Eur J Biochem 269, 5459–5473 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03244.x Hemocyanin from the keyhole limpet Megathura crenulata (KLH) carries a novel type of N-glycans with Gal(b1–6)Man-motifs Tomofumi Kurokawa1,2, Manfred Wuhrer2, Gunter Lochnit2, Hildegard Geyer2, Jurgen Markl3 ă ă and Rudolf Geyer2,4 Pharmaceutical Discovery Center, Pharmaceutical Research Division, Takeda Chemical Industries, Ltd, Osaka, Japan; Institute of Biochemistry, University of Giessen, Giessen; and 3Institute of Zoology, Johannes-Gutenberg University of Mainz, Mainz, Germany Keyhole limpet (Megathura crenulata) hemocyanin (KLH), an extracellular respiratory protein, is widely used as hapten carrier and immune stimulant Although it is generally accepted that the sugar constituents of this glycoprotein are likely to be implicated in the antigenicity and biomedical properties of KLH, knowledge of its carbohydrate structure is still limited Therefore, we have investigated the N-linked oligosaccharides of KLH Glycan chains were enzymatically liberated from tryptic glycopeptides, pyridylaminated and separated by two-dimensional HPLC Only neutral oligosaccharides were obtained and characterized by carbohydrate constituent and methylation analyses, MALDI- TOF-MS, ESI-ion trap-MS and sequential exoglycosidase digestion The results revealed that KLH is carrying high mannose-type glycans and truncated sugar chains derived thereof As a characteristic feature, a number of the studied N-glycans contained a Gal(b1–6)Man-unit which has not been found in glycoprotein-N-glycans so far Hence, our studies demonstrate that this marine mollusk glycoprotein is characterized by a unique oligosaccharide pattern comprising, in part, novel structural elements Hemocyanins are oxygen-transporting proteins found in many arthropod and mollusc species [1] Binding of oxygen is mediated by binuclear copper-binding sites, resulting in the characteristic blue color of the oxygenated molecule The hemocyanin of the Californian giant keyhole limpet Megathura crenulata, a marine gastropod, has been further recognized as a potent immunoactivator [2] Based on these immunostimulatory properties, keyhole limpet hemocyanin (KLH) is widely used in research and clinical studies Present fields of application include: (a) its use as a highly immunogenic antigen in order to assess the immune competence of an organism [3,4]; (b) immunotherapy of bladder cancer [5,6], whereby its efficacy is assumed to be due to the expression of Gal(b1–3)GalNAc-determinants as cross-reacting epitopes [2,7]; and (c) its frequent use as a carrier of low molecular mass haptens, such as oligosaccharides, gangliosides or (glyco)peptides, designed, for example, as anticancer vaccines [8–11] In addition, it has been demonstrated that KLH shares a cross-reacting oligosaccharide epitope with glycoconjugates from Schistosoma mansoni [12–14], thus allowing the diagnosis of infections with S mansoni [15–17], Schistosoma haematobium [18] and Schistosoma japonicum [19] by enzyme-linked immunosorbent assay Furthermore, KLH has been reported to be of potential value for vaccination against these pathogens [19,20] Due to this widespread use of KLH, its molecular structure has been analyzed in detail [2,21,22] KLH consists of two structurally and physiologically distinct isoforms, KLH1 and KLH2, each being based on a subunit with a molecular mass of approximately 400 kDa Every subunit comprises eight different functional, i.e oxygen binding units of about 50 kDa At the level of the quaternary structure, KLH1 occurs as a cylindrical didecamer, whereas KLH2 exists as a mixture of didecamers and tubular multidecamers [2,21,22], thus leading to molecular masses of roughly