analysis of glycan structures on the 120 kda aminopeptidase n of manduca sexta and their...

8/3/2019 Analysis of Glycan Structures on the 120 kDa Aminopeptidase N of Manduca Sexta and Their Interactions With Bacil

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Insect Biochemistry and Molecular Biology 34 (2004) 101112www.elsevier.com/locate/ibmb

Analysis of glycan structures on the 120 kDa aminopeptidase NofManduca sexta and their interactions with Bacillus thuringiensis

Cry1Ac toxin

Peter J.K. Knighta, Joe Carroll b, David J. Ellar c,

a Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, BC, Canada V6T 1Z4b Medical Research Council, Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, UK

c Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK

Received 12 May 2003; accepted 18 September 2003

Abstract

The Bacillus thuringiensis Cry1Ac toxin specifically binds to a 120 kDa aminopeptidase N (APN) receptor in Manduca sexta.The binding interaction is mediated by GalNAc, presumably covalently attached to the APN as part of an undefined glycanstructure. Here we detail a simple, rapid and specific chemical deglycosylation technique, applicable to glycoproteins immobilizedon Western blots. We used the technique to directly and unambiguously demonstrate that carbohydrates attached to the 120 kDaAPN are in fact binding epitopes for Cry1Ac toxin. This technique is generally applicable to all putative Cry toxin/receptor com-binations. We analysed the various glycans on the 120 kDa APN using carbohydrate compositional analysis and lectin binding.The data indicate that in the average APN molecule, 2 of the 4 possible N-glycosylation sites are occupied with fucosylated pauci-mannose {Man23(Fuc12)GlcNAc2-peptide} type N-glycans. Additionally, we identified 13 probable O-glycosylation sites, 10 ofwhich are located in the Thr/Pro rich C-terminal stalk region of the protein. It is likely that 56 of the 13 sites are occupied,probably with simple {GalNAc-peptide} type O-glycans. This O-glycosylated C-terminal stalk, being GalNAc rich, is the most

likely binding site for Cry1Ac.# 2003 Elsevier Ltd. All rights reserved.

Keywords: Bacillus thuringiensis; Cry1Ac; Receptor; Insect glycosylation; Deglycosylation; Aminopeptidase N

1. Introduction

The bacterium Bacillus thuringiensis (BT) expresses a

wide variety of protein delta-endotoxins (Cry toxins;

Crickmore et al., 1998, 2002) that exhibit toxicity to a

range of invertebrates, and are used worldwide as bio-logical insecticides. It is generally accepted that follow-

ing ingestion by susceptible insect larva, the (pro)toxins

are simultaneously solubilised and proteolytically acti-

vated in the insect midgut. Activated toxins bind to

specific receptors in the midgut epithelial brush border

membrane and insert into the membrane, leading to

the formation of a pore or ion channel that ultimately

kills the insect (reviewed in Schnepf et al., 1998).

However, many of the details of toxinreceptor interac-

tions and pore formation are imperfectly understood

and are the subject of active research.

Corresponding author. Tel.: +44-1223-333651/766001; fax: +44-1223-766043/766002.

E-mail address: [email protected] (D.J. Ellar).Abbreviations: 2-AA, 2-aminobenzoic acid; AAA, Aleuria aurantiaagglutinin; APN, aminopeptidase N; BBMV, brush border membranevesicles; BT, Bacillus thuringiensis; ConA, Concanavalin A (Canavaliaensiformis agglutinin); DSA, Datura stramonium agglutinin; Fuc,Fucose; Gal, Galactose; GalN, galactosamine; GalNAc, N-acetylgalactosamine; GC-MS, gas chromatography/massspectrometry; Glc, Glucose; GlcN, glucosamine; GlcNAc, N-acetylglucosamine; GNA, Galanthus nivalis agglutinin; GPI, glycosyl-phosphatidyl-inositol (anchor); HPLC, high pressure liquidchromatography; HRP, horseradish peroxidase; LacNAc, N-acetyllactosamine; PNA, peanut agglutinin (Arachis hypogeaagglutinin); SBA, soybean agglutinin (Glycine max agglutinin); SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis;TFA, trifluoroacetic acid; Xyl, xylose.

0965-1748/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.ibmb.2003.09.007


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A number of putative Cry toxin receptors have beenidentified in various insect midgut epithelia. In fiveinsect species, Manduca sexta, Heliothis virescens,Lymantria dispar, Plutella xylostella and Bombyx mori,members of the aminopeptidase N (APN) family(EC3.4.11.2) have been identified as putative receptorsfor the Cry1A subfamily of toxins (Knight et al., 1994;Sangadala et al., 1994; Gill et al., 1995; Lee et al.,1996; Denolf et al., 1997; Luo et al., 1997; Yaoi et al.,1997). A single APN may bind multiple toxins, or con-versely a single toxin may bind multiple APNs (Denolfet al., 1997).

One member of the Cry1A toxin subfamily, Cry1Ac,appears to specifically bind to N-acetylgalactosamine(GalNAc) residues covalently linked to the 120 kDaAPNs of M. sexta and L. dispar, and the 170 kDaAPN of H. virescens (Oddou et al., 1993; Knight et al.,1994; Sangadala et al., 1994; Gill et al., 1995; Valaitiset al., 1995; Lee et al., 1996). This conclusion is sup-

ported by two lines of evidence. Firstly, Cry1Ac bind-ing to these APNs is specifically inhibited by GalNAc,but not by other sugars. This has been demonstratedwith numerous assay systems, mostly using the M.sexta APN, including in vitro cell assays (Knowleset al., 1984), in vivo brush border membrane vesicles(BBMV) binding assays (Knowles et al., 1991), ligandblots (Garczynski et al., 1991), SPR experiments(Cooper et al., 1998) and vesicle swelling assays (Car-roll et al., 1997). These studies have been invaluable inuncovering the importance of GalNAc in the Cry1AcAPN interaction. Secondly, Cry1Ac domain III

mutants that are defective in GalNAc binding are alsounable to bind to the M. sexta APN (Burton et al.,1999).

