Galactofuranoic-oligomannose N-linked glycans of alpha-galactosidase A from Aspergillus niger

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Eur. J. Biochem. 268, 4134±4143 (2001) q FEBS 2001

Galactofuranoic-oligomannose N-linked glycans of a-galactosidase A from Aspergillus niger Gregg L. F. Wallis1,2, Richard L. Easton3, Karen Jolly1, Frank W. Hemming2 and John F. Peberdy1 Schools of 1Biological and 2Biomedical Sciences, University of Nottingham, Nottingham UK; 3Department of Biochemistry, Imperial College of Science, Technology and Medicine, London UK

Extracellular a-galactosidase A was purified from the culture filtrate of an over-producing strain of Aspergillus niger containing multiple copies of the encoding aglA gene under the control of the glucoamylase (glaA) promoter. Endoglycosidase digestion followed by SDS/PAGE, lectin and immunoblotting suggested that glycosylation accounted for < 25% of the molecular size of the purified protein. Monosaccharide analysis showed that this was composed of N-acetyl glucosamine, mannose and galactose. Mild acid hydrolysis, mild methanolysis, immunoblotting and exoglycosidase digestion indicated that the majority of the galactosyl component was in the furanoic conformation (b-d-galactofuranose, Galf). At least 20 different N-linked oligosaccharides were fractionated by high-pH anion-exchange chromatography following release

a-Galactosidase (a-d-galactoside galactohydrolase; EC is an exoglycosidase that catalytically removes a-linked terminal nonreducing galactose residues from small oligosaccharides such as melibiose and raffinose and larger galactopolysaccharides and galactolipids. It is widely distributed in biological systems [1] and the genes encoding a-galactosidases have been isolated from microorganisms, plants and animals and classified into three well-conserved families based on sequence similarity ([2]; Prosite entry no. PDOC00443). Several species of filamentous fungi produce extracellular a-galactosidases. They have been identified and

Correspondence to G. L. F. Wallis, Meristem-Therapeutics, 8 rue des Freres Lumiere, 63100 Clermont-Ferrand, France. Fax:. 1 33 473986819, Tel.: 1 33 473986810, E-mail: [email protected] Abbreviations: AMM, Aspergillus minimal medium; b-Galfase, b-galactofuranosidase; EBA-2, peroxidase-conjugated monoclonal antibody against Galf-containing glycoconjugates; Endo-H, endo N-acetylglucosaminidase H; Galf, b-d-galactofuranose; GAM, glucoamylase; GNA, Galanthus nivalis agglutinin; HPAEC, high pH anion-exchange chromatography; GlcNAc, N-acetyl-glucosamine; PNGase F, peptide-N-glycosidase F; DD20, A. niger strain DD20[pAB-6S]#37. Enzymes: a-d-galactoside galactohydrolase (EC; peptide-N-glycosidase F (EC; endo N-acetylglucosaminidase H (EC; a-galacto(pyrano)sidase (EC; b-galacto(pyrano)sidase (EC; bovine pancreatic trypsin (EC (Received 7 February 2001, revised 25 May 2001, accepted 27 May 2001)

from the polypeptide by peptide-N-glycosidase F. The structures of these were subsequently determined by fast atom bombardment mass spectrometry to be a linear series of Hex7226HexHAc2. Indicating that oligosaccharides from GlcNAc2Man7, increasing in molecular size up to GlcNAc2Man24 were present. Each of these were additionally substituted with up to three b-Galf residues. Linkage analysis confirmed the presence of mild acid labile terminal hexofuranose residues. These results show that filamentous fungi are capable of producing a heterogeneous mixture of high molecular-size N-linked glycans substituted with galactofuranoic residues, on a secreted glycoprotein. Keywords: Aspergillus niger; a-galactosidase; N-linked oligosaccharide; galactofuranose.

purified from many Aspergillus species including A. niger [3,4], A. nidulans [5], A. tamarii [6], A. awamori [7] and A. ficuum [8]. The majority of these enzymes are glycoproteins. However, only limited glycan structural data is available [7,9]. The corresponding encoding genes have also been identified in A. niger [10,11], Trichoderma reesei [12], Penicllium sp. [13,14]. and Mortierella vinacea [15]. There is accumulating evidence that these organisms are capable of producing more than one a-galactosidase. A. niger [4,10,11], A. tamarii [6,16], T. reesei [12,17] and P. simplicissimum [13] can probably produce at least two different polypeptides (encoded by separate genes). These are probably induced by, and subsequently degrade different, galactose-containing substrates within the extracellular environment [11]. a-Galactosidase A (melibiase) from A. niger is the product of the aglA gene ([10]; Swiss-prot accession no. P28351) and represents a minor activity when strain N402 is grown on a galactomannan substrate. The mature, extracellular and purified enzyme possesses an SDS/ PAGE-determined molecular mass of < 20 kDa greater than that suggested by the predicted amino acid sequence. There are also seven potential sequons for N-linked glycan attachment and a C-terminal domain that is rich in hydroxy amino acids [10]. These data suggested that the translated polypeptide undergoes considerable posttranslational modification during its passage along the secretory pathway. In this paper, a-galactosidase A secreted during the exponential growth phase by an A. niger strain containing multiple copies of the aglA gene (under the control of the homologous glucoamylase promoter), was purified to

