were purified and radiolabelled by reduction with tritiated sodium borohydride (6-15 Ci/mmol, New England Nuclear). A detailed methodology and Bio-Gel F-4 chromatography conditions have been reported previously [14].

Oligosaccharide analysis Reduced oligosaceharides were subjected to high voltage paper electrophoresis to separate anionic from neutral oligosaccharides. The neutral oligosaccharides were eluted and applied to a Bio-Gel P-4 high-resolution gel filtration column. All chromatographic runs were standardized with a partial dextran hydrolysate. All digests were performed on 1.0-0.05 - 106 cpm of 3H-labelled oligosaccharides (approx, 1.0-0.05 nmol) at 37°C for 18 h under a toluene atmosphere. Jack bean /3-galactosidase, jack bean a-mannosidase, jack bean /3-he×osaminidase, Achatina fidica /3-mannosidase, Arthr6bacter ureafaciens neuraminidase and Aspergillus phoenicus a-mannosidase sources, purification, specificities and digest conditions have been previously reported [15]. Charonia lampas ot-fucosidase digests were performed in 10 p,I of 50 mM sodium acetate b~:fi~-r a,~ ~H 4~5 ,vith _~50mM ~.,xliuw c:~oride at 6 units/ml, the puriheation procedure will shortly be reported elsewhere (Butters et al., unpublished data). The following enzymes were used direct from source. Bovine kidney a-fucosidase (Boehringer-Mannhelm) digests were performed in 20 /~1 of 200 mM sodium acetate buffer (pH 5.0) at 10.0 units/ml. Sweet almond fl-glueosidase (Boehringer-Mannheim) digests were performed in 20 p,I of 100 mM citrate buffer (pH 4.5) at 20 units/ml. Bovine epididymal a-fucosidase (Sigma) digests were performed in 10 ttl of 200 mM citrate phosphate buffer (pH 6.0) at 2 units/ml. Streptomyces plicams endoglycosidase H (Boehringer-Mannheim) digests were performed in 20 /~1 of 50 mM acetate buffer (pH 5.5) at 100 munits/ml. High performance anion-exchange chromatography (ItPAEC) was used to analyse fucose linkage isomers, this consisted of a Dionex gradient pump and a PA-I column run isocratieally in 3.75 mM sodium hydroxide. Acetolysis was performed on the tentatively identified oligomannose structures from the LSP2 preparation. Individual peaks were pooled and then re-chromatographed to determine accurate initial elution volumes. 100-200 pmol of each structure was aeetylated

148 in 40 p,I acetic anhydride and 40 /.LI of pyridine at 100°C for 20 min, and then subjected to partial acetolysis by the method of Zamze et al. [16], with the slight modification of 3.5-h and 7-h time points, Results High voltage paper electrophoresis (HVE) was performed on the oligosaccharides derived from the two preparations of D. melanogaster as shown in Fig. 1. The 3rd instar larvae preparation (Fig. la) showed a single peak that co-migrated with [3H]lactitol standard, very small amounts of anionic material could be seen in the LSP2 sample (Fig. lb). FPLC analysis using a Pharmacia Mono-Q column confirmed that single- and double-negatively charged species existed (data not shown), and in conjunction with the relatively large migration distances on H V E it seems most likely that these charged oligosaccharides are small. This was confirmed in a D. melanogaster embryo preparation where considerable anionic components were observed (unpublished observation). These structures were partially susceptible to neuraminidase and the resulting neutral sugars eluted as mono- or disaccharides on Bio-Gel P-4 (peaks at 1.5 g.u. and 2.3 g.u.). It is therefore possible that small sialylated oligosaccharides exist in D. melanogaster, this observation is at present under further investigation. The neutral fractions from 3rd instar larvae (Fig. la), and LSP2 (Fig. lb) preparations gave complex profiles containing several major peaks when subjected to Bio-Gel P-4 high-resolution gel permeation chromatography (Fig. 2a and b, respectively). To characterize these oligosaccharides further the 3rd instar larval preparation was analysed in detail using endo-