Many members of the Cry toxin family show highsequence similarity (Crickmore et al., 2002). Crystal-lographic solutions for the Cry3A (Li et al., 1991) andCry1Aa toxins (Grochulski et al., 1995) suggest thatthey also share a common three-dimensional structure,consisting of three domains (I, II and III). Compara-tive modeling studies indicate that domain II of theCry1Aa toxin has a b-prism structural fold, similar tothat found in the carbohydrate-binding protein VMO-1(Shimizu et al., 1994), and the lectins KM+ from Arto-

corpus integrifola (Rosa et al., 1999), jacalin (Sankar-anarayanan et al., 1996), and Maclura pomiferaagglutinin (Lee et al., 1998). In the latter three cases,the b-prism structural fold has been shown by X-raycrystallography to be directly involved in carbohydratebinding, and in the latter two cases the carbohydrateconcerned is the O-glycan Galb1,3GalNAc-O. Simi-larly, domain III of the Cry3A toxin has a strikingresemblance to the N-terminal cellulose-bindingdomain of the 1,4-b-glucanase (CenC) protein from thebacterium Cellulomonas fimi (Johnson et al., 1996; Bur-ton et al., 1999). Both proteins contain a b-jellyroll

structural motif, also called the lectin fold since it isan invariant feature of the legume lectin family. Thissuggests that the insecticidal specificity of the Cry1Atoxin family (and possibly other Cry toxins) could bedetermined by two lectin-like domains (II and III) act-ing in concert or independently.

Despite the fact that most members of the Cry toxinfamily show high sequence homology and appear toshare a common three-dimensional structure, it is notclear whether covalently attached carbohydrates are acommon binding paradigm for BT toxins. This isbecause the competition experiments used to demon-strate the involvement of a glycan moiety require pre-existing knowledge of the monosaccharide involved,and are impractical if the glycan moiety is a di- or tri-saccharide, or if two separate glycan structures are co-operatively involved in the interaction. We reasonedthat a method that selectively modifies or destroys pre-existing glycan residues on glycoproteins would be a

direct experimental approach that circumvents theseproblems.

The objectives of this study were twofold. First, toestablish experimental conditions by which a mildchemical deglycosylation method, periodate oxidationfollowed by borohydride reduction, can be generallyapplied to any putative receptor (glyco)protein todirectly demonstrate the importance or otherwise ofglycan structures to BT toxin binding. Based on ourprevious work, we used Cry1Ac toxin and the 120 kDaAPN from M. sexta as a model system. Second, toinvestigate the nature and distribution of glycan struc-

tures on the M. sexta APN putative receptor using lec-tin blots and carbohydrate compositional analysis, inorder to determine which glycan structures contain Gal-NAc and can thus be considered Cry1Ac toxin epitopes.

2. Materials and methods

2.1. Chemicals

All common reagents were of analytical grade, andwere purchased from a variety of suppliers. Mono-saccharides and peroxidase-labeled lectins were pur-

chased from Sigma. Digoxigenin-labeled lectins wereobtained from Boehringer Mannheim. Centricon andCentriprep ultrafiltration units were from Amicon.The PVDF-based membrane Immobilon-P was fromMillipore.

2.2. Toxin purification and activation

BT strain kurstaki HD-73 was obtained from theBacillus Genetic Stock Culture Collection (Columbus,OH). Bacterial growth (Stewart et al., 1981) and purifi-cation of Cry1Ac inclusions (Thomas and Ellar, 1983)

102 P.J.K. Knight et al. / Insect Biochemistry and Molecular Biology 34 (2004) 101112


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was performed as previously described. Purified

Cry1Ac crystals were solubilised at a concentration of

1 mg ml1 in freshly prepared 50 mM Na2CO3/HCl,

pH 10.5, 10 mM DTT at 37v

C for 1 h. Insoluble

material was removed by centrifugation at 13,000g at

room temperature for 5 min. Soluble Cry1Ac protoxin

was activated with either 5% (v/v) Manduca sexta gutextract, or with TPCK-treated trypsin (50:1, w/w,

enzyme/toxin), for 1530 min at 37v

C (Knowles et al.,

1991). Following centrifugation at 13,000g for 5 min

at room temperature, the supernatant was used as the

activated toxin preparation.

2.3. Isolation of M. sexta BBMV

BBMV were prepared from either fresh or frozen

insect midguts by the Mg2+ precipitation/differential

centrifugation method (Wolfersberger, 1993), with the

following additional steps. Isolated midguts were sus-

pended in 9 their weight of 300 mM mannitol, 5 mM

EGTA, 17 mM Tris/HCl, pH 7.5, cut into pieces and

homogenised in a Dounce homogeniser for 20 strokes.