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homogeneity and the N-linked glycan component was analysed.

M AT E R I A L S A N D M E T H O D S Fungal strains and cultivation A. niger strain N402 derived from wild-type strain A. niger van Tiegham, is a morphological mutant with short conidiophores [18]. A. niger strain DD20[pAB-6S]#37 (referred to as DD20) was constructed using plasmid pAB1-6S which contains multiple copies of the aglA gene under the control of the A. niger glucoamylase (glaA) promoter [10]. Southern analysis suggested that the DD20 strain contained < 10 copies of the aglA gene (P. J. Punt, Dept of Applied Microbiology and Gene Technology, Zeist, the Netherlands, personal communication). Both strains were routinely maintained on plates of Aspergillus minimal medium (AMM) or Vogel's minimal medium with glucose (1% w/v) as the carbon source. Spore suspensions from these plate cultures were prepared as described previously [19]. Shake-flask cultivation was performed with 1-L shake-flasks containing 200 mL AMM, 1% maltodextrin (13±17 dextrose units in size; Aldrich) and 1  107 spores´ml21. These were grown on an rotary shaker (180 r.p.m.) at 28 8C for up to 3 days. The culture filtrate was removed from the biomass by filtration through Mira cloth (CN Biosciences, Nottingham, UK), filtered once more through Whatman No.1 filter paper and then dialysed consecutively against distilled water and 30 mm Tris/HCl, pH 8.0 at 4 8C. Purification of a-galactosidase A Dialysed culture filtrate (approx. 1 L volume at pH 8.0) was first concentrated and partially fractionated by anionexchange chromatography on Sepharose-Q fast flow (Pharmacia), as described previously for invertase [19]. Fractions possessing a-galactosidase activity were desorbed with 0.2 m NaCl in 50 mm Tris/HCl pH 8.0, dialysed against distilled water, pooled and then lyophilized. These were then fractionated further sequentially by two FPLC steps; anion-exchange chromatography on Neobar AQ using a similar protocol as described previously for glucoamylase (GAM) purification [20], followed by chromatofocusing on Mono-P HR 5/20 (Pharmacia) using a pH gradient of 6±4 (as reported in [19]). Fractions possessing a-galactosidase activity eluted with a NaCl concentration of 100±150 mm from the former and at an eluate pH of 5.0 from the latter. These were pooled, dialysed against distilled water at 4 8C and then lyophilized. SDS/PAGE and tryptic digestion followed by reversephase HPLC and Edman automatic peptide sequencing were used for purity estimation. Enzyme assays a-Galactosidase activity was assayed by following the hydrolysis of p-nitrophenyl-a-d-galactopyranoside (Sigma). A 200-mL volume containing the sample and 1 mm substrate in 0.2 m sodium acetate buffer (pH 4.5) was incubated at 37 8C for 30 min. The reaction was terminated by the addition of 500 mL 0.25 m NaOH and the

