Báo cáo khoa học: The N-linked oligosaccharides of aminopeptidase N from Manduca sexta pptx

The N-linked oligosaccharides of aminopeptidase N from Manduca sexta Site localization and identification of novel N-glycan structures Elaine Stephens 1 , Jane Sugars 2 , Sarah L. Maslen 1 , Dudley H. Williams 1 , Len C. Packman 2 and David J. Ellar 2 1 Department of Chemistry and 2 Department of Biochemistry, University of Cambridge, UK Mass spectrometric studies on the N-linked glycans of aminopeptidase 1 from Manduca sexta have revealed unusual s tructures not previously observed on any insect glycoprotein. Structure elucidation of these oligosaccha- rides was carried out by high-energy collision-induced dissociation (CID) using a matrix-assisted laser desorp- tion/ionization time-of-flight/time-of-flight (MALDI-TOF/ TOF) tandem mass spectrometer. These key experiments revealed that three out of the four N-linked glycosylation sites in this protein (Asn295, Asn623 and Asn752) are occupied with highly fucosylated N-glycans that possess unusual d ifucosylated cor es. Cross-ring fragment ions and ÔinternalÕ fragment ions observed in the CID spectra, showed that these fucoses are found at the 3-position of proximal GlcNAc and at the 3-position of distal GlcNAc in the chitobiose unit. The latter substitution has only been previously observed in nematodes. In addition, these core structures can be decorated with novel fucosylated antennae composed of Fuca(1-3)GlcNAc. Key frag- ment ions revealed that these antennae are predominantly found on the upper 6-arm of the core mannose. The paucimannosidic N-glycan (Man 3 GlcNAc 2 ), commonly found on other insect glycoproteins, is the predominant oligosaccharide found at the remaining N-glycosylation site (Asn609). Keywords: aminopeptidase N; fucose; high-energy CID; insect glycosylation; MALDI-TOF/TOF. As in other eukaryotic cells, many of the proteins in insect cells are covalently modified b y N-glycosylation. The protein N-glycosylation pathway in insect cells is similar tothatobservedinmammaliancells(reviewedin[1–3]). Each begins with the c otranslational transfer of a dolichol- linked precursor oligosaccharide to a specific recognition sequence (Asn-X-Ser/Thr; where X can be any amino acid except Pro) within newly synthesized glycoproteins. Exo- glycosidases and glycosyltransferases i n the endoplasmic reticulum and Golgi apparatus catalyze trimming and elongation reactions to yield th e common intermediate, GlcNAcMan 3 GlcNAc 2 -N-Asn. In mammalian cells, ter- minal glycosyltransferases can elongate this common inter- mediate into the more elaborate structures of hybrid and complex-type N-glycans. In contrast, insect cells have only extremely low levels of the terminal glycosyltransferase activities and, in most cases, have a competing exoglyco- sidase that can r emove the terminal N-acetylglucosamine (GlcNAc) residue from the G lcNAcMan 3 GlcNAc 2 -N-Asn. Hence, the major processed N-glycan produced by insect cells is usually the paucimannosidic structure, Man 3 Glc- NAc 2 -N-Asn. T his is the case for t he honeybee glycoprotein phospholipase A 2 (PLA 2 ), where the major N-glycan structures are composed of the common paucimannosidic structure Man 3 GlcNAc 2 -N-Asn an d t he truncated stru cture Man 2 GlcNAc 2 -N-A sn. However, a sma ll proportion of th e N-linked glycan population on this protein was shown to be substituted with fucosylated lacdiNAc [GalNAcb1- 4(Fuca1-3)GlcNAc] a ntennae a nd difucosylated c ores [4,5]. The discovery of these structures demonstrated that insect cells are capable of producing more elaborate N-glycan structures. However, this is one of the f ew endogenous insect glycoproteins that has been rigorously characterized and the full structural diversity of the N-glycan core and terminal structures that exist on insect glycoproteins remains to be established. It is imp ortant to understand insect protein glycosylation because insects occupy an important evolutionary position among eukaryotic o rganisms. Additionally, i nsect cells play an important biotechnological role as hosts for t he produc- tion of recombinant mammalian glycoproteins [3,6]. In the context of this application, it is crucial t o realize the differences between insect and mammalian N-glycan pro- cessing pathways because N-glycans are known to influence many different protein properties and functions, which include enzymatic activity, conformation and immuno- genicity. For example, the Fuca(1-3)GlcNAc substitution, Correspondence to E. Stephens, Department o f Chemistry, U niversity of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK. Fax: +44 1223 336913; Tel.: +44 1223 336688; E-mail: es287@cam.ac.uk Abbreviations: APN, aminopeptidase N; CHCA, a-cyano- 4-hydroxycinnamic acid; CID, collision-induced dissociation; DHB, 2,5-dihydroxybenzoic acid; GPI, glycosyl phosphotidylinositol; PLA 2 , phospholipase A 2 ;PNGaseA,peptideN-glycosidase A; Hex, hex ose; HexNAc, N-acetylhexosamine; Fuc, fucose; GlcNAc, N-acetylgluco- samine. Enzymes: trypsin (EC 3.4.21.4); PNGase A (EC 3.5.1.52); PNGase F (EC 3.5.1.52); a- L -fucosidase (EC 3.2.1.51); b-N-acetylhexosamini- dase (EC 3.2.1.30). (Received 2 2 July 2004, revised 6 September 2004, accepted 8 September 2004) Eur. J. Biochem. 