Refenti0n ffme(rnin) Fig. 2. Bio-Oel P4 profiles of neutral oligosaccarides from; (a) 3rd instar larvae, numbered bars (1) refer to pools mentioned in text, and

(b) LSP2. The numbers at the top of each profile in this and future figures indicate the elution positions of non-radioactive standard glucose oligomers(g.u.) detected by refractive index monitor (model ERC 7510 HPLC Technology,U.K.). The radioactivityoligosaccharide alditols were detected by a Berthold LBS03 radioactive flow monitor (Berthold, U.K.). and exo-glycosidases. The profile (Fig. 2a) can be divided into three main sections 12.5-8.9 g.u. (I), 8.2-6.5 g.u. (ll) and 6.0-4.0 g.u. (liD. The structures eluting between 12.5-8.9 g.u. were pooled and digested with jack bean a-mannosidase, a single product peak at 5.5 g.u. co-migrating with the standard oligosaccharide Manf14GlcNAc/34OlcNAc-ol, was found. (Fig. 3a). The 12.5-8.9 g.u. pool was also digested with A. phoenicus ot-mannosidase, an enzyme that cleaves M a n a l - 2 linkages, and again, a single product eluting at 8.9 g.u co-migrated with the standard oligosaccharide

Mana3(Mana6)Mana6(Mana3)Man~4GlcNAc134.

LSP2

Migrationdisl'nnce(cm) Fig. 1. High voltage eletrophoretogramsof all-labelled insect oligosaccharide alditols. The oligosaccharideswere subjected to high voltage electrophoresis (80 V cm-l) in pyridine/acetic acid/water (3: 1:387, by volume)pH 5.4. (a) 3rd instar larvae and (b) LSP2. The arrows indicate the positions of [aH]laetitol (L), 6'(3')-sialyl[3Hllactitol (SL) and Bromophenolblue (BPB) markers.

GIcNAc-ol (see Fig. 3h). The 12.5-8.9 g.u. pool when treated with endoglycosidase H (see Fig. 3c), gave a single product eluting at 2.5 g . u (i.e., GlcNAc-ol). This data taken together with the initial Bio-Gel P-4 elution positions, which correspond to known standard elution volumes [17], strongly suggests that these structures are an oligomannose series. Digestion of the 8.2 g.u. peak (Fig. 2a) with jack bean a-mannosidase yielded three components (see Fig. 4a). The resultant 6.5 g.u. peak, when digested with bovine epididymal, Charonia lampas or bovine kidney ct-fucosidase gave a product eluting at 5.5 g.u. (Fig. 4b). In addition the 6.5 g.u. peak was susceptible to digestion with A. fulica fl-mannosidase followed by jack bean/3-hexosaminidase giving a product at 3.5 g.u. (Fig. 4c). Treatment of this product with bovine epididymal a-fucosidase yielded a final product at 2.5 g.u.

149

12 1110

9 8 ?

could not be digested with any of the enzymes used on the similarly resistant 8.2 g.u. peak. The structures eluting at 6.5 g.u. (see Fig. 2a) were partially susceptible to jack bean a-mannosidase (Fig. 5c). With a product eluting at 5.5 g.u. indicating the loss of a single a-mannose residue. The remainder at 6.5 g.u. was also resistant to jack bean /3-hexosaminidase, A. fulica /3-mannosidase, almond .8-glucosidase and bovine epididymal a-fucosidase.

11 ~I 9

lallll

8

7

0

S

Z,

i

i

I

3 l

2 i

b Refenti0~ time Lnin}

Fig. 3. Bio-Gel P4 profiles showing sequence data for pool (|) from Fig. 2a. Digestion with (a) jack bean a-mannosidase, (b) A. phocnicus a-mannosidase and (c) endoglyeosidase H.