absorbance recorded at 405 nm. One unit of a-galactosidase activity is defined as the amount of enzyme releasing 1 mmol of the substrate per min. In situ activity following nonreducing SDS/PAGE was determined by the method of Chen et al. [21] except, following electrophoresis gels were incubated with 0.25 m raffinose in 0.1 m sodium acetate buffer (pH 5.0) for 1 h at 37 8C, prior to staining. GAM activity was determined as described previously [20]. Glycan isolation and analysis Monosaccharide composition and Western blot analysis were performed according to Wallis et al. [20]. N-linked glycans were isolated from a-galactosidase A by endoglycosidase digestion with either peptide-N-glycosidase F (PNGase F; EC or endo N-acetylglucosaminidase H (Endo-H; EC (both from Oxford Glycosciences, Oxford, UK) at 37 8C for 24 h. Both methods were conducted following prior denaturation of the protein sample by heating at 100 8C for 3 min in the presence of mercaptoethanol and/or SDS as described in manufacturer's instructions. Oligosaccharides were isolated from these digestion mixtures by a modification of the acetonic precipitation method of Verostek et al. [22]. Following acetonic precipitation the N-linked oligosaccharides were specifically solubilized by extraction with 60% methanol (v/v) and 60% ethanol (v/v). When electrophoretic analysis of the reaction was used the reactions were terminated by the addition of an equal volume of SDS/PAGE sample buffer (2  normal concentration) and a further denaturation step. O-Linked glycan was released by reductive b-elimination as described by Wallis et al. [20]. Exoglycosidase digestions with a- and b-galatopyranosidase (from green coffee beans, EC and bovine testes, EC (both from Oxford Glycosciences) and a-mannosidase (EC, jack bean) were performed at 37 8C for 24 h, as per manufactures' protocols. b-Galactofuranosidase (b-Galfase) was purified from the culture filtrate of A. niger strain N402 [23]. Digestions of protein or glycan with b-Galfase were performed at pH 4.5, 37 8C for 24 h (unless stated otherwise). Product analysis was conducted either directly by SDS/PAGE, or by high pH anion-exchange chromatography (HPAEC) analysis following precipitation of protein with 80% ethanol. High pH anion-exchange chromatography/pulsed amperometric detection analysis Neutral monosaccharides (dissolved in nanopureTM water) were separated on a CarboPac PA1 pellicular anionexchange column at 30 8C (Dionex) with pulsed amperometric detection, attached to a Dionex BIO-LC system [24]. Separation was achieved with an isocratic elution with 15±18 mm NaOH and identification by comparison with a mixture of monosaccharide standards. Oligosaccharides were fractionated with a linear gradient of 30±200 mm sodium acetate in 150 mm NaOH. Oligosaccharides of known structure from ribonuclease B and yeast invertase were used as standards. Chromatographic data were collected and evaluated by means of labchrom software (version 2.1; LabLogic, Sheffield, UK).

4136 G. L. F. Wallis et al. (Eur. J. Biochem. 268)

Mass spectrometry Pure glycoprotein was subject to reduction and carboxymethylation followed by digestion with trypsin (bovine pancreas, EC; Sigma). N-linked oligosaccharides were released by PNGase F (Flavobacterium meningosepticum, recombinantly expressed in Escherichia coli, Glyko), fractionated from the polypeptide by Sep-Pak reverse phase chromatography (Sep-Pak C-18, Waters), permethylated and further purified by Sep-Pak reverse phase chromatography, and eluted sequentially with 15, 35, 50, 75 and 100% acetonitrile, essentially as described by

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Dell et al. [25]. The dried permethylated N-glycans were then dissolved in methanol and analysed by FAB MS on a ZAB 2SE 2FPD double focussing fast-atom bombardment mass spectrometer fitted with a caesium ion gun operating at 30 kV. Data was acquired and processed using Micromass Ltd analytical opus software (Wythenshaw, Manchester, UK). Partially methylated alditol acetates were prepared from the permethylated N-glycans as described [26] and analysed by GC-MS using a MD800 (Fisons) instrument. Chromatographic separation was performed using a 30 m  0.25 mm inner diameter RTX-5 fused silica

Fig. 1. Time course showing the secretion of a-galactosidase A by A. niger strains N402 (X) and the aglA expressing transformant DD20 (W). Replicate 1-L flasks containing 200 mL AMM 1 1% maltodextrin were inoculated with 1  107 spores´ml21 and incubated at 28 8C. At the times indicated, 10 mL of culture filtrate were decanted and centrifuged (1000 g, 5 min), the amount of biomass was estimated as a percentage of the total volume (A). The resultant supernatant was dialysed overnight against distilled water at 4 8C and then assayed for soluble protein (B), GAM (C) and a-galactosidase activity (D), as described. Electrophoretic analysis of the proteins secreted during cultivation by N402 and DD20 are shown in panels E and F, respectively. One millilitre of the dialysed culture filtrate from each time point was lyophilized and the dried residues were then analysed by SDS/PAGE on a 9% gel, stained with Coomassie blue. The position of molecular mass markers is indicated.

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capillary column (Thames Restek Corp., Windsor, Berkshire, UK). The sample was dissolved in hexanes prior to injection onto the column. The column was at a temperature of 65 8C for injection and was maintained at this temperature for 1 min followed by an increase to 290 8C at a rate of 8 8C´min21. Analytical procedures Protein concentration was measured by the Biorad method using GAM (data from Fig. 1 only) or Yeast invertase as the standard. Mild acid hydrolysis was carried out by incubation of the sample in 0.1 m trifluoroacetic acid for 1 h at 100 8C, followed by lyophilization or drying under nitrogen gas. Mild methanolysis of galactofuranosides and their analysis by HPAEC was as described by Hemming et al. [27]. Mycelial growth in shake-flasks was estimated using a wet settled volume technique [19].