eight million Daltons for each didecamer [2] From related molluscan hemocyanins, detailed structural information is available that is applicable to KLH [22]: the X-ray structure ˚ ˚ of a functional unit at 2.3 A resolution [23], a 12 A reconstruction of the didecamer from electron microscopical images [24] and the gene structure of the subunit [25] Moreover, a variety of functional units has been sequenced [26,27], including those from KLH [22] In contrast to this wealth of data on features like molecular architecture and amino acid sequence, information regarding the Correspondence to Rudolf Geyer, Biochemisches Institut am Klinikum der Universitat Giessen, Friedrichstrasse 24, D-35392 Giessen, ¨ Germany Fax: + 49 641 9947409, Tel.: + 49 641 9947400, E-mail: Rudolf.Geyer@biochemie.med.uni-giessen.de Abbreviations: dHex, deoxyhexose; endoH, endo-b-N-acetylglucosaminidase H from Flavobacterium meningosepticum; Hex, hexose; HexNAc, N-acetylhexosamine; IT, ion trap; KLH, keyhole limpet hemocyanin; MS/MS, tandem mass spectrometry; PA, 2-aminopyridine; PGC, porous graphitic carbon; PNGase A, peptide-N4-(N-acetyl-b-glucosaminyl)asparagine amidase A from almond; PNGase F, peptide-N4-(N-acetyl-b-glucosaminyl)asparagine amidase F from Flavobacterium meningosepticum; RP, reversed phase; TPCK, tosylL-phenylalanine-chloromethylketon Enzymes: b-N-acetyl-D-hexosaminidase (EC 3.2.1.52); a-L-fucosidase (EC 3.2.1.51); endo-b-N-acetylglucosaminidase H (EC 3.2.1.96); a-D-galactosidase (EC 3.2.1.22); b-D-galactosidase (EC 3.2.1.23); a-D-mannosidase (EC 3.2.1.24); peptide-N4-(N-acetyl-b-glucosaminyl)asparagine amidase A (EC 3.5.1.52); peptide-N4-(N-acetyl-b-glucosaminyl)asparagine amidase F (EC 3.5.1.52); trypsin (EC 3.4.21.4) Note: a website is available at http://www.uniklinikum-giessen.de/bio (Received 25 July 2002, accepted 10 September 2002) Keywords: keyhole limpet hemocyanin; carbohydrate structure analysis; mass spectrometry; N-glycans Ó FEBS 2002 5460 T Kurokawa et al (Eur J Biochem 269) carbohydrate structure of this glycoprotein is rather limited, although it is widely acknowledged that oligosaccharide constituents are likely to be of prime significance for the antigenicity and biomedical functions of KLH The carbohydrate content of total KLH has been calculated to amount approximately 4% by mass [28] Both isoforms, KLH1 and KLH2, were found to contain mannose, galactose, Nacetylglucosamine, N-acetylgalactosamine and fucose in differing amounts [29; Wuhrer, unpublished results] Furthermore, lectin binding studies provided evidence for the presence of N-linked or N-linked plus O-linked glycans in KLH1 or KLH2, respectively [29] In contrast to hemocyanins from other mollusc species such as Helix pomatia [30,31] or Lymnaea stagnalis [32,33], KLH has been reported to contain neither xylose nor 3-O-methylhexose moieties [28] Structural analyses, however, have not yet been performed We have therefore initiated a detailed investigation of KLH carbohydrates The isolation and characterization of the N-linked glycans, performed in this study, revealed in part novel structural motifs which might contribute to the pronounced immunogenicity of this gastropod glycoprotein EXPERIMENTAL PROCEDURES Materials KLH (VacmuneÒ) was provided by Biosyn Company, Fellbach, Germany The glycoprotein sample had been purified to homogeneity from M crenulata hemolymph by anion-exchange chromatography and contained both KLH isoforms in their native oligomeric states in a proportion of approximately : (KLH1/KLH2); the purity of this material was controlled by nondenaturing gel electrophoresis [2,34] Purified KLH can be stored at °C for at least year without detectable proteolytic degradation Isomaltosyl oligosaccharides