271, 4241–4258 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04364.x found in a small proportion of N-glycans on honeybee PLA 2 is the major allergenic determinant in this protein [4,5,7], and individuals who are allergic to honeybee venom produce high levels of antibodies to the 3-linked fucose moiety [7,8]. In the present study, w e have characterized the N-glycans attached to the insect protein aminopeptidase N (APN1) from the lepidopteran Manduca sexta (tob acco hornworm) by mass spectrometry. This protein, which is found in the brush border m embrane of the larval midgut, is attached to the cell m embrane via a glycosyl phosphotidylinositol (GPI) anchor [9]. Amino a cid sequence a nalysis shows four possible N-glycosylation sites in APN1 (at asparagines 295, 609, 623 and 752) as well as several likely sites for O-glycosylation, with 10 of these being in the C-terminal region of the protein [10]. Importantly, the N- and/or O-linked glycans on this membrane-associated protein are thought to mediate the binding of the delta-endotoxins (Cry toxins) produced as crystalline inclusions by the Gram-positive bacterium Bacillus thuringiensis (Bt) [9,11]. These Cry toxins are environmentally friendly insecticides and currently provide protection against a wide range of insects, from beetle and caterpillar crop pests to mosquito larvae [9,12]. The sequences of four distinct APN isoforms (APN1–4) from M. sexta larval midguts are available. Whilst APN2 is known also to bind Cry1 toxins [13,14], no binding partners have yet been identified for either APN3 or APN4. The total monosaccharide composition of APN (inclu- ding the N-linked glycans, O-linked glycans and the GPI anchor) has been shown to include large amounts of GlcNAc and Man with lesser amounts of GalNAc and Fuc [10]. In addition, APN1 appeared mainly resistant to deglycosylation with peptide N-glycosidase F (PNGase F), indicating that the majority of glycans are substituted with a1-3 linked fucose at the Asn-linked GlcNAc residue [15]. Furthermore, lectin binding studies have suggested the presence of fucosylated paucimannose-type N-glycans [10]. However, a detailed site-specific structural characterization of the N-linked glycans on APN has not been carried out, nor is i t known which part of the glycoprotein interacts with the toxin. The initial purpose of the present s tudy was to provide a thorough site-specific identification of the N-glycan struc- tures on APN1 in order to reveal possible b inding sites for Cry 1Ac. However, these studies revealed the presence of extremely unusual complex-type glycan structures that can contain up to four antennae composed of Fuca(1-3)Glc- NAc, which have not been observed previously on any glycoprotein. These novel antennae decorate the triman- nosyl core, which is itself unusually difucosylated, with one fucose attached to each of the c hitobiose G lcNAc residues. Mass spectrometric analysis of N-linked glycopeptides showed that these novel glycans are the major structures at three out of the four N-glycosylation sites on APN1. Experimental procedures Purification of aminopeptidase N The 120 kDa APN membrane protein was purified from M. sexta brush border membrane vesicles as described previously [9]. Alkylation and tryptic digestion Approximately 200 lg of purified APN and 1 mg o f honeybee phospholipase A 2 (PLA 2 , Sigma) were reduced, carboxymethylated and digested with trypsin as described in [16]. HPLC separation The glycopeptide/peptide mixtures were purified by reverse- phase HPLC on a Hewlett-Packard 1050 HPLC equipped with a Jupiter C18 column (250 · 4.6 mm, Phenomenex, Macclesfield, Cheshire, UK). Solvent A was 0.1% (v/v) trifluoroacetic acid in H 2 OandsolventBwas90% acetonitrile containing 9.90% H 2 O and 0.1% trifluoroacetic acid (v/v/v). The column was equilibrated with 100% A, and the gradient was initiated 10 min after injection and increased linearly to 80% B ove r 120 min. The flow rate was 1mLÆmin )1 , and the elution was monitored at 214 nm. Fractions were collected at 1 min intervals and dried down. These fractions were redissolved in 4 0 lL o f 5 0% ace tonit- rile/H 2 O (v/v) containing 0.1% (v/v) trifluoroacetic acid, and 1 lL aliquots were screen ed for glycopeptides and peptides by MALDI-TOF MS. Deglycosylation of glycopeptides Pooled HPLC fractions containing purified glyco peptides from PLA 2 were dige sted with PNGase F (Roche) in ammonium bicarbonate buffer ( 50 m M ,pH8.4)for24hat 37 °C using 3 U of enzyme. The released glycans w ere separated from PNGase F-resistant glycopeptides by puri- ficationusingaSep-PakC18(WatersLtd,Elstree, Hertfordshire, UK). The PLA 2 glycopeptide-containing fraction and purified glycopeptides from APN1 were digested with peptide N-glycosidase A (PNGase A; Roche) in ammonium acetate buffer (50 m M ,pH5.0),for16hat 37 °C using 0.2 mU of enzyme. The p roducts were purified using a Sep-Pak C18. Exoglycosidase digestions These were carried out on glycopeptides using the following enzymes