R E S U LT S A N D D I S C U S S I O N Production of a-galactosidase A A time course for the cultivation of A. niger strains N402 and DD20 in AMM and a maltodextrin carbon source showed the accumulation of both protein, GAM and a-galactosidase activity in the culture filtrate of both strains, during the exponential growth phase (30±60 h) (Fig. 1). The growth rate (determined by biomass production) and the secretion of GAM activity were slightly higher in N402 than in DD20 (Fig. 1A and C). For example, after 64 h cultivation, the extracellular GAM activities were 301 ^ 1.5 mU´mL21 for N402 and 231 ^ 2.6 mU´mL 21 for DD20. In contrast, the levels of soluble protein (74 ^ 3 mg´mL21 and 156 ^ 7 mg´mL21) and a-galactosidase activity (36 ^ 6 mU´mL21 and 103 ^ 7 mU´mL 21) detected in the culture filtrate of the transformed DD20 strain were two- and threefold greater, respectively, than those in the untransformed N402 strain (Fig. 1 B and D). A. niger strain N402 was the parental (wild-type) strain from which the a-galactosidase A protein was originally purified (for N-terminal sequencing and ultimately gene cloning) and can be assumed to possess low levels of expression under these growth conditions [10]. However, a-galactosidase activity was observed at unexpectedly high levels (40 mU´mL culture filtrate21) in the late exponential/early stationary growth phase of N402 cultivation (Fig. 1D). This activity probably originated from the other endogenous galactosidases produced by A. niger [4,11] and/ or cell lysis from older mycelium. Electrophoretic analysis of

the total extracellular protein secreted during this experiment clearly showed the appearance of an extra broad protein band (75±90 kDa) in the culture filtrate of the DD20 strain which was absent from N402 (Fig. 1 E and F). This was later confirmed to be a-galactosidase A. A comparison of the secretion kinetics of GAM and a-galactosidase A by strain DD20 suggested that the former glycoprotein may be accumulating in the culture filtrate slightly earlier (Fig. 1 C, D and F). During the midexponential phase (< 38 h) the levels of GAM in the culture filtrate were 50% of those in the stationary phase (maximal value) while comparatively only 21% of the total a-galactosidase had been secreted at the same time. The simplest explanation is that other a-galactosidases were being secreted during the stationary phase which were contributing to the enhanced activity. However, it is also possible that the processing time (from translation to secretion) could be different for these two secreted glycoproteins, resulting in a faster secretion of GAM. The extent and type of glycosylation attached to these two proteins is substantially different: GAM has predominantly O-linked glycans [28] and a-galactosidase A has mainly N-linked glycans. A recent study [29] showed that the secretion of the model glycoprotein, cellobiohydrolase I (which possesses many O-linked glycans) was complete within only 15 min. Purification of a-galactosidase A a-Galactosidase A was purified to homogeneity from the late-exponential phase (50 h) culture filtrate of strain DD20. This harvesting point was chosen to minimize any unspecific liberation of other galactosidases by autolysis in the stationary growth phase, as observed in the parental strain N402 (Fig. 1D). Purification was achieved with a three-step protocol (Table 1). The major contaminating proteins observed were the two forms of GAM which were removed completely by the two anion-exchange steps (Sepharose Q and Neobar AQ). An unknown but glycosylated contaminant was further removed by chromatofocusing over a pH range of 6±4. a-Galactosidase A desorbed from the Mono-P column at an eluate pH of 5.0, which is close to the expected isoelectric point of 4.8 [10]. Purity of the a-galactosidase A was confirmed by SDS/PAGE analysis, which gave a broad band of < 75±90 kDa. Furthermore, under nondenaturing conditions a single band (< 120 kDa) was similarly observed which also possessed in situ activity against raffinose. Although direct comparison of molecular size estimations by SDS/PAGE and native electrophoresis of glycoproteins is unreliable, this data suggested that a-galactosidase A

Table 1. Purification of extracellular a-galactosidase A secreted by A. niger strain DD20. Purification was achieved with a consecutive protocol consisting of two anion-exchange and one chromatofocusing steps.