(Fig. 4d) confirming the location of the fucose residue to the reducing terminal GIcNAc-ol. The resultant 2.5 g.u. peak was identified as GlcNAc-ol by borate electrophoresis (data not shown). The major component of the 8.2 g.u. peak would therefore be consistent with t h e structure ( M a n a ) 2 M a n / 3 4 G l c N A c ~ 4 ( F u c a 6 ) GlcNAc-ol. The 8.2 g.u. peak that was resistant to jack bean a-mannosidase was also resistant to jack bean /3-hexosaminidase, A. fulica /3-mannosidase, almond /3-glucosidase and bovine epididymai a-fucosidase and consequently has not been assigned. The structure giving rise to the 5.5 g.u product following jack bean a-mannosidase treatment would be consistent with the structure (Mana)3 Man fl4GIcNAcfl4GlcNAc-ol. The structures eluting at 7.2 g.u. (see Fig. 2a) when digested with jack bean a-mannosidase yielded three main peaks (Fig. 5a). The major peak at 5.5 g.u. lost two a - m a n n o s e residues, while the 6.5 g.u. peak lost one a - m a n n o s e residue and could be further digested with bovine epididymal a-fucosidase to a product eluting at 5.5 g.u. (Fig. 5b). The 7.2 g.u. region therefore contains structures consistent with (ManoDzMan~4 GIcNAe~4GIcNAc-ol and (Manot)~Man,a4GleNAc/34 (Fuea6)GIcNAc-ol. The activity remaining at 7.2 g.u.

Retention time (rain)

Fig. 4. Sequence data for the 3rd instar larvae derived 8.2 g.u. region. (a) shows the 8.2 g.u. region post jack bean a-mannosidase; (b) the 6.5 g.u. region derived from Fig. 4a post bovine epididymal ~fucosidase: (c) is a further aliquot of the 6.5 g.u. region post snail //-mannosidaseand jack bean/3-bexosaminidase;and (d) the activity at 3.5 g.u. digestedwith bovineepididymalcz-fucosidase.

150

,0o00

i;

n

1~00" 10000-

~

.

sooo. •

.

200, 1500 1ooo. .~ sop ~-

o

,

C

3000

20OO 1500 1000

5Oo7

.

'°°l 5~

R e t e n t ~ time (r~n)

Fig. 4a) could be digested with C. lampas ot-fucosidase, bovine epididymal t~-fucosidase and bovine kidney afucosidase. Dionex high-performance anion-exchange chromatography was used to separate authentic o~-3 and a-6 linked core fucosylated standards (see Fig. 6a, b and c). Man/34GlcNAcf14(Fuct~6)GlcNAc-ol was prepared by sequential digestion of N-linked oligosaccharides from human immunoglobulin G with A. ureafaciens neuraminidase, jack bean fl-galactosidase, jack bean hexosaminidase, and jack bean ct-mannosidase. Manfl4GlcNAcfl4(Fucoc3)GlcNAc-ol was prepared by d i g e s t i o n of Mant~6(Mana3) Manfl4GlcNAcfl4(Fuca3)GlcNAc-ol from HRP with jack bean a-mannosidase. The 6.5 g.u. peak obtained from the jack bean a-mannosidase digest of the 8.2 g.u. fraction from the 3rd instar larvae preparation when run against these standards on the PAl column, confirmed that the 3rd instar larvae sample contained a core fucose linked pal-6 (Fig. 6d). Table l summarizes

i

I

.

d

3OO 200

Fig. 5. Bio-Oel P4 elution profiles of the 7.2 and 6.5 g.u. region derived from the 3rd instar lan,ae preparation (sec Fig. 2a). (a) 7.2 g.u. post jack bean a-mannosidase,(b) 6.5 8.u. region from Fig. 5a post bovine epididymal a-fucosidase,and (c) the 6.5 g.u. region from Fig. 2a postjack bean ct-mannosidase.