and conditions: a- L -fucosidase (from bovine kid- ney; Sigma), 0.2 U in 100 lLof50m M ammonium acetate buffer, pH 5; b-N-acetylhexosaminidase (from Jack b ean; GlykoÒ, Prozyme, San Leandro, CA, USA), 60 mU in 50 m M sodium citrate phosphate buffer, pH 5. Both digestions were incubated at 3 7 °C for 24 h, after which, the reactions were terminated by boiling for 1 min. The products were dried down a nd analyzed by nanoLC- MALDI-TOF MS. NanoLC-MALDI-TOF MS NanoLC was carried out using an LC-Packings Ultimate HPLC (Dionex, Leeds, UK), which was used to generate the g radient that flowed at 150 nLÆmin )1 . T he glycopeptide containing fraction, produced from sequential exoglycosi- dase digestions, was dried down, resuspended in 40 lLof 0.1% (v/v) trifluoroacetic acid in H 2 Oand5lLofthe sample was loaded onto a nanoLC column (C18, 4242 E. Stephens et al.(Eur. J. Biochem. 271) Ó FEBS 2004 75 lm · 5 cm; LC-Packings, Dionex) and the glycopep- tides were eluted with increasing organic concen tration. Solvent A was 0.1% (v/v) trifluoroacetic acid in H 2 Oand solvent B was 90% acetonitrile containing 9.90% H 2 Oand 0.1% trifluoroacetic acid (v/v/v). The column was equili- brated with 100% A, and the gradient was increased linearly to 80% B over 60 min. The column effluent passed through a UV detector (monitoring at 214 nm) directly to a Probot sample fraction system (Dionex). This system mixed the LC effluent with matrix [a-cyano-4-hydroxycinnamic acid (CHCA);Sigma;5mgÆmL )1 in 50% acetonitrile/0.1% trifluoroacetic acid (v/v)] prior to s potting onto a 4700 MALDIplateat30sintervals.Thematrixspotswere allowed to air dry prior to MALDI-TOF MS. Chemical derivatization Permethylation of PNGase A released glycans was per- formed using the sodium hydroxide procedure and subse- quently purified by Sep-Pak using an acetonitrile gradient as described in [17]. MALDI-TOF MS and MALDI-MS/MS All mass spectra were recorded on a 4700 Proteomics analyzer with TOF/TOF optics (Applied Biosystems, Foster City, CA, USA). This MALDI tandem mass spectrometer used a 200 Hz frequency-tr ipled N d:YAG l aser operating at a wavelength of 355 nm. The average of 1000 laser shots was used t o obtain the MS and MS/MS spectra using CHCA matrix. The MS/MS spectra obtained using 2,5- dihydroxybenzoic acid (DHB; Fluka, Dorset, UK) matrix were acquired using 5000–10 000 laser shots. For protein identification experiments, database searching was carried out against the NCBI protein database using GPS EXPLORER (Applied Biosystems). The mass spectrometric data used for protein identification was provided by automated MS and MS/MS acquisitions of peptides in HPLC purified fractions. For MS/MS experiments, the collision e nergy, which is defined by the potential difference between the source acceleration voltage (8 kV) and the floating collision cell (7 kV), was set at 1 kV. Inside the collision cell the selected precursor io ns w ere c ollided with argon or air at a pressure of 2 · 10 )6 Torr. Glycopeptide and p eptide samples p rovi- ded in HPLC fractions were mixed 1 : 1 (v/v) CHCA solution [10 mgÆmL )1 in 50% acetonitrile/H 2 O (v/v) con- taining 0.1% (v/v) triflu oroacetic acid] a nd spotted directly onto the target plate and allowed to air dry. Permethylated glycans for MS/MS were analyzed using DHB matrix [10 mgÆmL )1 in 50% (v/v) methanol/H 2 O]. Results General strategies for glycopeptide characterization The general mass spectrometric strategy for the character- ization of the glycans and the identification of all glycos- ylated sites of APN1 is summarized in Fig. 1. Reduced and alkylated A PN was digested with t rypsin and the proteolytic peptides were separated by reverse-phase HPLC. A small aliquot of each HPLC fraction was analyzed by MALDI- TOF MS in linear and reflectron mode, a nd those f ractions containing glycopeptides from APN1 were identified. A distinct feature of g lycans is their h eterogeneity. As a result, glycopeptides tend to yield a series of low intensity peaks in a MALDI-TOF MS spectrum, with mass differences corresponding to sugar residues. Where possible, these putative glycopeptides w ere identified b y high-energy collision-induced dissociation (CID) of selected signals [M + H] + , using a MALDI-TOF/TOF tandem mass spectrometer. The resulting CID mass spectra gave minor signals for fragment ions derived from the peptide portion, which facilitated the identification of the g lycosylated peptide. Those fractions containing unusual glycopeptides from APN1 were pooled and treated with PNGase A. The released glycans and peptides were separated, and the peptide-containing fraction was analyzed b y MALDI-TOF MS to co nfirm t he presence of the deglycosylated p eptides. The glycan-containing fraction was permethylated and major oligosaccharide signals were subjected to MALDI- TOF/TOF high-energy CID. Fragment ions derived glycosidic and high-energy cross-ring cleavages revealed carbohydrate sequence and linkage information. Peptide mapping The HPLC chromatogram o f the tryptic digestion of APN exhibited a low UV absorbance at 214 nm indicating that subpicomolar quantities of peptides were present (data not shown). Despite this, a nalysis of each H PLC fraction of the APN d igest b y M ALDI-TOF MS revealed tryptic peptides that covered  70% of t he sequence of APN1. Among these peptides, two gave very minor signals at m/z 2707.324 and m/z 2240.107, corresponding to tryptic peptides spanning potential N-g lycosylation sites at Asn295 and Asn752 (data not shown). These data indicate that these glycosylation sites are either not occupied or partially glycosylated. Fig. 1. Procedure for the site-specific characterization of APN N-gly- cosylation. Ó FEBS 2004 Novel insect N-glycans from Manduca sexta (Eur. J. Biochem. 