Purification step

Volume (mL)

Protein (mg)

Enzyme activity (mU)

Specific activity (mU´mg21)

Yield (%)

Fold purification

Culture filtrate Sepharose-Q Neobar-AQ Mono-P

850Š 100Š 30Š 11Š

42Š.4 20Š.1 18Š.5 7Š.5

11050Š 10650Š 8460Š 5840Š

261 525 557 779

100Š 96Š 77Š 53Š

1 2Š.0 2Š.7 3Š.0

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q FEBS 2001 Fig. 2. Glycosidase digestion of a-galactosidase A. (A). Incubation of 10 mg a-galactosidase A in the presence (PF) and absence (C) of 0.6 U PNGase F under denaturing conditions at 37 8C for 24 h. The reaction was terminated by the addition of SDS/PAGE loading buffer and the mixture was analysed on a 9% gel. (B±D) 20 mg of denatured a-galactosidase A (0.2% SDS, 100 8C, 3 min) was digested with either; 3 U Endo-H (EH), 5 U b-Galfase (GF) or a combination of both glycosidases (EH 1 GF), in 0.1 m sodium citrate phosphate buffer at pH 5.5, 4.5 and 4.8, respectively, for 24 h at 37 8C. The reactions were terminated as before. Electrophoretic analysis was performed using an 8% gel and bands were visualized for protein with Coomassie blue (10 mg protein loaded per lane, panel B), or Western blotted and either stained with GNA-lectin for the presence of terminal mannose residues (5 mg, panel C) or immunostained with EBA-2 (anti-Galf) monoclonal antibody (2.5 mg, panel D). Undigested a-galactosidase A was loaded in the lane labelled C and the positions of the molecular weight marker proteins are as indicated.

probably exists as a monomer in its native state. Tryptic digestion followed by reverse-phase HPLC and Edman automatic sequencing of two of the resultant peptides (324LLTVLNK and 469WTYPVTGNLK) confirmed the identity and purity of the a-galactosidase A preparation. The purified enzyme hydrolysed melibiose, raffinose and p-nitrophenyl-a-d-galactopyranoside, but not p-nitrophenylb-d-galactopyranoside. Monosaccharide composition Monosaccharide compositional analysis by HPAEC (of a trifluoroacetic acid hydrolysate of the protein) showed that a-galactosidase A contained; N-acetylglucosamine (GlcNAc), galactose and mannose in the approximate molar proportions of 1 : 3.5 : 9. This data was collated from replicate acid hydrolyses and represents an average composition. However, the susceptibility and stability to acid hydrolysis is different for each monosaccharide and the conditions used here probably result in an underestimation of GlcNAc. Variation in the galactose : mannose ratio (from 1 : 1 to 1 : 4) between different batches of the purified protein was also observed. This may represent small differences between each cultivation and possibly the differential activity of the endogenous b-Galfase [23]. Small amounts of glucose were also found but as this is a common contaminant of biological samples this can probably be disregarded. Electrophoretic and glycosidase analysis Western analysis of a-galactosidase A, followed by detection with lectin and anti-glycan antibodies confirmed that this was a glycoprotein (Fig. 2). The blotted bands were

visualized by Galanthus nivalis agglutinin (GNA)-lectin (indicating the presence of terminal a-linked mannose residues; Fig. 2C) and a monoclonal antibody EBA-2 [suggesting the presence of galactofuranoic components, b-d-galactofuranose (Galf); Fig. 2D]. EBA-2 is a rat monoclonal antibody raised against an immunogenic polysaccharide secreted by A. fumigatus that recognizes b1±5-linked Galf residues [30,31]. It has been used previously by us to reveal the presence of these unusual glycan modifications in GAM secreted by A. niger [20,23] and invertase from A. nidulans [27]. Lectins that bind to galactopyranoic-containing glycans such as DSA (Datura stramonium agglutinin, b1±4-linked Gal) and RCA (Ricinus communis agglutinin, b-linked Gal) did not bind to a-galactosidase A on Western blots (data not shown). Digestion with endoglycosidases such as PNGase F (Fig. 2A) and Endo-H (Fig. 2B, lane C and EH) led to mobility shifts of < 25 and < 15 kDa, respectively, of the a-galactosidase A protein band upon subsequent SDS/ PAGE analysis. The molecular size of the PNGase F digested band, approximately 55±60 kDa, is close to the expected size (60 148 Da) of the unprocessed precursor [10]. Furthermore, as this precursor possesses a signal sequence of < 2700 or < 3300 Da the translated polypeptide probably has a size of < 57 kDa. A time course PNGase F digestion showed that staining by GNA-lectin disappeared after an incubation of only 1 h, whereas reactivity to EBA-2 was still present (at a reduced level) after 18 h (data not shown). This suggested either incomplete digestion or the presence of O-linked glycans. Digestion with Endo-H, b-Galfase and a double digest containing both enzymes all led to increases in SDS/ PAGE mobility of a-galactosidase A (Fig. 2B). The