The fucose linkage to the reducing terminal Nacetylglucosamine was of key interest especially in the context of a potential anti-HRP cross reacting epitope. The 8.2 g.u. fucosylated structure (product at 6.5 g.u.

.

o

2~

/,8 72 % 120 I~ I ~ Retenh0n time {sees}

Fig. 6. Core fucose linkage analysis by H P A E C of the 6.5 g.o. region from Fig. 4a. a-d show the elution positions of authentic a - 6 linked core fucose, authentic a-3 [inked core fucose, co-injection of the

previous two standards and injection of the 6.5 g.u. region, respectively.

the structures determined from the 3rd instar larvae preparation. The smaller oligosaccharides eluting between 6.0-4.0 g.u. (Fig. 2a) are still being analysed. Initial observaTABLE 1 N-linked olisosaccharide structures determined from 3rd instar larvae Bio-Gel Structure % of P-4 elution Fraction position/ fraction 12.5-8.9 (MantY)s-4Man/34GlcNAc/34GIcNAc-ol > 95 8.2 (Mana)3Mao/34GlcNAcf14GIcNAc-ol 23 (Manta)2Man/]4GIcNAc/34(Fuca6)GIcNAc-ol 42 7.2 (Mana)2Man~4GIcNAc~4OIcNAc-ol 50 6.5

M a n a ~4an/34GIcNAc/34(Fuc o~6)GlcNAc-o[

26

Mana Man/34GlcNAc,84GlcNAc-ol

55

151 tttttti)

t t

t

t

The data indicate that the oligomannose structures on LSP2 comprise one isomer of Man,)GIcNAc/34 GIcNAc-oL two isomers of MansGlcNAc/34GIcNAc-ol (82% - D ? . 18% - D 1 ) , and one main isomer of Man 7(31cNAcfl4GIcNAc-ol, Man6GIcNAc/34GIcNAcol and MansGlcNAc/34GlcNAc-ol. Some minor heterogeneity was observed in the MansGlcNAc/34GlcNAc-ol and Man6GIcNAc~4GIcNAc-ol samples but this accounted for less than 10% of the totals. Assignment of these structures is dependant upon the structures being compatible with the known parent Manta2

t

it)frYe|tit

~.1 i i i

Mana6(Mana2Mana3)Mana6(Mana2Mana2Mana3) ~ltttt)

t t ) t

t

ttil,~lli('

t t t

i

)

Man/34GIcNAc/34GIcNAc-ol structure. Discussion

Retention hme (mini

Fig. 7. Bio-Gel P4 elution profiles of LSP23H-labelled oligomannose series after partial acetol~,sis,a, e, e, g and i. show the (Man)s_,~GIcNAcGleNAc-ol fragments after 3.5 h aeetolysis,respectively,and b, d, f, h and j, the same structures after the more complete 7 h acetolysis. Bold arrows indicate original elutior, position of starting material. tions, however, suggest that they may be a novel series o f / 3 1 - 4 linked glucose oligomers. The LSP2 Bio-Gel P-4 profile (Fig. 2b) shows the presence of structures eluting between 12.5-8.9 g.u. all these structures (as found previously for 3rd instar larvae) were susceptible to jack bean a-mannosidase and probably represented an oligomannose series. In order to confirm the presence of this series and determine the isomeric structures present these fractions were subjected to partial acetolysis. The acetolysis Bio-Oel P-4 chromatograms are shown in Fig. 7, and Table II shows the observed elution positions for the partial acetolysis products of the Mang_sGleNAe/34GlcNAc-ol structures. Computer simulation of the acetolysis results allows unambiguous assignment of the isomers shown in Table I1, when compared to a data set of all 42 daughter fragm e n t s o f Mana2Mana6(Mana2Mana3)Mana6(Man a 2Man a 2Man a3)Man/~4GIcNAc/34OIcNAc-ol.