After adding an equal volume of 24 mM MgCl2 made

up in 300 mM mannitol, 5 mM EGTA, 17 mM Tris/

HCl, pH 7.5, and standing on ice for 15 min, the mix-

ture was centrifuged at 2500g for 15 min at 4v

C. The

pellet was resuspended in the same volume of 300 mM

mannitol, 5 mM EGTA, 17 mM Tris/HCl, pH 7.5 and

the process repeated. The two supernatants were then

pooled and the method continued exactly as describedby Wolfersberger et al. (1987). BBMV were collected as

the final pellet from a second high speed spin at

30,000g for 30 min at 4v

C. BBMV were stored in

half-strength 300 mM mannitol, 5 mM EGTA, 17 mM

Tris/HCl, pH 7.5 at an approximate concentration of 5

mg ml1 at 80v

C after freezing in liquid nitrogen.

2.4. Mild periodate oxidation

Western blots of M. sexta BBMV were oxidised with

periodate as described by Woodward et al. (1985).

Briefly, blots were equilibrated in 50 mM sodium acet-

ate buffer, pH 4.5, then exposed to 10 mM periodate

(Sigma, periodic acid) dissolved in the same buffer for 1

h in the dark at 23v

C. After extensive washing with

water, the blots were reduced with 50 mM sodium bor-

ohydride in phosphate-buffered saline (PBS), pH 7.4

for 30 min at 23v

C. After extensive washing with PBS,

blots were blocked and incubated with lectins, toxin or

antibodies as described below. Control blots were

treated identically except that no periodate was added

to the pH 4.5 buffer at the oxidation stage.

2.5. Purification of APN

The 120 kDa APN membrane protein was purified

from M. sexta BBMV by detergent solubilisation fol-

lowed by Cry1Ac protoxin affinity chromatography

(affinity-purified APN, as used in Fig. 2), followed

by anion exchange chromatography (affinity/IEX-

purified APN, as used in Figs. 1 and 3), as previously

described (Knight et al., 1994).

2.6. Western blotting

Proteins separated by sodium dodecyl sulphate-poly-

acrylamide gel electrophoresis (SDS-PAGE) were elec-

trophoretically transferred to nitrocellulose or PVDF-

based membranes (Immobilon-P or Pro-Blot) by the

method of Towbin et al. (1979), using a semi-dry blot

apparatus (Bio-Rad or LKB Novablot). The transfer

buffer was 25 mM glycine, 192 mM Tris, 10% (v/v)

methanol. Transfer was performed at 23 mA cm2 for

60 min at room temperature. Following transfer, filters

were washed in TBS (154 mM NaCl, 10 mM Tris/HCl,

Fig. 1. Deglycosylation by periodate oxidation/borohydridereduction. M. sexta BBMV and affinity/IEX-purified 120 kDa puta-tive receptor were fractionated by SDS-10%-PAGE and blotted tonylon membranes. Experimental membranes (B and D) were oxidisedwith periodate, whilst control membranes (A and C) were not. Allmembranes were subsequently reduced with borohydride, as detailedin the text. Putative receptors were visualised by Cry1Ac toxin immu-noblotting (A and B), whilst GalNAc-containing glycoproteins werevisualised by lectin blotting with SBA (C and D). The 120 kDa APNis marked with an arrow head (>). Lane 1, molecular mass markers(116, 66, 45, 36 and 29 kDa); lane 2, M. sexta BBMV; lane 3, affin-ity-purified 120 kDa putative receptor; lane 4, trypsin-activatedCry1Ac toxin.

P.J.K. Knight et al. / Insect Biochemistry and Molecular Biology 34 (2004) 101112 103


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pH 7.4) prior to incubation with toxin, antibodies orlectins as detailed below.

2.6.1. Immunodetection of putative receptorsBlots were incubated overnight with 3% (w/v) bov-

ine hemoglobin in TBS (blocking buffer) to block non-specific binding sites. The filter was then sequentially

incubated with activated Cry1Ac toxin (0.21l

g ml

1

,60 min), primary (rabbit anti-toxin) antiserum (0.05%,v/v, 60 min), and secondary (peroxidase-conjugatedgoat anti-rabbit) antiserum (0.05%, v/v, 60 min), all inblocking buffer (Knowles et al., 1991). The filter wasextensively washed with TBS between each stage.Bound antibodies were detected by incubation with 30mg 4-chloro-1-napthol in 10 ml methanol, 50 ml TBSand 20 ll hydrogen peroxide (Hawkes et al., 1982).

2.6.2. Lectin blotsDigoxigenin-labelled lectins ConA, GNA, DSA,

AAA and PNA were from Boehringer Mannheim, and

were used on ligand blots according to the manu-facturers instructions. Peroxidase-labeled lectin SBA(Sigma) was incubated with blots at 0.16 purpurogallinunits ml1 as described (Knowles et al., 1991). Non-specific binding to membranes used in lectin blots wasblocked with Boehringer Mannheim blocking reagent(Cat. No. 1-096-176).

2.6.3. Anti-HRP blotsThe anti-horseradish peroxidase (HRP) antibody was

supplied by Sigma. Blots were blocked with 3% bovineserum albumin in TBS prior to incubation with anti-

HRP antibody (0.1%, v/v, 60 min) followed by the sec-

ondary antibody and development as described for the

immunodetection of putative receptors.

Fig. 3. Anti-HRP blots. Rat BBMV, M. sexta BBMV, HRP, Fetuinand affinity/IEX-purified 120 kDa putative receptor were fractio-nated by SDS-13%-PAGE and blotted to nitrocellulose membranes.Panel A is a Coomassie-Blue stained gel. Panel B is a nitrocellulosemembrane, developed with an anti-HRP antibody. The 120 kDaAPN is marked with an arrow head (>). Lane 1, Rat BBMV; lane 2,molecular mass markers (116, 66, 45, 36, 29, 24 and 20.5 kDa); lane3, M. sexta BBMV; lane 4, HRP; lane 5, Fetuin; lane 6, affinity-purified 120 kDa putative receptor.