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to Galf. The in situ removal of the Galf led to more efficient cleavage of the N-linked glycans by Endo-H and a corresponding further increase in mobility compared to that with the two glycosidases acting alone (Fig. 2B±D, lane EH 1 GF). Thus N-linked glycans containing b-galactofuranoic components were relatively (compared to those without Galf) resistant to Endo-H digestion. This situation was less apparent with PNGase F. The double digested band also possessed a very weak reactivity to GNA and has an apparent molecular mass of 65 kDa. Additional evidence for galactofuranoic residues

Fig. 3. HPAEC analysis of N-linked glycans from a-galactosidase A. N-glycans were released from 160 mg of denatured a-galactosidase A by PNGase F for 24 h at 37 8C. The reaction was terminated and the products precipitated with cold 80% acetone as described. The released oligosaccharides were dissolved in 50 mL nanopureTM water. 15 mL (equivalent to approx. 50 mg protein) were analysed on a CarboPac PA1 column (Dionex) with pulsed amperometric detection, attached to a Dionex BIO-LC system (chromatogram B). Elution was started with 30 mm sodium acetate in 150 mm NaOH for 10 min followed by a linear gradient up to 200 mm sodium acetate over 35 min (shown as a dotted line on chromatogram C). The N-linked glycans released from ribonuclease B (5 mg glycan loaded) are shown in chromatogram A for comparison. In order of elution they are Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2 (two isomers), Man8GlcNAc2 and Man9GlcNAc2.

double digest with Endo-H and b-Galfase was possible as the pH optima of these two enzymes are similar at around pH 4±5 (manufacturers' instructions and [23]). Following Endo-H digestion the a-galactosidase A band (Fig. 2B±D, lane EH) contained considerably less terminal mannose (as shown by a decrease in staining by GNA), but surprisingly the levels of the galactofuranoic staining-component (EBA-2) were still quite high. Digestion of a-galactosidase A with b-Galfase alone produced a more compact protein band suggesting that the Galf component may be responsible for the majority of the electrophoretic heterogeneity observed by this glycoprotein (Fig. 2B±D, lane GF). This exoglycosidase removed the majority of the exposed Galf (Fig. 2D, lanes C and GF) revealing more terminal mannose residues (Fig. 2C, lane GF). This data suggested that the Galf components are probably attached to mannose residues, although it does not rule out the presence of other linkage combinations, e.g. Galf linked

The galactosyl glycan component of a-galactosidase A could be selectively released by chemical treatments such as mild acid hydrolysis and mild methanolysis and by digestion with the A. niger b-Galfase, but was resistant to both a-galactosidase (from green coffee bean which specifically cleaves a1±3, a1±4 and a1±6 links) and b-galactosidase (bovine testes; cleaves b1±4 and b1±3 links). Mild acid hydrolysis has been used before as a diagnostic tool for the presence of Galf residues [9,20,23,32]. Recently, we have used mild methanolysis coupled with HPAEC as an unambiguous assay for Galf residues [27]. Thus, this data supported by that obtained by Western blotting indicated the presence of a galactofuranoic composition on this glycoprotein. Furthermore, as both EBA-2 and the galactofuranosidase recognize the b-conformation and mild methanolysis resulted in the synthesis of a-methyl galactose (mild methanolysis results in an initial inversion of anomeric configuration [27]), it can be concluded that this Galf is present in the b-conformation. O-linked glycosylation Reductive b-elimination on both the intact protein and on material previously digested with PNGase F did not give any alditol oligo- or monosaccharides when analysed by HPAEC (data not shown). The conditions used for this cleavage have previously been successful for the removal of O-linked glycans from yeast invertase (Gregg L. F. Wallis, unpublished data) and A. niger GAM [20,23]. Thus, despite the presence of a putative S/ T-rich region in the polypeptide sequence of a-galactosidase A, we could find no evidence that O-linked glycans were an important component of this protein. These findings are in agreement with those obtained on other galactosidases from A. niger [4] but are in contrast with some other fungal a-galactosidases, e.g. A. awamori [7] and T. reesei [33]. HPAEC analysis of N-linked oligosaccharides The N-linked glycans released from a-galactosidase A following digestion with PNGase F were recovered by the acetonic precipitation method of Verostek et al. [22]. HPAEC analysis of the endoglycosidase-released oligosaccharides did not give easily interpretable chromatograms (Fig. 3B). Up to 25 different peaks were observed, although, seven or eight peaks accounted for 80% of the total composition (based on peak area). These oligosaccharide peaks eluted (with a sodium acetate gradient