Oligosaccharide analysis revealed that the major N-linked species were oligomannose, this is in agreement with the observation from mosquito cell lines [5]. We find little evidence for conventional complex, or hybrid-type structures, although a number of oligosaccharides were not characterized due to resistance to available exoglycosidases, these structures will be the subject of future studies. Further studies on the anionic species will also be necessary to document the anionic groups present (i.e., carboxyl, phosphate, sulphate, etc.). It must be stressed however that we looked at total protein fractions which may have hidden small amounts of more unusual structures, especially if their expression is restricted temporally or to a specific minor tissue. On the other hand to look at individual proteins could limit the range of oligosaccharides detected. For example, one of the major larval serum proteins LSP-2 is known to be glycosylated by the incorporation of labelled N-acetylglucosamine into the protein by fat bodies in vitro (Roberts, unpublished results). We analysed the oligosaccharides associated with the purified protein and found almost exclusively an oligomannose series and no fucosylated mannosides as found in 3rd instar larvae. It is not possible to determine oligomannose structures unambiguously from their monosaccharide composition, exoglycosidase sensitivities and acetolysis profiles. For example, their are 1428 isomers of (Mana2/3/6)sMan~4GIcNAc~4GIcNAc-ol and of these, only one gives an acetolysis Bio-Gel P-4 profile that is unique within this set of structures. This structural set becomes even larger if a l - 4 or fl linkages are considered, or if the basic core structure is not retained. An unambiguous structural determination, however, can be achieved if consideration is limited to a structural subset which contains the 42 daughter fragments of Mana2Manot6(Mana2Mana3)Mana6-

(Mana2Mana2Mana3)ManB4GlcNAcB4GIcNAc-ol, eight of which also occur in the (Mana2/3/6)s Man/34GIcNAe/~4OlcNAc-ol structural set discussed

152 TABLE II Obserced P4 eh~tion positions for the major acetolysis dericed fragments from the LSP2 oligomannose series

Man,~GIcNAc,ol (12.5) "

MansGIcNAc~ol (11.7)

Man 7GIcNAc2ol (10.8)

Man6GlcNAe2ol (9.9)

11.0 ~'

-Dj - Dz

-Di2

-DI23

7."6

10.010.0

7.6 8.6

9.2

7.6

9.2

7.6

MansGIcNAe2ol(8.9) -CDI: 3 8.2 6.6

~' Numbers in brackets are initial Bin-Gel P-4 elution positions in g.u. h The paired numbers represent the Bio-Oel P-4 elution positions in g.u. of the acetolysisfragments.The nnmbered letters, i.e., - D z refer to the mannose residue(s) missingfrom a particular isomer, see the Man~GIcNAc2olstructure drawn below. The followingisomers have been unambiguouslydetected given the constraints mentioned in the text. Manal-2Man al,,, o~

.

e Mana1%

Maflal.2M~Agal/3

/ ~ Manpl-4GIcNAo pl- 4GIcNAc-ol

Man Manal 01 al-2Marlal-2 c

Mana1-2Maria1%6 Manal ~., /3 6 Manl~l-4GIcHAcp1-4GtcNAc.ol / 3

Mana1-2Manal

Marl~l-2 Manal -D 1

MansGIcNAc-ol2

Manal-2Manal '%6 3 Manal %6 Maria1/ / a Man~l*4GIcNAc~1-4GIcNAe-ol Manal-2Manal-2 Maria1 -D 2