271) 4243 Signals f or peptides spanning the remaining N-glycosylation sites (Asn609 and Asn623) were not observed, suggesting that these sites are f ully glycosylated. In a ddition to signals corresponding to peptides from APN1, many major signals from the digest mixture could not be mapped onto the APN1 amino acid sequence. Consequently, each HPLC fraction was reanalyzed by M ALDI-TOF MS and auto- mated MS/MS was carried out on the five most abundant peaks in each HPLC f raction. These MS and MS/MS data were submitted for database searching against the NCBI protein database using GPS EXPLORER software. Among the  100 peptides that were chosen for MS/MS, 38 peptides were from APN1 (a ccession number 2499901). T he remain- ing peptides w ere derived from aminopeptidas e 4 ( M. sexta; accession number 20260704; 20 peptides), aminopeptidase 3 (M. sexta; accession number 20279109; 10 pept ides) a nd the Bacillus thuringiensis Cry 1Ac protein (accession number 143126; 20 peptides). The latter protein (Cry 1Ac) is a likely contaminant as this protein is used to purify the APN protein by affinity chromatography. However, the presence of the two M. sexta aminopeptidase proteins (APN3 and APN4) in the digestion m ixture complicated the character- ization of the N-glycans on the major APN1 protein, as inspection of their amino acid sequences revealed seven consensus sequences for N-glycosylation in APN3 and five in APN4. Therefore, these proteins are likely to be heavily glycosylated and careful analysis of any glycopeptides detected was necessary to ensure the correct assignment was given. The following sections describe the detailed mass spectrometric characterization of glycopeptides from APN1, which is the major protein in the digestion mixture. Glycopeptide analysis of APN1 Residue N295. MALDI-TOF MS analysis of HPLC fraction number 6 8 in linear m ode revealed a s eries of signals (from m/z 3700 to m/z 4800) (Fig. 2A). The mass differences between these peaks were 146 Da (deoxyhexose) and 203 Da (N-acetylhexosamine; HexNAc) that are char- acteristic of glycopeptides. Subtraction of the mass of the peptide LLLAMENYTAIPYYTMAQNLDMK(289–311) (m/z avg 2709.24 [M + H] + ) from the molecular masses of the glycopeptides observed, provided the oligosaccharide residue compositions and stoichiometry summarized in Table 1. Previous experiments in our laboratories have shown that fucose is the only deoxyhexose found on APN [10]. Taking this into account, these data suggest that the N-linked glycans at Asn295 have compositions consis- tent with fucosylated paucimannosidic core structures (Fuc 1)2 Hex 2)3 HexNAc 2 ) and a difucosylated structure bearing one short antenna composed of HexNAc (Fuc 2 Hex 3 HexNAc 3 ). These glycans have been observed previously in insect glycoproteins [3,6,18]. However, abun- dant oligosaccharide components consisting of Hex 3 Hex- NAc 3 Fuc 3 (m/z 4243.82 [M + H] + ), Hex 3 HexNAc 4 Fuc 3 (m/z 4447.14 [M + H] + )andHex 3 HexNAc 4 Fuc 4 (m/z 4593.33 [M + H] + ) were surprising because they corres- pond to no known N-glycan s tructures. These assignments were corroborated by analysis of the released peptide after treatment with PNGase A. After treatment with this enzyme, the deglycosylated peptide is one mass unit larger than the expected mass of its unglycosylated counterpart because PNGase A converts Asn (114 Da) (to which the oligosaccharides had been attached) to Asp (115 Da) [15]. The MALDI-TOF mass spectrum shown in Fig. 2B exhibits signals for the released peptide spanning residues L289–K311. This peptide contains three methionine resi- dues, which have all partially oxidized during storage to give four signals at m/z 2708.3313 [M + H] + , m/z 2724.3005 [M + H] + , m/z 2740.3098 [M + H] + and m/z 2756.3022 [M + H] + . The expected monoisotopic masses for the Fig. 2. MALDI-TOF MS of HPLC fraction number 68. (A) Linear MALDI mass spectrum of the glycopept ides spanning residu es Leu289–Lys311 from APN1, which contain the consensus sequence for N-linked glycosylation at Asn295. Mass differences corresponding to fucose (146 Da) and HexNAc (203 Da) are observed. (B) Reflectron MALDI mass spectrum of methionine oxidized peptides spanning residues Leu289–Lys311 after tr eatment with PNGase A. Table 1. Glycopeptides L289–K311 spanning Asn295 observed by MALDI-TOF M S. Average masses are i ndicated a nd m ajor c ompo- nents are highlighted in bold text. Observed m/z [M + H] + Measured carbohydrate residue mass (Da) Calculated carbohydrate residue mass (Da) Carbohydrate assignment 3732.01 1022.77 1022.96 Hex 2 HexNAc 2 Fuc 2 3748.21 1038.97 1038.96 Hex 3 HexNAc 2 Fuc 3894.05 1184.81 1185.10 Hex 3 HexNAc 2 Fuc 2 4097.60 1388.36 1388.30 Hex 3 HexNAc 3 Fuc 2 4243.82 1534.58 1534.44 Hex 3 HexNAc 3 Fuc 3 4447.14 1737.90 1737.64 Hex 3 HexNAc 4 Fuc 3 4593.33 1884.09 1883.78 Hex 3 HexNAc 4 Fuc 4 4244 E. Stephens et al.