Fig. 2. Lectin blots. M. sexta BBMV and affinity-purified 120 kDa putative receptor were fractionated by SDS-10%-PAGE and blotted to nylon

membranes. Panel A is a Coomassie-Blue stained gel. The remaining panels are nylon membranes, developed with Cry1Ac toxin to visualise puta-tive receptors (B), and with the digoxigenin-labelled lectins GNA (C), ConA (D), AAA (E), DSA (F), and PNA (G) and with peroxidase-labeledSBA (H). The 120 kDa APN is marked with an arrow head (>). Lane 1, molecular mass markers (116, 66, 45, 36 and 29 kDa); lane 2, M. sextaBBMV; lane 3, affinity-purified 120 kDa putative receptor; lane 4, Cry1Ac protoxin.



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2.7. Carbohydrate compositional analysis

Elemental monosaccharide analysis was performedon purified APN desalted into H2O using a P30 spincolumn (Bio-Rad). Monosaccharides released by acidhydrolysis (15% v/v trifluoroacetic acid, TFA, 100

v

C,4 h) were labeled with a fluorescent 2-aminobenzoicacid (2-AA) tag and cleaned-up using the Signal 2-AAlabeling kit and Glycoclean S column (Oxford Gly-cosciences) as per manufacturers instructions. Thelabeled material was separated on a GlycoSep R col-umn (Oxford Glycosciences) coupled to an HP1090HPLC and HP1046A fluorescence detector (HewlettPackard). The fluorescent signal was quantified using amixture of six monosaccharide standards (Dionexmonostandards; GalN, GlcN, Gal, Glc, Man, Fuc).

3. Results

3.1. Chemical deglycosylation

Several lines of evidence suggest that GalNAc isdirectly involved in the binding interaction betweenCry1Ac toxin and its APN receptor in M. sexta midgutepithelium (Knowles et al., 1991; Knight et al., 1994;Sangadala et al., 1994). If so, destruction or loss ofGalNAc moieties should abolish binding to the recep-tor. Therefore, a mild chemical method, periodate oxi-dation followed by borohydride reduction, was used toselectively destroy glycan moieties on APN.

Periodate oxidation is a well established reaction incarbohydrate chemistry (reviewed in Bobbitt, 1956).Periodic acid oxidises vicinal diols associated with sialicacids and neutral sugars to aldehydes, which are thenreduced by borohydride to primary alcohols. Whenapplied to glycoproteins under appropriately controlledconditions the reaction can be highly selective, destroy-ing carbon-3 unsubstituted sugars within the oligo-saccharide chain, with minimal core peptide degradation(Gerken et al., 1992). Chemical deglycosylation usingthis technique has been used to distinguish betweencarbohydrate- and protein-binding monoclonal anti-bodies (Woodward et al., 1985; Swords and Staehelin,

1993).M. sexta BBMV, affinity/IEX-purified M. sexta

APN and Cry1Ac toxin were fractionated by SDS-PAGE then blotted onto PVDF membranes whichwere subjected to mild periodate oxidation/borohy-dride reduction as described in Materials and methods.Membranes were then immunostained with eitherCry1Ac toxin or the lectin SBA. In trial experiments(data not shown) periodate concentrations and oxi-dation times were optimised, using ligand blotting withCry1Ac to monitor both proteincarbohydrate interac-tions (with the 120 kDa APN) and proteinprotein

interactions (between the primary antibody andCry1Ac toxin immobilized on the membrane). We wereable to establish conditions which abolished the for-mer, but left the later interactions unchanged.

Coomassie-Blue staining of membranes before andafter the oxidation/reduction showed that there is aslight loss of protein from the blot during the course ofthe procedure. To preclude the possibility that loss ofbinding of Cry1Ac following periodate oxidation/bor-ohydride reduction might simply be caused by loss ofthe putative receptor from the blotting membrane, con-trols consisting of duplicate membranes treated identi-cally except for the periodate oxidation step werealways included.

The results are shown in Fig. 1. Using 10 mM per-iodate for 60 min resulted in total loss of binding ofboth Cry1Ac and SBA to the 120 kDa APN (Fig. 1Band D, lanes 2 and 3), whilst binding of anti-Cry1Acpolyclonal antibody to Cry1Ac toxin was unaffected

(Fig. 1B, lane 4). With control membranes treated inparallel but without periodate, both Cry1Ac and SBAbound to the 120 kDa APN as expected ( Fig. 1A andC, lanes 2 and 3). This result is a direct demonstrationthat the selective destruction of carbohydrate residueson APN is sufficient to disrupt binding of Cry1Actoxin.