Maria1%'6 Man(x1 /3 '%6ManJ$1-4GIcNAcJ31-4GIcNAc-01 /3

Manal

Manal-2 Manal -D1 =s

above. Computer simulations of the acetolysis results for these 42 structures gives unique Bio-Oel P-4 profiles for all structures. The isomers tabulated in Table II are consistent with a controlled processing pathway. The data also show similarity with the processing patl'way in Trypenosoma brucei [16], with the same isomers being present although differences in the quantities of the MansOlcNAcOlcNAc-ol isomers were observed, i.e., - D 1 40% - D 2 60% in T. brucei and - D 1 18% - D 2 82% in D. melanogaster. The detection of fucose on some of the smaller oligosaccharides is also of interest. It was anticipated that the fucose might be linked oL1-3 to the reducing terminal GlcNAc residue and hence provide an epitope explaining the cross-reactivity between D. melanogaster and horseradish peroxidase. Only a l - 6 linked fucose was found in D. melanogaster, since these two different core fucose linkages have yet to be reported in the same organism, we assume therefore that core fucosylation in D. melanogaster is exclusively a l - 6 . The identity of the cross-reacting carbohydrate epitope in D. melanogaster (especially as we have not detected any xylose in our preparations, a sugar that has also been implicated in the epitope), remains un-

Manal-2Manal '%6 Milnat.% hal ,,3 6 Manl~1-4GtcNAcp1-4GlcNAc-oI Ma

/3

Mana':.-2 Manal -I)12

Manal %6 Mgnal Mafia1 / 3 %.6Man!~1.4GIcNAC[~I-4GICNAC.Ol /gan~l "CD123

der investigation. In m a m m a l s [18], and plants [19], the substrate for the fucosyl-transferase has been proposed to carry a terminal N-acetylglucosamine residue on the otl-3 mannose arm. No evidence of such outer-arm substitution was found in 1). melanogaster, implying that this residue may be removed rapidly after fucosyiation, as appears to be the case in plants [20]. Alternatively the D. melanogaster fucosyl-transferase may have a different substrate specificity. The (Man)2_3GlcNAc /~4(Fuc0_~)GlcNAc-ol structures have been reported previously [21], and are possibly the result of as yet undefined a-mannosidases. Post-translational addition (as opposed to co-translational) of pre-processed dolichol substrates by oligosaccharide specific oligosyltransferases, however, cannot be ruled out [22]. If the biosynthesis of oligosaccharides follows the same initial pathway in D. melanogaster as in yeast, plants and mammals then to synthesize the structures reported here requires a minimum of 20 enzymes. D. mdanogaster has about 10000 genes. If a similar number of enzymes are involved in O-linked glycosylation and in the synthesis of glycolipids then up to 1% of the genes are dedicated to some part of the glycosylat,.'on process. This is similar to the number required for metameric pattern in D. melanogaster [24]. Yet so far

153 n o t h i n g is k n o w n o f the f u n c t i o n o f glycosylation in D. melanogaster a n d little is k n o w n o f its f u n c t i o n in o t h e r organisms. In c o n c l u s i o n , this initial s t u d y has s u r v e y e d the m a j o r o l i g o s a c c h a r i d e species p r e s e n t in D, melanogaster. W e a r e c u r r e n t l y investigating the g e n e s c o d i n g f o r s o m e o f the e n z y m e s in t h e s e p a t h w a y s , with a view to c l o n i n g the c o d i n g s e q u e n c e a n d using this to m a p the g e n e cytogenetically. O n c e m a p p e d classical Drosophila g e n e t i c s allows the selection o f m u t a n t s o f t h e c o d i n g r e g i o n a n d t r a n s f o r m a t i o n - r e s c u e experim e n t s will allow t h e identification o f m u t a n t s o f the appropriate gene.

Acknowledgements W e wish to t h a n k Mrs. J e a n M a t t h e w s for p r e p a r a tion o f t h e fly m e d i u m . D . B . R . a c k n o w l e d g e s a g r a n t f r o m t h e E.P. A b r a h a m C e p h a l o s p o r i n F u n d w h i c h a l l o w e d t h e w o r k o n D. melanogaster to get s t a r t e d . A u t h e n t i c H R P o l i g o s a c c h a r i d e w a s p r e p a r e d by Dr. J e a n H a r t h i l l . T h e O x f o r d G l y e o b i o l o g y U n i t is s u p p o r t e d by the M o n s a n t o C o m p a n y , U S A .

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