(Eur. J. Biochem. 271) Ó FEBS 2004 native, m ono-, di- and tri-oxidized peptide (in which Asn295 has been converted to A sp) are m/z 2708.3025 [M + H] + , m/z 2724.2974 [M + H] + , m/z 2740.2920 [M + H] + ,and m/z 2756.2872 [M + H] + , respectively, giving a mass accuracy of less than 11 p.p.m. in each case. These assignments were confirmed by MALDI-CID of the most abundant species at m/z 2740.3098 (data not shown). Residue N609. The MALDI-TOF ma ss spectrum of the HPLC fraction number 54 exhibited two signals that differed in mass corresponding to a hexose r esidue (Fig. 3A). Subtraction of the calculated monoisotopic mass of the peptide VNYDNTTWGLITR(605–617) (m/z mono 1552.7760 [M + H] + ) from the measured masses of the two glycopeptides observed, provided the oligosaccha- ride residue compositions Hex 2 HexNAc 2 (m/z 2283.06 [M + H] + )andHex 3 HexNAc 2 (m/z 2445.14 [M + H] + ). These compositions correspond to nonfucosylated pauci- mannosidic structures that have frequently been observed as major constituents on other insect glycoproteins [3,18–20]. These putative assignments were corroborated by fragmentation of the glycopeptide at m/z 2445.14 Fig. 3. MALDI-TOF MS of H PLC fraction number 54. (A) Reflectron MALDI mass spectrum of the glycopeptides spanning residues Val605– Arg617 from APN1, which contain the consensus sequence for N-linked glycosylation at Asn609. A mass difference corresponding to hexose (162 D a) is observed. The s ignal at m/z 2441.14 corresponds to a nonglycosylated tryptic peptide Asn182–Arg203 from APN3. (B) MALDI-TOF/ TOF fragment ion m ass spec trum of the major glycopeptide Hex 3 HexNAc 2 -(Val605–Lys617) at m/z 2445.1389 [M + H] + .Theformationofthe 0,2 X 0 fragment is shown in the insert (R ¼ He x 3 HexNAc). Ó FEBS 2004 Novel insect N-glycans from Manduca sexta (Eur. J. Biochem. 271) 4245 (Hex 3 HexNAc 2 -[V605–R617]) by MALDI high-energy CID (Fig. 3B). The resulting CID spectrum is dominated by fragment ions at high mass, which a re derived from the cleavage of glycosidic linkages in the oligosaccharide portion. The major signal at m/z 1 635.92 corresponds to a 0,2 X 0 fragment (Fig. 3B, insert), which has been observed previously in the high-energy CID spectra of Asn-linked glycopeptides [21]. Other signals at low mass are derived from cleavage of the peptide portion and reveal peptide sequence information (V605–R617). Residue N623. MALDI-TOF MS analysis of two HPLC fractions (fractions 43 and 44) gave a set of signals, which differ by masses corresponding to 203 Da (HexNAc) and 146 Da (Fuc) (Fig. 4). Many sig nals w ere c ommon t o e ach HPLC fraction (e.g. m/z 2770, m/z 2916, m/z 3322, etc.), which s uggested that one set of heterogeneous glycopeptides had e luted i nto t wo HPLC fractions. Surprisingly, MALDI- CID of the two most intense signals in each fraction revealed the presence of glycopeptides consisting of two different peptide moieties. These peptides differ by a mass that corresponds to a HexNAc residue (203 Da), which significantly complicated the glycopeptide assignments. Consequently, the other signals in each fraction were subjected to MALDI-CID in order to confirm the identity of each glycopeptide constituent. The CID spectra of the smaller glycopeptide signals in fraction 43 exhibited non- glycosylated peptide fragment ions that indicated the peptide portions consist to SANRTVIHELSR(621–632) (Fig. 5A) from APN1. However, MALDI-C ID of the larger glycopeptides in that fraction (m/z 3322.55 [M + H] + , Fig. 5B) revealed the presence of a glycopep- tide consisting of AFRNNNTLVPVNAR(739–752) from a contaminating isoform of aminopeptidase (APN3). Subse- quent MALDI-CID of the glycopeptides in HPLC fraction 44 revealed that the majority of signals are due to the latter glycopeptide from APN3 (A739–R752) (Fig. 5C). How- ever, a small proportion of the smaller, less polar glyco- peptides in fraction 44 were shown to be from APN1 (e.g. m/z 2568.19, m/z 2770.3 and m/z 2916.35). Consequently, subtraction of the mass of the tryptic peptides span- ning residues S621–R632 (APN1, m/z mono 1382.7504 [M + H] + ) and A739–R752 (APN3, monoisotopic mass 1585.8563 [M + H] + ) from the measured mass of each designated glycopeptide yielded the glycan compositions summarized in Tables 2 and 3 for APN1 and APN3, respectively. These data indicate that the N-linked glycans at Asn623 on APN1 are similar in structure to those unusual, highly fucosylated oligosaccharides found at Asn295. Interestingly, most compositions for the N- glycans from APN3 (Table 3) are the same as those found on APN1, suggesting that similar structures are present. However, one major glycopeptide at m/z 3176.49 [Hex 3 Hex- NAc 4 Fuc 2 -(A739–R752)] is composed of a difucosylated glycan moiety that is