3.2. Lectin-binding analysis

Lectins were used in conjunction with Western blotsto identify carbohydrate structures covalently attached

to the M. sexta APN. Samples of M. sexta BBMV andaffinity-purified M. sexta APN were fractionated bySDS-PAGE, transferred to nitrocellulose and probedwith various peroxidase- or digoxigenin-labelled lectins.The results are shown in Fig. 2. Note that the APNsample was the product of protoxin affinity chromato-graphy only, and thus still contains some activatedCry1Ac toxin leeched off the column that shows up asadditional (non-receptor) bands in the Cry1Ac blot(Fig. 2, panel B, lane2). Of the six lectins tested, fourwere able to recognise the 120 kDa APN. The Galan-thus nivalis lectin (GNA) binds to non-reducing ter-minal mannose (Man) residues, with binding affinities

in the order a1; 3 > a1; 6 > a1; 4 (Hester and Wright,1996). The Canavalia ensiformis lectin (ConA) alsobinds with high affinity (but with less linkage specificityrequirements) to non-reducing terminal Man residues(Osawa and Tsuji, 1987), and in addition (but withlower affinity) to terminal a-linked glucose (Glc) andGalNAc. Both lectins bound the 120 kDa APN (Fig. 2,panels C and D, respectively), and to multiple otherproteins in M. sexta BBMV. In keeping with the strin-gent linkage specificity requirements displayed byGNA, relatively fewer glycoproteins in BBMV boundto GNA than to ConA. The Aleuria aurantia lectin



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(AAA) binds fucose (Fuc) residues, with linkage speci-ficities in the order a1; 6 > a1; 3 > a1; 4 (Yamashitaet al., 1985). AAA binds to many glycoproteins inBBMV, including the 120 kDa APN (Fig. 2, panel E).This is not surprising since fucosylation is commonlyfound in insect (and plant) glycoproteins, as a modifi-

cation either to the proximal N-acetylglucosamine(GlcNAc) residue of N-linked glycans, and/or toantennae structures (Staudacher et al., 1992b; Stau-dacher and Marz, 1998). The Datura stramonium lectin(DSA) does not bind to the aminopeptidase (Fig. 2,panel F). DSA recognises Gal b1-4GlcNAc and{GlcNAcb1-4 GlcNAc} oligomers, typically part ofhybrid or complex N-linked glycans (and also of someO-linked glycans) usually found in the antennae ofhybrid or complex N-linked glycans but also in O-gly-cans (Crowley and Goldstein, 1982; Cross, 1990). Thebinding pattern of these four lectins suggests that M.sexta 120 kDa APN contains at least one fucosylatedN-glycan of the high-mannose or hybrid type, and mayin addition contain other N-glycans.

The last two lectins tested bind preferentially to O-linked oligosaccharides. The Arachis hypogea (peanut)lectin (PNA) did not bind the 120 kDa APN (Fig. 2,panel G), whilst the Glycine max (soybean) lectin(SBA) did (Fig. 2, panel H). PNA binds to O-glycansconsisting of Gal (b13) GalNAc (a13) Ser/Thr butnot to GalNAc (a13) Ser/Thr, whilst with SBA thebinding pattern is reversed. In addition, SBA bindsstrongly to GalNAc (a13) Gal (b13) GlcNAc (b13)Gal (b14) Glc, provided that this structure is not

fucosylated (Sueyoshi et al., 1988). The binding of SBAindicates that M. sexta APN probably contains O-linked glycans.

3.3. Ant-HRP antibody-binding analysis

Anti-HRP antibodies have been shown to bindspecifically to N-glycans containing a1,3 linked Fucand/or b1,3 Xyl residues in N-glycans (Kurosaka et al.,

1991; Faye et al., 1993). Since Xyl is not known toexist in insect glycoproteins, any binding by anti-HRPantibodies to insect glycoproteins can be attributed toa1,3 linked Fuc. In view of the binding of the lectinAAA to the 120 kDa APN, indicating the presence ofa1,3, a1,4 or a1,6 linked Fuc (see above), we decided

to use anti-HRP antibody binding as a means of dis-criminating between these possible Fuc linkages.Samples of rat BBMV, M. sexta BBMV, HRP (posi-

tive control), fetuin (negative control), and affinity/IEX-purified M. sexta APN were fractionated by SDS-PAGE, transferred to nitrocellulose and probed withanti-HRP antibody. The results are shown in Fig. 3.The positive and negative controls (Fig. 3, lanes 4 and5) behaved as expected. There was no binding to ratBBMV (Fig. 3, lane 1), but significant binding to mul-tiple bands in M. sexta BBMV (Fig. 3, lane 3), and tothe 120 kDa affinity-purified APN (Fig. 3, lane 6).

The results obtained with anti-HRP match thoseseen with the lectin AAA, and indicate that a1,3 linkedFuc is a common modification to many M. sextaBBMV glycoproteins, including the 120 kDa APN. Thepresence of Fuc in a1,3 linkage on the APN moleculecould explain its apparent insensitivity to PNGase F,whose action is known to be blocked by the presenceof this modification on the proximal GlcNAc residueof N-glycans (Chu, 1986; Maley et al., 1989).

3.4. Carbohydrate compositional analysis

APN was isolated from M. sexta BBMV using pro-

toxin affinity chromatography followed by anionexchange chromatography (Knight et al., 1994). A pur-ified sample of APN was hydrolysed with TFA tocleave glycosidic linkages. The released mono-saccharides were then labeled with fluorescently tagged2-AA and separated by reverse-phase HPLC. Mono-saccharide standards (Man, Gal, Glc, GlcN, GalN andFuc) were treated in parallel and used to calibrate theHPLC elution profile, allowing identification and

Table 1Carbohydrate compositional analysis of APN

GalN(GalNAc)

GlcN(GlcNAc)

Gal Glc Man Fuc Inositol

APN/GC-MS 2.1 8.3 0 1.9/8.3 6.4/10.8 nd 6.4/3.8BBMV 2.9 9.1 0 0 7 6.6 ndAPN 5:8 0:3 9:6 0:3 0 0 7:3 0:2 3:1 0:1 nd

Minus GPI anchor core(Man4GlcN)

1 3

Minus 2 (GlcNAc2Man2)N-glycans

4 4

Remaining residues 5.8 4.6 0 0 0.3 3.1

Values are mol/mol APN (S.D.). The two values for APN/GC-MS samples represent acetylation and no acetylation post-hydrolysis andpre-GC-MS, respectively, nd: not done.