4140 G. L. F. Wallis et al. (Eur. J. Biochem. 268)

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Fig. 5. Electron impact mass spectrum of furanosylhexose present in PNGase F released N-glycans from A. niger a-galactosidase A tryptic glycopeptides. Permethylated N-glycans were converted to their partially methylated alditol acetates and analysed by GC-MS. A peak eluting with a retention time of 19.07 min possessed this fragmentation pattern. An identical analysis performed after mild acid hydrolysis of released N-glycans prior to permethylation showed an absence of this peak.

of 30±200 mm) with retention times greater than those from the glycoprotein ribonuclease B (Man529GlcNAc2; Fig. 3A), and at a similar elution to some of the `inner-core' N-glycans from yeast invertase (Man8214GlcNAc2 [34,35], data not shown). Direct identification of each oligosaccharide was not possible; however, the smallest structure was equivalent in elution position to Man7GlcNAc2 and they increased to approximately the size of Hex20GlcNAc2. The complexity of the chromatograms could be explained by a combination of the much lower sensitivity of the pulsed amperometric detector for oligosaccharides (compared with monsaccharides) and the high resolving power of the HPAEC system for different oligosaccharide isomers. Thus, the system has separated the heterogeneous mixture of N-linked oligosaccharides isomers found on a-galactosidase A into numerous separate peaks, which were sometimes difficult to distinguish from the background. However, both mild acid hydrolysis and b-Galfase digestion led to a simplification of this oligosaccharide profile and a decrease in retention times (data not shown). This reflected the known influence of Galf residues upon the elution of neutral oligosaccharides during HPAEC [32], and suggested that these residues were probably attached to different positions in the molecule resulting in structural isomers and a complex elution pattern. Mild acid hydrolysis followed by incubation with a-mannosidase (jack bean) led to the disappearance of these peaks with a corresponding appearance of mannose and galactose when the hydrolysed samples were re-analysed for monosaccharide composition by HPAEC (data not shown). MS of N-linked oligosaccharides a-Galactosidase A was reduced, carboxymethylated and digested with trypsin. The N-linked oligosaccharides were released from glycopeptides using PNGase F. These were isolated from peptides by Sep-Pak purification, permethylated, fractionated by a second Sep-Pak step and then analysed by FAB-MS. The data showed the presence of

hexose containing N-glycans of considerable size variation from: Hex7HexNAc2 (m/z 1988) to Hex14HexNAc2 (m/z 3417) detected in the 50% acetonitrile (Sep-Pak) fraction (Fig. 4A), Hex9HexNAc2 (m/z 2396) up to Hex25HexNAc2 (m/z 5663), in the 75% (Fig. 4C) and Hex12HexNAc2 (m/z 3008) up to Hex26HexNAc2 (m/z 5867) in the 100% acetonitrile fraction (data not shown). The most abundant components were Hex10215HexNAc2, with a progressive decrease in abundance with increasing size. Although, there is an apparent loss of sensitivity at higher mass range [25]. In a second experiment the released N-glycans were split into two aliquots. One fraction was permethylated and analysed as above, and the other was subjected to mild acid hydrolysis prior to permethylation. FAB-MS data showed a shift in the profile of one (50% acetonitrile fraction, Fig. 4B) or two to three (75% acetonitrile fraction, Fig. 4D) hexose residues, compared to the nonhydrolysed sample. A comparison of the relative signal intensities shows that (in the 50% acetonitrile fraction) Hex7 to Hex9HexNAc2 increased two- to threefold, Hex10HexNAc2 was unchanged and Hex11 to Hex14HexNAc2 decreased by about one-half following mild acid hydrolysis (Fig. 4A and B). Similarly, Hex9 and Hex10HexNAc2 increased by four- and twofold, respectively, Hex11HexNAc2 was unchanged and Hex12 to Hex23HexNAc2 decreased in intensity to only one-half or 30% of the un-hydrolysed value (Fig. 4C and D). No oligosaccharides greater in size then Hex23HexNAc2 were found after hydrolysis. These data indicate that the mild acid treatment had removed one, two or possibly three hexose monosaccharides from the N-glycans. In corroboration with our other data (monosaccharide composition, Western analysis, b-Galfase digestion, etc.) this suggested that up to three Galf residues were present on each N-linked oligosaccharide. Linkage analysis was performed on the partially methylated alditol acetates derived from both hydrolysed and unhydrolysed a-galactosidase A N-glycans. Comparison of the two sets of data showed a peak eluting at 19.07 min in the latter sample that was absent from the

Fig. 4. FAB mass spectra of PNGase F released N-glycans from A. niger a-galactosidase tryptic glycopeptides with or without mild acid hydrolysis. Released N-glycans were either permethylated, fractionated and purified by Sep-Pak reverse-phase chromatography (A and C), or, hydrolysed in 0.1 m trifluoroacetic acid at 100 8C for 1 h prior to permethylation and Sep-Pak fractionation (B and D). The 50% (A and B) and 75% (C and D) acetonitrile fractions were analysed by FAB-MS. The signals at m/z 1947, m/z 2151, m/z 2355 and m/z 2559 arise from partial hydrolysis of the core GlcNAcb1±4GlcNAc bond in the major components.