not observed in APN1. It is interesting to note that the major 0,2 X 0 fragment ion, which was observed in the fragment ion spectrum of Hex 3 HexNAc 2 - (Val605–Arg617) (Fig. 3B) is not detected in the high- energy CID spectra of the fucosylated glycopeptides (Fig. 5). This cross-ring cleavage may be prevented by the presence of fucose on the Asn-linked GlcNAc residue, whichisindicatedineachspectrumbymajorfragments corresponding to the peptide bearing HexNAcFuc. Residue N752. M ALDI-TOF MS analysis o f fraction number 63 revealed a series of signals (from m/z 3400 to m/z 4400) (Fig. 6A) that differ by masses corresponding to sugar residues (146 Da and 203 Da). Subtraction of the calculated monoisotopic mass of the peptide spanning res- idues NGSFIPANMRPWVYCTGLR(752–770) (m/z mono Fig. 4. MALDI mass spectra of HPLC fractions 43 (A) and 44 (B) containing the g lycopeptides spanning residues S er621–Arg632 (from APN1) and Ala739–Arg752 (from APN3), respectively. Mass differe nces correspon ding to fucose (146 Da) and H exNAc (203 Da) are observed. 4246 E. Stephens et al.(Eur. J. Biochem. 271) Ó FEBS 2004 Fig. 5. MALDI-TOF/TOF fragment i on mass s pe ctra of ( A) the APN1 glycopeptide in f raction 43 [ Hex 3 HexNAc 3 Fuc 3 -(Ser621–Arg632)] at m/z 2916.32 [M + H] + , (B) the APN3 glycopeptide [Hex 3 HexNAc 4 Fuc 3 -(Ala739–Arg752)] in fraction 43 at m/z 3322.52 [M + H] + and ( C) the A PN3 glycopeptide [Hex 3 HexNAc 3 Fuc 2 -(Ala739–Arg752)] in fraction 44 a t m/z 2973.39 [M + H ] + . Ó FEBS 2004 Novel insect N-glycans from Manduca sexta (Eur. J. Biochem. 271) 4247 2240.0745 [M + H] + ) revealed the glycan compositions summarized in Table 4. These assignments were corro- borated by MALDI-CID o f selecte d signals, where fragment ions from the peptide backbone confirmed the sequence of the peptide portion (Fig. 6B). These data indicate that the N-linked glycans at Asn752 are similar to those highly fucosylated oligosaccharides found at Asn295 and Asn623. Analysis of the released peptide fraction after treatment with PNGase A r evealed minor signals for the native and methionine oxidized deglycosylated peptides N752–R770 with Asn752 converted to Asp. However, major signals for its glycosylated counterparts were also observed indicating that these glycopeptides are m ostly resistant to glycopeptidase A digestion (data not shown). This is probably due to the presence of the free amino group at Asn752 [22,23]. Structure elucidation of released N-glycans by MALDI-TOF/TOF tandem mass spectrometry The PNGase A-released glycan fractions containing un- usual, highly fucosylated oligosaccharides from Asn295 and Asn623 were pooled and analyzed by MALDI-TOF MS after permethylation (Fig. 7 and Table 5). In addition to the major glycan components (Fuc 2-4 Hex 3 HexNAc 2)5 ), these data reveal minor amounts of large, highly fucos- ylated N-glycans (Fuc 5–6 Hex 3 HexNAc 5)6 ), which were not detected in the g lycopeptide data. Many of these N-glycan compositions (Fuc 3-6 Hex 3 HexNAc 3)6 ) indicate that difucosylated trimannosyl core structures, decorated with fucosylated antennae, are likely to be present on APN1.Detailedstructuralcharacterization o f the novel highly fucosylated N-glycans was achieved by high-energy CID of the major oligosaccharide signals using a MALDI-TOF/TOF tandem mass spectrometer. Previous work on native and permethylated N-glycans have shown that this instrument can reveal unambiguous oligosaccha- ride sequence, as well as linkage and branching informa- tion [24–26]. Characterization of core fucosylation In order to locate the fucose substitutions on the chito- biose core, MALDI high-energy CID was carried out on the glycan at m/z 1519.8 [M + Na] + ,whichis consistent with a difucosylated paucimannosidic struc- ture (Fuc 2 Hex 3 HexNAc 2 ) (Fig. 8A). As depicted in Scheme 1A, structurally informative fragment ions include 1,5 X 1a and Y 1a (Domon & Costello nomenclature [27]), which show that only one fucose is attached to the reducing-end GlcNAc in the chitobiose core. Previous studies have shown t hat the N-glycans on APN are resistant to PNGase F, suggesting that this fucose is likely to be attached to the C-3 position of proximal GlcNAc. However, the presence of glycans bearing 6-linked fucose on Asn-linked GlcNAc cannot be ruled out. Importantly, the cross-ring fragment ion, 1,5 X 2a , indicates that one other fucose is attached to distal GlcNAc of the chitobiose unit. This assignment is verified by two majo r ÔinternalÕ fragment ions at m/z 671.32 and m/z 880.38. The former ion (m/z 671.32) is the likely product of a Z-type cleavage (Z 2 ) followed by the elimination of fucose from C-3 of distal GlcNAc [24]. The latter ion (m/z 880.38) is likely to be derived from a C-type cleavage (C 3 ) followed Table 2. Glycopeptides fr om APN1 (S621–R632) spanning Asn 623 observed by MALDI-TOF MS. Major components are noted in bold text and monoisotopic masses are indicated. m/z [M + H] + Measured carbohydrate residue mass (Da) Calculated carbohydrate residue mass (Da) Carbohydrate assignment HPLC fraction 2567.191 1184.441 1184.443 Hex 3 HexNAc 2 Fuc 2 43 a +44 2770.279 