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quantification of individual monosaccharide content,expressed as nanomoles present in the volume injected.Experimental conditions were evaluated using the gly-coprotein fetuin. A preliminary analysis was also per-formed on the 120 kDa band evident in BBMV (seeFig. 2A, lane 2), after fractionation by SDS-PAGE andblotting to a PVDF membrane.

The results shown in Table 1 are the mean values(S.D.) obtained from four HPLC runs on mono-saccharides released from a single APN sample (APN).The preliminary analysis from the same 120 kDa bandin BBMV is also shown (BBMV). For the purposes ofcomparison, the gas chromatography/mass spec-trometry (GC-MS) monosaccharide results obtained bySangadala et al. (2001) are also shown (APN, GC-MS),with the reported mol% values converted to mol/molAPN. There are two key differences between our analy-sis and that of Sangadala et al. (2001). Firstly, we didnot de-lipidate the APN or BBMV samples prior toacid hydrolysis. Secondly, we did not re-N-acetylatesamples after acid hydrolysis, which destroys N-acetylgroups (e.g. in GalNAc and GlcNAc), but insteadassumed that the GlcN and GalN detected afterhydrolysis were equal to GlcNAc and GalNAc presentpre-hydrolysis.

All three sets of results are reasonably consistent inmonosaccharide content and amounts, showing largeamounts of GlcNAc and Man, lesser amounts of Gal-NAc, and no Gal associated with the APN. They differin the amounts of GalNAc present, with the purifiedAPN data set almost twice that of the BBMV andAPN/GC-MS data sets. Given that affinity purification

of the APN requires GalNAc elution (Knight et al.,1994), and despite desalting step prior to acid hydroly-sis (see Materials and methods), it is possible that ourpurified APN sample is contaminated with GalNAc.Other differences are that this study determined theamount of Fuc present, and that Sangadala et al.(2001) detected low levels of Glc in their APN/GC-MSsample, where none was detected in our APN orBBMV samples. This could be a methodological arti-fact, or may represent a real difference in glycosylationpotential between different M. sexta insect colonies.The results are unusual in that all three analyses report

large amounts of N-acetylated sugars, particularly

GlcNAc, on the 120 kDa APN. As discussed later, this

is difficult to reconcile with known insect glycans.

3.5. Prediction of N- and O-linked glycan motifs in

APN

We previously cloned a 3095 nucleotide (990 amino-acid) APN cDNA from M. sexta (Knight et al., 1995,

Genbank X89081). N-terminal amino acid sequencing

of the purified (mature) protein shows that 34 residues

(F1R34) are proteolytically removed from the N-

terminus, and the presence of a glycosyl-phosphatidyl-

inositol (GPI) anchor (Knight et al., 1995; Lu and

Adang, 1996) implies the removal of the predicted C-

terminal GPI signal sequence (residues S969A990).

The mature polypeptide would then be 934 residues

long (numbered D1G934), with a calculated molecular

mass of 105 kDa. The predicted amino acid sequence

of the mature (proteolytically processed) APN has fourpotential N-glycosylation motifs (N-X-S/T) at N261,

N575, N589 and N718.A more sophisticated analysis of probable N-

glycosylation sites was conducted using the predictive

program NetNglyc 1.0 (http://www.cbs.dtu.dk/ser-

vices/NetNGlyc). This predicts potential N-glycosyla-

tion sites using artificial neural networks that have

been trained using the sequence context of N-X-S/T

motifs in human proteins. The results, shown in

Table 2, predict only two N-glycosylation sites in the

M. sexta APN, at N261 and N589.Probable mucin-type O-glycosylation sites (GalNAc-

a-O-S/T) in the APN were predicted using a similar

program, NetOGlyc 2.0 (Hansen et al., 1998, http://

genome.cbs.dtu.dk/services/NetOGlyc). Thirteen

potential O-glycosylation sites were identified, as

shown in Table 2. Two are very close to the N-ter-

minus of the mature protein, and 10 are tightly clus-

tered in the Thr- and Pro-rich C-terminus. Their

distribution supports the presence of a predicted O-gly-

cosylated stalk region at the C-terminus of the M.

sexta APN (Knight et al., 1995).

Table 2Prediction of APN glycosylation sites

1 2 3 4 5 6 7 8 9 10 11 12 13

NetNGlyc 1.0 predictionof occupiedN-glycosylation sites

N261 N589

NetOGlyc 2.0 predictionof occupiedO-glycosylation sites

S3 T9 T359 T906 T907 T914 T915 T917 S919 T920 T924 T929 T930

Mature APN protein (D1G934, numbering described in text), derived from Genbank nucleotide sequence X89081.