4142 G. L. F. Wallis et al. (Eur. J. Biochem. 268)

former. The fragmentation pattern of this component, 2,3,5,6-tetra-O-methyl galactitol, was consistent with that for a terminally linked hexofuranose sugar (Fig. 5). Identically linked mannose residues were detected in both samples. The structural data indicate that a-galactosidase A contains a heterogeneous mixture of high-molecular mass oligomannose N-glycans (Man7224GlcNAc2) substituted with one, two or three terminal b linked Galf monosaccharides. However, other batches of protein had two- to threefold greater levels of Galf (based on monosaccharide composition). Biological significance The initial synthesis of N-linked glycans is relatively conserved amongst eukaryotes. This is in contrast with the processing and maturation steps which vary at a genus or species level and are also subject to additional and poorly understood environmental factors. An overall view of N-linked glycosylation in the ascomyceteous yeasts shows that there is an inner core of Hex8215GlcNAc2 to which is usually attached an extended poly(a1,6mannosyl) backbone. This in turn is substituted with a variety of differently linked-mannose, galactose and mannose phosphate diesters to give an outer chain of Hex502200GlcNAc2 in size [36]. The addition of such large oligomannose glycans is thought not occur in the filamentous fungi. Although, the number of studies using glycoproteins from the latter is small, a consensus opinion is that these N-linked glycans undergo a limited removal of glucose and a1,2-linked mannose followed by substitution with galactose, galactofuranose or mannosyl phosphate. Resulting in an average oligosaccharide size of Hex5210GlcNAc2 [37]. The N-linked oligosaccharides attached to a-galactosidase A are generally larger than those reported previously. We have a core structure of Man627GlcNAc2, which has probably resulted from a-1,2 mannosidase trimming [38] of the Man9GlcNAc2 precursor. The addition of up to 17 extra a-linked mannose residues, and one to three terminal b linked Galf residues has extended the chain to give the mature oligosaccharide. This provides evidence and, supports a previous study [32], that limited polymannosyl addition to N-linked oligosaccharides, similar to that found in the yeasts does also occur in some filamentous fungi. Furthermore, both the smallest (Hex8HexNAc2) and the largest oligosaccharide (Hex26HexNAc2) existed predominantly (there may be a small amount without galactofuranosylation) in the galactofuranosylated form, GalfMan7GlcNAc2 and Galf3Man23GlcNAc2, respectively (Hex7HexNAc2 was more abundant after hydrolysis and Hex23HexNAc2 the largest following hydrolysis). This suggested that all of these N-glycans were substituted with at least one terminal b-Galf residue. It is possible that these residues serve as a stop signal for further mannose addition (N-linked glycans larger than the core Man9GlcNAc2 would have additional mannose added), in a situation analogous to that proposed for a1,3-linked terminal mannose in Saccharomyces cerevisiae [39]. Glycoconjugates and polysaccharides containing galactose in the furanoic conformation have been found in

q FEBS 2001

bacteria and the lower eukaryotes (fungi and protozoa) only but not yet in mammalian systems [40]. However, the biological significance of this monosaccharide has yet to be elucidated. b-Galactofuranosyl residues have been shown to be a component of the galactomannan produced by Aspergillus spp. [31,41], the O-linked glycan of A. niger GAM [20,23], invertase secreted by A. nidulans [27] and part of other fungal glycoproteins [32,42,43]. Takayanagi et al. [9] also observed the, to date, novel structure of a-linked Galf residues on a different a-galactosidase produced by A. niger. The potential presence of galactofuranosylation and its associated antigenicity is an important consideration as A. niger is the most important fungal species currently involved in commercial enzymes for food production and its potential for recombinant protein production is under detailed investigation [44,45].

ACKNOWLEDGEMENTS This investigation was partly supported by a grant BIO4CT96-0535 (Eurofung) from the EU. We would like to thank P. Punt for supplying the A. niger strain DD20, and M. Tabouret and D. B. Archer for their kind gift of EBA-2 and anti-GAM antibodies, respectively. K. Bailey (Biopolymer structure and analysis unit, University of Nottingham) performed the peptide sequencing.

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