1387.529 1387.512 Hex 3 HexNAc 3 Fuc 2 43 a +44 a 2916.323 1533.573 1533.570 Hex 3 HexNAc 3 Fuc 3 43 a +44 a 3119.425 1736.675 1736.650 Hex 3 HexNAc 4 Fuc 3 43 a 3265.452 1882.702 1882.708 Hex 3 HexNAc 4 Fuc 4 43 a a Assignments corroborated by MALDI-CID of glycopeptides. Table 3. Glycopeptides from APN3 (A739–R752) spanning Asn743 observed by MALDI-TOF MS. Monoisotopic ma sses are i ndic ated and m ajor components are noted in bold t ext. m/z [M + H] + Measured carbohydrate residue mass (Da) Calculated carbohydrate residue mass (Da) Carbohydrate assignment HPLC fraction 2770.310 1184.450 1184.443 Hex 3 HexNAc 2 Fuc 2 44 a 2973.388 1387.530 1387.512 Hex 3 HexNAc 3 Fuc 2 44 a 3119.492 1533.64 1533.570 Hex 3 HexNAc 3 Fuc 3 44 a 3176.489 1590.630 1590.590 Hex 3 HexNAc 4 Fuc 2 43 a +44 a 3322.550 1736.68 1736.650 Hex 3 HexNAc 4 Fuc 3 43 a +44 a 3468.630 1882.77 1882.708 Hex 3 HexNAc 4 Fuc 4 43 + 44 a 3671.749 2085.890 2085.790 Hex 3 HexNAc 5 Fuc 4 43 + 44 a a Assignments corroborated by MALDI-CID. 4248 E. Stephens et al.(Eur. J. Biochem. 271) Ó FEBS 2004 by the elimination of fucose from C-3 (Scheme 2). These mechanisms are probable b ecause previous mass spectro- metric studies on native and permethylated glycans have shown that there is a preferential loss of fucose from the C-3 position of GlcNAc [17,24,25,28,29]. These assign- ments were corroborated by comparing the fragment ions observed to those formed (under the same conditions) from a similar permethylated glycan purified from honey- bee PLA 2 . This paucimannosidic glycan has the same monosaccharide composition as the glycan from APN1 at m/z 1519.8 (Hex 3 HexNAc 2 Fuc 2 ). However, this structure, which was previously characterized by NMR [30], has a difucosylated core with the two fucose residues linked via C-6 and C-3 to the Asn-linked GlcNAc. The resulting high-energy CID spectrum (shown in Fig. 8B) exhibited different fragment ions from those observed for the APN1 glycan. In particular, the signals for the monofucosylated Y 1a (m/z 474.23) and 1,5 X 1a (m/z 502.22) fragments were absent. Instead, major signals were observed at m/z 648.25 and m/z 676.26 for the difucosylated Y 1a and 1,5 X 1a fragments, respectively. Moreover, the B 3 ion shows t hat the distal GlcNAc residue is not fucosylated (Scheme 1B) and the major Ôinternal Õ ion at m/z 671.32 in Fig. 8A (produced by elimination of fucose from the C-3 position of the Z 2 ion) is absent. However, a major signal for the ÔinternalÕ fragment at m/z 880.3 is still observed. This ion Fig. 6. MALDI-TOF M S of HPLC fraction number 63. (A) MALDI-TOF m ass spectrum exhibiting signals fo r the t ryptic glycopeptides spanning residues Asn752–Arg770 from APN1, which contain the consensus sequence for N-linked glycosylation at Asn752. (B) MALDI-TOF/TOF fragment ion mass s pectrum of the major g lycopeptide Hex 3 HexNAc 3 Fuc 3 -(Asn752–Arg770) at m/z 3773.63 [M + H] + . Ó FEBS 2004 Novel insect N-glycans from Manduca sexta (Eur. J. Biochem. 271) 4249 is likely to be formed by the same mechanism as shown in Scheme 2 but, in this case, methanol is eliminated from the C-3 position of GlcNAc instead of fucose. Together, these data unequivocally establish the attachment of fucose at the 3-position of distal G lcNAc in the difucos- ylated paucimannosidic structure from APN1. Characterization of fucosylated antennae The monosaccharide compositions of the larger N-glycans observed (Fuc 3)6 Hex 3 HexNAc 3)6 ), suggested that these structures are decorated with antennae consisting of Hex- NAc and fucose. Previous studies on the glycans from honeybee PLA 2 have shown that fucosylated lacdiNAc [GalNAcb1-4(Fuca1-3)GlcNAc] antennae are present o n a small population of N-glycans [4,5]. Antennae composed solely of GlcNAc-Fuc were not observed. However, the monosaccharide composition of the major N-glycan from APN1 at m/z 1939.0 (Fuc 3 Hex 3 HexNAc 3 ) indicates that the latter novel antennae (GlcNAc-Fuc) is present on this structure. Consequently, the existence of this antenna was investigated by high-energy CID of this glycan and the resulting fragment ion spectrum is shown in Fig. 9A. The presence of two fucose residues on both GlcNAc residues in the chitobiose unit (Scheme 3A) was demonstrated by the characteristic fragment ions Y 1a , 1,5 X 1a ,and 1,5 X 2a .In addition, this spectrum gave signals for 3,5 A 5 and 3,5 A 6 , which show that both fucoses are linked via C-3 to each GlcNAc residue in the core. Other important, structurally informative fragment ions include the B 2a ion (m/z 456.32) and the cross-ring fragment a t m/z 1329.38 ( 1,5 X 3a/b ). These Table 4. Glycopeptides Asn752–Arg770 spanning Asn752 observed by MALDI-TOF MS. Monoisotopic masses are indicated and major constituents are noted in b old text. m/z [M + H] + (monoisotopic) Measured carbohydrate residue mass (Da) Calculated carbohydrate residue mass (Da) Carbohydrate assignment 3424.5145 1184.440 1184.443 Hex 3 HexNAc 2 Fuc 2 3627.5753 1387.501 1387.512 Hex 3 HexNAc 3 Fuc 2 a 3773.6285 1533.554 1533.570 Hex 3 HexNAc 3 Fuc 3 a 3976.7195 1736.645 1736.650 Hex 3 HexNAc 4 Fuc 3 4122.7215 1882.647 1882.708 Hex 3 HexNAc 4 Fuc 4 4325.7823 2085.708 2085.790 Hex 3 HexNAc 5 Fuc 4 a Assignments corroborated by MALDI-CID of glycopeptides. Fig. 7. MALDI-TOF mass s pectra exhibiting major signals for permethylated glycans f rom Asn295 and Asn6 23 from APN1. N-glycans were r eleased from APN1 tr yptic glycopeptid es by d ige stion with PNGase A. The released N -gly cans were permethylated and p urifi ed by Se p-Pa k. The 35% (v/v) (A) and the 50% (v/v) (B ) a cetonitrile fractio ns w ere s creened b y M ALDI-TOF MS. T he insert shows an expanded r egion exhibiting m inor signals for the high m ass components. Signals marked withanasteriskareglucosepolymercontaminants. 