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4. Conclusion and discussion

In this study we demonstrate that, under appropri-ately controlled conditions, selective destruction ofcarbohydrate residues on M. sexta APN is sufficient todisrupt binding of Cry1Ac toxin. Several other authorshave reported the use of periodate oxidation to investi-

gate toxinreceptor interactions, without providingexperimental details of the procedure (Oddou et al.,1991; Garczynski and Adang, 1995). Ligand (Western)blotting is the most common technique employed toidentify new toxin receptors, and the periodate oxi-dation/borohydride reduction procedure is easily andrapidly applicable to this methodology. We proposethat periodate oxidation/borohydride reduction mayserve as a general and direct method of investigatingthe possible involvement of covalently linked glycans intoxinreceptor interactions, without the requirementfor any pre-existing information on the glycan

involved.An alternative to chemical deglycosylation is to use

deglycosylating enzymes (Maley et al., 1989; Umemotoet al., 1977). However, care must be exercised in inter-preting the results from glycosidase digestion experi-ments, since the steric inaccessibility of otherwisesusceptible glycan structures can render them resistantto cleavage. In this study we were unable to achieveany measurable deglycosylation of APN with PNGaseF or O-glycosidase (data not shown). Enzymatic degly-cosylation as a technique is not readily applicable toligand blotting, usually requires a purified sample of

the putative receptor glycoprotein and is significantlymore expensive than chemical deglycosylation. A pref-erable strategy is to use chemical deglycosylationapplied to ligand blots as a first approach, perhapsbacked up by enzymatic deglycosylation in the event ofa positive result.

The information obtained in this study from lectinligand blots and carbohydrate compositional analysis,coupled with published information on glycan bio-synthesis in insects (reviewed in Altmann et al., 1999),enables us to propose probable structures for both N-and O-linked glycoconjugates of the 120 kDa APN ofM. sexta (see Fig. 4). We note that, by analogy to

other mammalian and insect glycoproteins, it is prob-able that the M. sexta 120 kDa APN exists as a seriesof glycoforms, with alternative glycan structurespresent on individual sub-populations of the protein.Thus glycan structure predictions based on the evi-dence in this paper can only reveal the glycosylationstate of an average APN molecule.

The lectin ConA binds mannose residues, and has ahigh affinity for the pentasaccharide core {Man-a1,3(Mana1,6)Manb1,4GlcNAc2; see Fig. 4A}. It ismost often indicative of a high mannose type N-gly-can, Man49GlcNAc2 or its trimmed derivatives. The

binding of lectin GNA supports this conclusion, sinceit binds non-reducing terminal mannose residues with aspecificity of a1; 3 > a1; 6 > a1; 4 (Hester and Wright,1996). Mannose residues fitting this description couldbe present as part of the pentasaccharide core structure(Fig. 4A) or in the antennae (Fig. 4A, #1) of an N-glycan. Binding of AAA lectin indicates the presence ofFuc (Yamashita et al., 1987), which in insect cells couldbe in a16, a13, or both (difucosylated) linkages tothe proximal GlcNAc of the N-linked glycans (Stau-

dacher et al., 1992a, 1995; Staudacher and Marz,1998). This is supported by the binding of anti-HRPantibody, which confirms the presence of a1,3 Fuc(Faye et al., 1993; Fig. 4A, paucimannosidic core).

Carbohydrate compositional analysis indicates anaverage of 7.3 mol Man/mol APN and 3.1 mol Fuc/mol APN (Table 1). Assuming all APN molecules con-tain a GPI anchor, and allowing 3 Man residues forthe core glycan structure of that anchor (Fig. 4C),leaves an average of 4 Man and 3 Fuc residues perAPN molecule. Thus, the average APN moleculeprobably contains two paucimannose-type N-glycans

Fig. 4. Predicted glycan structures on 120 kDa APN. Core glycanstructures for which firm experimental evidence exists are shown inbold. Possible additional structures are numbered (#1,. . . #x), boxedin with dotted lines and italicized. Probable lectin-binding sites are

enclosed in grey bubbles.



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{Man23GlcNac2-Asn; Fig. 4, paucimannosidic core},either a1,3 mono-fucosylated or a1,3(a1,6) di-fucosy-lated on the core GlcNAc residue. This is consistentwith previous studies on insect glycoproteins, in whichpaucimannose structures are usually found to be theprevalent N-glycan (Marz et al., 1995; Altmann et al.,

1999). It is also consistent with the NetNGlyc 1.0 pre-dictions of two high probability N-glycosylation sites inthe APN, at N261 and N589.

Insect cells are known to have an array of transfer-ase and glycosidase enzymes required to process N-glycans into more complex structures (Altmann et al.,1995a; Staudacher and Marz, 1998; Altmann et al.,1999), and our results from lectin blotting and carbo-hydrate compositional analysis indicate that some ofthese more processed structures are present in the APNpopulation. Carbohydrate compositional analysis indi-cates an average of 9.6 mol GlcNAc/mol APN, but noGal or Glc residues. This unusual richness in GlcNAcresidues (relative to Gal and Glc) could be due to theirpresence in the antennae of N-glycans (hybrid- or com-plex-type), or in O-glycans. Both options are discussedbelow, although our data does not enable us to dis-tinguish between them.

At some stage, GlcNAc residues must have been inb1,2 linkage to the a1,3(a1,6)Man residues of the N-glycan pentasaccharide cores, both because this structureis a precursor in N-glycan biosynthesis (Altmann et al.,1999, 2001), and because the GlcNAcb1,2Mana1,3Manmotif (Fig. 4A, #2) is a pre-requisite for the action of thetwo distinct fucosyltransferase enzymes responsible for

core fucosylation (Staudacher and Marz, 1998). Parti-cularly in insects (Wagner et al., 1996), these GlcNAcresidues are often removed by the actions of N-acet-ylglucosaminidases (Altmann et al., 1995b). It is possiblethat on APN, one or both of the GlcNAc residues in theantennae of individual N-glycans escape this trimmingreaction to leave the structure shown in Fig. 4A, #2.