4250 E. Stephens et al.(Eur. J. Biochem. 271) Ó FEBS 2004 [...]... substitution is provided by the dominant signal at m/z 850.40 that is derived from the elimination of mannose from the C-3 position of the C4 ion, by a similar mechanism shown in Scheme 2 Similar fragment ions (nonreducing cross-ring fragments and internal ions), which indicate the presence of a structural isomer composed of one antenna attached to the 3-arm of core mannose were not observed, indicating that... fragment ions at m/z 1749.76 and m/z 850.39 indicate that one antenna (consisting of Fuc-HexNAc) can be linked to the 3-arm of core mannose The latter ion (m/z 850.39) is an ÔinternalÕ ion derived from a C4 cleavage followed by the elimination of FucGlcNAcMan from the C-3 position of core mannose Together, these high-energy CID data indicate that the major N- glycan structures on APN1 possess unusual... analyses of endogenous insect glycoproteins are generally lacking, in contrast to those of recombinant mammalian glycoproteins expressed in insect cells Therefore, the discovery of new structures widens our understanding of the glycosylation of these lower animals In addition, the relatively large quantities of the fucosylated mono- and bi-antennary N- glycans are unusual as compared to other insect glycoproteins... paucimannosidic structure (Man3GlcNAc2) is the major component at Asn609 in APN1 This glycan is the most common N- glycan found in insects or cultured insect cells [3,19,20,32] The other three sites on APN1 (Asn295, Asn623 and Asn752) were shown to contain very unusual, highly fucosylated N- glycans, with the most abundant glycoform consisting of Hex3HexNAc3Fuc3 These highly fucosylated glycans were... Subsequent exoglycosidase digestions of intact glycopeptides revealed the anomeric configurations of the residues in the antennae as Fuca(1-3)GlcNAc Together, these data show that the majority of the glycans on APN1 (75%) consist of novel, highly fucosylated structures that are depicted in Fig 11 The presence of the unusual fucosylated N- glycans in M sexta is of considerable interest The glycosylation analyses... 268.12 in Fig 9A provides strong evidence for a Fuc(1-3)HexNAc linkage in the antenna In addition, the A-type cross-ring fragment ions, 0,4A4 and 3,5A4, show that this short fucosylated antenna is linked to the 6-arm of core mannose This assignment is confirmed by the presence of a minor signal for 1,3A4, which shows that only one mannose residue is attached to C-3 of core b-mannose Further verification of. .. mannose [33] It is interesting to speculate on a possible functional role for these highly fucosylated N- glycans on APN They are unlikely to be involved in binding the Cry 1Ac toxin, as previous studies have shown that GalNAc mediates this binding event [9] GalNAc has been found in the terminal structures of lacdiNAc [GalNAcb1-4(Fuca13)GlcNAc] containing N- glycans from honeybee PLA2, but this terminal... proportion of the Fuc(1-3)GlcNAc moiety can be linked to the 3-arm of the core mannose residue Furthermore, molecular ion signals observed for the released permethylated glycans suggest that putative tri- and even tetra-antennary glycans can exist However, the signals for these multiantennary components were too small to gain fragment ion spectra and alternative assignments for these minor structures cannot... glycoprotein preparation, purified by Cry 1Ac affinity chromatography, contained three aminopeptidase proteins: APN1 (111 kDa), APN3 (108 kDa) and APN4 (114 kDa), with the APN1 protein being the most abundant Inspection of the amino acid sequence of each protein revealed four potential N- glycosylation sites in APN1, seven in APN3 and five in APN4 Thus, the peptide digest mixture could contain up to 16 sets of. .. for the ions observed in Fig 9B are correct Other key fragment ions in the CID spectrum of the APN1 glycan are depicted in Scheme 3B Structurally informative fragment ions include 1,5X3a, and Y3a, which indicate that only one arm of the trimannosyl core is substituted with the HexNAcFuc antennae Importantly, the minor signal for 3,5A4 shows that these two Ó FEBS 2004 Novel insect N- glycans from Manduca . GlcNAc antennae and the distal GlcNAc of th e chitobiose un it in N- glycans. Furthermore, the major N- glycan components on APN1 carry antennae linked to the. that these antennae are predominantly found on the upper 6-arm of the core mannose. The paucimannosidic N- glycan (Man 3 GlcNAc 2 ), commonly found on other