Whilst the first of these N-glycan antennae structuresmay exist, they cannot be the sole explanation for thehigh levels of GlcNAc observed in APN unless both ofthe predicted APN N-glycans are so modified on bothantennae, an unlikely eventuality. Alternative glycan

structures on the APN include the GPI anchor, and O-glycans. We consider it unlikely that the GPI anchorcontains significant amounts of GlcNAc, both becausethis is generally a very uncommon modification to GPIanchor core glycan structures, and because the compo-sitional analysis performed by Sangadala et al. (2001)indicates that there are on average 4 moles of ethanola-mine per mole of APN. Extra ethanolamine residuesare a common modification to the core glycan structureof GPI anchors (McConville and Menon, 2000), andtheir presence in these numbers would preclude alterna-tive side chains such as GlcNAc.

The remaining option is that GlcNAc is part of anO-glycan structure. The available evidence from bind-ing of the lectin SBA (with specificity for GalNAc-O-Ser/Thr; Fig. 2, panel H) and lack of binding of PNA(with specificity for Galb1,3GalNAc-O-Ser/Thr; Fig. 2,panel G), coupled with the results of monosaccharide

analysis, which indicates a lack of Gal residues inAPN, suggest that a major population of O-glycansassociated with the APN consist simply of GalNAc(Fig. 4Bi). This is consistent with previous examina-tions of insect O-glycans (Marz et al., 1995; Lopezet al., 1999). However, in mammals the structureGlcNAcb1,3GalNAca1-Ser/Thr is an important pre-cursor (Core 3 structure; Iwai et al., 2002) in O-glycanbiosynthesis, and most interestingly its occurrence inmammals is limited to mucins present in the stomach,small intestine and colon. Although such an O-glycanstructure has not hitherto been identified in insects, itmay be present in M. sexta midgut glycoproteins(Fig. 4Bii). It is not clear if such a structure would bean epitope for the lectin SBA.

We conclude that the average 120 kDa APN mol-ecule in M. sexta will have two N-glycosylation sitesoccupied with fucosylated (or di-fucosylated) pauci-mannose-type N-glycans of the type shown in Fig. 4A.Various combinations of the glycan modifications tothe a1,3Man and a1,6Man antennae indicated in Fig. 4are likely to represent the major sub-populations (gly-coforms) of the APN, although it is probable thatmore complex structures exist as rare glycoforms.

Of the 13 predicted mucin-type O-glycosylation sites,

it is most probable that five or six are occupied withGalNac-peptide or possibly GlcNAcb1,4GalNAc-pep-tide structures. Most of the predicted O-glycosylationsites are tightly clustered in the Thr and Pro rich C-ter-minus of the APN (Table 2) supporting the existence ofthe predicted C-terminal O-glycosylated tail of APN(Knight et al., 1994). This is highly likely to be thebinding site for the Cry1Ac toxin, since it is both richin GalNAc residues and in close proximity to the mem-brane. In the event that 2 or more GalNAc-O-S/T O-glycans are arranged with the appropriate spacing inthis structure, it is possible that they act co-operatively

to amplify both the affinity and specificity of binding toCry1Ac toxin.

The expression of the 120 kDa APN in Drosophilamelanogaster larval midgut membranes in vivo led tothe appearance of Cry1Ac toxin susceptibility andtissue specific damage in this normally non-susceptibledipteran host, providing evidence that the APN is infact a functional receptor for Cry1Ac, as well as beinga Cry1Ac-binding protein (Gill and Ellar, 2002). Thisdoes not preclude a functional role for additionalreceptors, either acting independently or synergisticallywith the 120 kDa APN.



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In this context, recent work using a BBMV lightscattering assay has revealed the presence of a secondand distinct Cry1Ac receptor in M. sexta (Carroll et al.,1997). Currently, the molecular entity involved has notbeen identified, but the toxinreceptor interactionapparently does not involve GalNAc. It is not knownwhether any other carbohydrate structures areinvolved. A group of insect cadherin-like proteins arealso strong candidate receptors for the Cry1A toxins(Vadlamudi et al., 1995; Francis and Bulla, 1997).Recently one such protein, BtR175, was expressed inHEK293 cells which subsequently swelled and died inthe presence of Cry1Aa toxin, demonstrating that cad-herin-like proteins can be functionally defined asCry1Aa receptors (Tsuda et al., 2003). The availableevidence does not implicate carbohydrates in the bind-ing interaction with Cry1Ab toxin (Gomez et al., 2001).

In this paper, we have demonstrated the utility of achemical deglycosylation technique as a general, direct

and rapid method of determining the importance ofcarbohydrate structures as binding epitopes for BT tox-ins. In addition, we have used lectin binding andcarbohydrate compositional analysis to deduce prob-able glycan structures attached to the 120 kDa APNreceptor, and shown that O-glycans associated with aC-terminal O-glycosylated stalk structure in theAPN molecule are the most likely site for Cry1Ac toxinbinding. This work will form the basis for more exten-sive structure/function studies to analyse the molecularbasis of receptortoxin recognition, which are urgentlyrequired in order to protect this natural resource from

the potential problem of insect resistance and ensurethe successful long term deployment of BT toxins asbiopesticides.

Acknowledgements

We thank Dr Elaine Stephens and Dr Jayne Sugarsfor illuminating discussions. We gratefully acknowl-edge the assistance of Dr Len Packman and the staff ofthe Protein and Nucleic Acid Chemistry Facility,Department of Biochemistry, Cambridge University.This work was funded by the Agriculture and Food

Research Council (P.J.K.K.) and the Biotechnologyand Biological Sciences Research Council (J.C.).

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analysis of glycan structures on the 120 kda aminopeptidase n of manduca sexta and their...

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