with 2–30 glucose units were prepared by partial hydrolysis (0.1 M HCl, 80 °C, h) of dextran (Serva, Heidelberg, Germany) and desalted by passage through a column containing mixed-bed ion-exchange resin (Amberlite AG MB-3; Serva) prior to pyridylamination a-Mannosidase from jack beans, a-galactosidase from green coffee beans, endo-b-N-acetylglucosaminidase H from Streptomyces plicatus (endoH), and peptide-N4-(N-acetylb-glucosaminyl)asparagine amidase F from Flavobacterium meningosepticum (PNGase F) were obtained from Roche Diagnostics (Mannheim, Germany) b-N-Acetylhexosaminidase from jack beans and a-fucosidase from bovine kidney were purchased from Sigma (Deisenhofen, Germany) Peptide-N4-(N-acetyl-b-glucosaminyl)asparagine amidase A from almond (PNGase A) was from Seikagaku (Tokyo, Japan) and b-galactosidase from jack beans was obtained from Glyco (Upper Herford, UK) Tryptic digestion of KLH Thirty milligrams of KLH were reduced with mmol of dithiothreitol (Sigma) in mL of 38 mM Tris/HCl buffer, pH 8.8, containing M guanidinium chloride (Sigma) and 0.38 mM EDTA for 3.5 h at 37 °C in the dark Iodoacetamide (2.2 mmol; Sigma) dissolved in mL of 50 mM Tris/ HCl buffer, pH 8.8, containing M guanidinium chloride and 0.5 mM EDTA was added to the reaction mixture and incubated at 37 °C for h in the dark After addition of mmol dithiothreitol and a further 15 min-incubation, excess reagents was removed by gel-filtration using a TSK-gel Toyopearl HW-40F column (2.6 · 14 cm, TosoHaas, Stuttgart, Germany) with 25 mM NH4HCO3 buffer, pH 8.5, containing M urea (Sigma) as running solvent The carboxymethylated KLH fraction was diluted twice with 25 mM NH4HCO3 buffer, pH 8.5, and digested with mg of tosyl-L-phenylalanine-chloromethylketon (TPCK) treated trypsin (Sigma) for 18 h at 37 °C The tryptic digest was desalted on a reversed-phase cartridge (C18ec; Macherey and Nagel, Duren, Germany) and the (glyco)peptides, ă eluted with 0.1% formic acid in 30% and 84% (v/v) aqueous acetonitrile, were lyophilized Isolation of oligosaccharides Oligosaccharides were released from the tryptic glycopeptides by sequential treatment with 4.5 nkat endoH, 0.8 nkat PNGase F and 0.08 nkat PNGase A overnight at 37 °C as outlined elsewhere [35,36] Incubation with PNGase F was repeated once After each treatment, the enzymatic digests were applied on a reversed-phase cartridge, and the released oligosaccharides, recovered in the flow through, were collected The bound glycopeptides were stepwise eluted with 0.1% formic acid in 30% and 84% (v/v) aqueous acetonitrile, lyophilized and subjected to the next enzymatic digestion Finally, residual glycopeptides were subjected to automated hydrazinolysis using the Glyco Prep 1000 from Oxford Biosystems (Abingdon, UK) in the so-called N + O mode resulting in the liberation of both N- and O-linked glycans In parallel, a total glycan fraction was prepared from intact KLH by hydrazinolysis using similar conditions Pyridylamination of oligosaccharides Chemically and enzymatically released oligosaccharides were pyridylaminated according to Kuraya et al [37] Excess 2-aminopyridine and reaction byproducts were removed by gel filtration using a TSK-gel Toyopearl HW-40F column (1.6 · 80 cm) at a flow rate of 15 mLỈh)1 with 10 mM ammonium acetate buffer, pH 6.0, as running solvent Pyridylaminated (PA)-oligosaccharides were monitored by fluorescence with an excitation wavelength of 320 nm and an emission wavelength of 400 nm MALDI-TOF-MS MALDI-TOF-MS data were obtained using a Vision 2000 apparatus (Finnigan MAT, Bremen, Germany), operating in the positive-ion reflectron mode Ions were formed by a pulsed ultraviolet nitrogen laser beam (k ¼ 337 nm) The matrix, 6-aza-2-thiothymine (5 mgỈmL)1; Sigma) and the PA-oligosaccharides (1–20 pmol) were mixed on the stainless steel target and dried in a cold air stream Mass spectra were obtained by averaging 5–30 single spectra External mass calibration was performed with the [M + Na]+ ions of PA-isomaltosyl oligosaccharides Average masses are given throughout Nano-liquid chromatography ESI-ion trap (IT)-MS The PA-oligosaccharides were separated on a porous graphitic carbon (PGC) column (7 lm, 75 lm · 100 mm; Ó FEBS 2002 ThermoHypersil, Kleinostheim, Germany) using an Ultimate nano-LC system from LC-Packings (Amsterdam, the Netherlands) and a Famos autosampler (LC-Packings) The system was directly coupled with an Esquire 3000 ESI-ITMS (Bruker-Daltonik, Bremen, Germany) equipped with an on-line nanospray source operating in the positive-ion mode For electrospray (1200–2500 V), capillaries (360 lm OD, 20 lm ID with 10 lm opening) from New Objective (Cambridge, MA, USA) were used The solvent was evaporated at 150 °C with a nitrogen stream of LỈmin)1 Ions from m/z 50 to m/z 2000 were registered The column was equilibrated with eluent A (H2O/acetonitrile 95 : 5, v/v, containing 0.1% formic acid) at a flow rate of 200 nLỈmin)1 at room temperature After injecting the sample, elution was performed with 100% eluent A for min, and a linear gradient to 25% eluent B (H2O/acetonitrile 20 : 80, v/v, containing 0.1% formic acid) in 28 followed by a final wash with 95% solvent B for The eluate was monitored by absorption at 236 nm Off-line ESI-IT-MS/MS Off-line ESI-IT-MS/MS experiments were performed employing an off-line nanospray source together with the same instrument as above A 2–5 lL aliquot of a solution of native PA-oligosaccharides (in distilled water or in methanol/0.1% aqueous formic acid : 1) or permethylated PA-glycans (in methanol) was loaded into a laboratory-made, gold-coated glass capillary and electrosprayed at a voltage of 700–1000 V The solvent was evaporated at 120 or 80 °C for native or permethylated PA-oligosaccharides, respectively, with a nitrogen stream of LỈmin)1 For each spectrum, 20–40 repetitive scans were averaged The skimmer voltage was set to 30 V, accumulation time amounted to 50 ms All MS/MS experiments were performed in the positive-ion mode using helium as collision gas N-Glycans of KLH (Eur J Biochem 269) 5461 28 and a final wash with 95% solvent B for The eluate was monitored by fluorescence with an excitation wavelength of 320 nm and an emission wavelength of 400 nm The resulting PA-glycan fractions were further separated by HPLC using an amino phase column (Nucleosil Carbohydrate, 4.0 mm · 250 mm; Macherey and Nagel) equilibrated with eluent A (200 mM acetic acid/ triethylamine, pH 7.3/acetonitrile 25 : 75, v/v) at a flow rate of 1.0 mLỈmin)1 [38] After injecting the sample, elution was performed with 100% eluent A for min, a linear gradient to 70% eluent B (200 mM acetic acid/triethylamine, pH 7.3/ acetonitrile 60 : 40, v/v) in 35 and a final wash with 100% solvent B for The elution was monitored by fluorescence with an excitation wavelength of 310 nm and an emission wavelength of 380 nm Reversed phase (RP)-HPLC PA-oligosaccharides H2-1, H2-2 and H2-3 were analyzed or subfractionated by HPLC using a Cosmosil 5C18-P column (5 lm, 0.46 · 15 cm; Phenomenex, Aschaffenburg, Germany) at pH 6.0 according to the method of Ohashi et al [39] Fraction F3-1 was analyzed using the same column at pH 4.0 as desribed by Hase and Ikenaka [40] PA-oligosaccharides were detected by fluorescence using an excitation wavelength of 320 nm and an emission wavelength of 400 nm Carbohydrate constituent analysis Samples were hydrolyzed in 100 lL of M aqueous trifluoroacetic acid (Merck, Darmstadt, Germany) at 100 °C for h, and dried under a stream of nitrogen Monosaccharides were converted into their anthranilic acid derivatives by reductive amination [41], resolved by RP-HPLC and detected by fluorescence as detailed elsewhere [42] Anion-exchange HPLC Methylation analysis The PA-oligosaccharides were separated by HPLC using a MonoQ HR 5/5 column (5 mm · 50 mm; Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany) according to the method of Hase [38] In brief, the column was equilibrated with aqueous ammonia, pH 9.0, at a flow rate of 1.0 mLỈmin)1 The elution was performed using a linear gradient from to 12% 0.5 M ammonium acetate, pH 9.0, during the first min, followed by a further increase up to 40% in the next 28 min, and to 100% in the last The eluate was monitored by fluorescence with an excitation wavelength of 310 nm and an emission wavelength of 380 nm PA-oligosaccharides were permethylated and hydrolyzed Partially methylated alditol acetates obtained after sodium borohydride reduction and peracetylation were analyzed by capillary GLC/MS using the instrumentation and microtechniques described elsewhere [43,44] Preparative separation of PA-oligosaccharides The PA-oligosaccharides were separated by preparative HPLC using a PGC column (Hypercarb, lm, 4.6 mm · 100 mm; ThermoHypersil) equilibrated with eluent A (H2O/acetonitrile 95 : 5, v/v, containing 0.1% formic acid) at a flow rate of 0.8 mLỈmin)1 After injecting the sample, elution was performed with 100% eluent A for min, followed by a linear gradient to 25% eluent B (H2O/ acetonitrile 20 : 80, v/v, containing 0.1% formic acid) in Digestion with exoglycosidases PA-oligosaccharides were degraded with a-mannosidase from jack beans (0.85 nkat), a-galactosidase from green coffee beans (0.85 nkat), b-galactosidase from jack beans (0.17 nkat), a-fucosidase from bovine kidney (0.07 nkat) and b-N-acetylhexosaminidase from jack beans (1.1 nkat) on a stainless steel MALDI-TOF-MS target as described elsewhere [45] All enzymes were dialyzed before use against 25 mM ammonium acetate buffer adjusted to the suggested pH for each enzyme (i.e pH 6.0 for a-galactosidase, b-N-acetylhexosaminidase and a-fucosidase (bovine kidney), pH 5.0 for a-mannosidase and pH 4.0 for the b-galactosidase) Aliquots (1–3 lL) of aqueous solutions of PA-oligosaccharides (1–20 pmol) and 0.8 lL of matrix solution were mixed on the target and dried in a gentle stream of cold air After determination of the molecular Ó FEBS 2002 5462 T Kurokawa et al (Eur J Biochem 269) mass by MALDI-TOF-MS, the sample spot was reconstituted with 2–3 lL of enzyme solution The target was incubated at 37 °C over night in a screw-capped jar containing the respective 25 mM ammonium acetate buffer for preventing solvent evaporation Subsequently, the spots were dried in a cold stream of air and the MALDI-TOF mass spectra were recorded Further sequential enzymatic digestions were performed in the same way RESULTS Carbohydrate constituent analysis of KLH Carbohydrate constituent analysis revealed that the KLH preparation investigated in this study contained about 3.3% (by weight) neutral carbohydrates N-Acetylglucosamine, N-acetylgalactosamine, galactose, mannose, and fucose were found in molar ratios of about 2.0 : 0.6 : 1.6 : 2.0 : 1.1 In agreement with literature data [28], methylation analysis of KLH-derived glycopeptides employing perdeuterated methyl iodide excluded the presence of methylated monosaccharide constituents Analysis of total KLH glycans In order to take an overview of the complexity of KLH glycosylation, intact glycoprotein was subjected to automated hydrazinolysis Resulting glycans were pyridylaminated and analyzed by on-line ESI-MS Both monitoring of absorbance at 236 nm (data not shown) as well as corresponding mass spectra revealed the presence of multiple, incompletely resolved oligosaccharides About 30 signals were assigned to molecular compositions of Hex0)7HexNAc2)3dHex0)3PA (Fig 1) As each of these molecular ion species may comprise a mixture of different isomeric or isobaric carbohydrate structures (see below), this result clearly demonstrated the vast heterogeneity of KLH glycosylation Signals reflecting the presence of complete, e.g diantennary, complex-type glycans with four (or more) HexNAc residues have not been registered Preparation of PA-oligosaccharide pools In order to facilitate oligosaccharide fractionation and subsequent structural analyses, glycans were sequentially released by enzyme treatment Following reduction and carboxymethylation, the glycoprotein was first digested with trypsin The total pool of tryptic glycopeptides obtained revealed a similar carbohydrate composition as intact KLH (i.e GlcNAc/GalNAc/Gal/Man/Fuc ¼ 2.0 : 0.6 : 1.5 : 2.1 : 1.0) N-Glycans were liberated by treatment with endoH, PNGase F and PNGase A, and separated from residual glycopeptides by reversed-phase chromatography after each step About 10% (by weight) of the total carbohydrates present in KLH were found in the endoH fraction, 20% were released by PNGase F and 5% were recovered by PNGase A treatment Residual glycopeptides were finally subjected to automated hydrazinolysis in analytical scale The four oligosaccharide fractions were separately pyridylaminated and designated endoH-PA, PNGaseF-PA, PNGaseA-PA and Hyd(HFA)-PA, respectively Analytical anion-exchange HPLC of the pyridylaminated oligosaccharide pools demonstrated the absence of negatively charged oligosaccharide derivatives in all fractions (data not shown) ESI-MS of fractions PNGaseF-PA, PNGaseA-PA and Hyd(HFA)-PA (Fig 2B–D) revealed the presence of PA-oligosaccharides, the dominating species of which comprised similar molecular compositions as total KLHderived glycans (Fig 1A) In the case of endoH-PA species, the monosaccharide compositions deduced from the prevailing molecular masses were Hex4)7HexNAc1PA (Fig 2A) due to the cleavage of the chitobiose core In order to further characterize the various glycans present in these oligosaccharide fractions, total KLH tryptic glycopeptides as well as endoH-PA, PNGaseF-PA, PNGaseAPA and Hyd(HFA)-PA glycans werde subjected to linkage analysis The results revealed, in part, significant differences between the individual oligosaccharide fractions studied endoH-released species comprised terminal, 2-substituted and 3,6-disubstituted mannosyl residues as well as small amounts of terminal Gal and 4- or 3-substituted GlcNAc (data not shown), thus demonstrating the presence of high mannose and, to less extent, hybrid-type glycans (cf Hex5)6HexNAc2PA species in Fig 2A) which might contain type-1 (Galß3GlcNAc-) or type-2 (Galß4GlcNAc-) N-acetyllactosamine antennae Oligosaccharides liberated by PNGaseF disclosed, in addition, terminal fucose, 6-substituted, and 2,4- and 2,6-disubstituted mannosyl residues as major constituents together with small amounts of 3-substituted GalNAc and 3,4-disubstituted GlcNAc (Fig 3F) In striking contrast, glycans recovered in fractions PNGaseA-PA and Hyd(HFA)-PA comprised significant amounts of internal, monosubstituted fucose (Fig 3C,D) [13] which could be identified as 4-substituted Fuc by electron impact mass spectrometry of the respective partially methylated monosaccharide derivative (Fig 3I) In addition, linkage analyses revealed the presence of increased levels of 3-substituted GalNAc as well as 3,4- and 4,6disubstituted GlcNAc (Fig 3G,H) in agreement with the data obtained in the case of total KLH glycopeptides (cf Fig 3A,E and [14]) Terminal GlcNAc residues were found in trace amounts only Hence, the results demonstrate that despite their similarity in ESI-MS, glycans released by PNGase F and PNGase A included obviously differing carbohydrate structures The small amounts of material recovered in fractions PNGaseA-PA and Hyd(HFA)-PA, however, precluded an unambiguous characterization of the respective glycans Therefore, this report is focused exclusively on the structural elucidation of the major carbohydrate compounds released by endoH and PNGase F Fractionation of PA-oligosaccharides endoH-PA and PNGaseF-PA oligosaccharide pools were separately fractionated on a PGC-column (Fig 4A,B) Subsequent MALDI-TOF-MS analyses still demonstrated a heterogeneous composition of most glycan fractions Therefore, further fractionation on an amino-phase column was performed, resulting in a large number of PA-oligosaccharide subfractions (referred to as H2-1 for subfraction of fraction H2, etc.) Representative elution profiles are given in Fig 4C,D Homogeneity of each subfraction was checked by MALDI-TOF-MS In total, more than 30 different PA-oligosaccharide subfractions were obtained, 15 of which, representing about 60% of the Ó FEBS 2002 N-Glycans of KLH (Eur J Biochem 269) 5463 Fig Positive-ion nano-LC-ESI-IT-MS analysis of total KLH-derived PA-oligosaccharides released by hydrazinolysis PA-oligosaccharides were separated on a PGC-column and monitored by their absorbance at 236 nm Spectra from m/z 50–2000 were recorded and those corresponding to PA-oligosaccharide peaks were summarized (A) Entire spectrum; (B–D) enlarged mass range details Deduced monosaccharide compositions are assigned to the pseudomolecular [M + H]+ ions of the respective PA-derivatives H, hexose; N, N-acetylhexosamine; F, deoxyhexose (fucose) recovered total N-glycans, were subjected to structural analysis Structural analysis of individual PA-glycans The purified PA-oligosaccharides were investigated by MALDI-TOF-MS for their molecular masses, whereas their carbohydrate compositions were estimated by constituent analysis (Table 1) Monosaccharide linkage positions were determined by methylation analysis (Table 2) Due to reductive amination of the GlcNAc residue at the reducing end, this monosaccharide has been neither registered in carbohydrate constituent analyses nor in methylation studies Therefore, no information could be obtained in the case of this particular residue with regard to its substitution pattern Native or permethylated PA-oligosaccharides were further analyzed by off-line ESI-IT-MS/MS (cf., for example, Fig and Table 3) Monosaccharide sequencing and determination of the anomeric configurations of the corresponding glycosidic linkages were performed by degradation with exoglycosidases (Fig and Table 4) All glycans containing galactosyl residues were found to be sensitive towards digestion with b-galactosidase from jack beans but resistant to a-galactosidase treatment Some glycans were core-fucosylated at the innermost GlcNAc residue as shown by the detection of terminal fucose in methylation analysis and the presence of dHex1HexNAc1PA fragment ions at m/z 446 (native state) or m/z 544 (after permethylation) in the ESI-IT-MS/MS spectra (Table 3) All core-fucosylated PA-oligosaccharides released by PNGase F were sensitive towards a-fucosidase from bovine kidney (Table 4) corroborating that these oligosaccharides contained (a1–6)-linked fucosyl residues 5464 T Kurokawa et al (Eur J Biochem 269) Ó FEBS 2002 Fig Positive-ion nano-LC-ESI-IT-MS analysis of separate KLH-derived PA-oligosaccharide fractions Glycans were sequentially released by endoH, PNGase F, PNGase A and hydrazinolysis After pyridylamination, PA-oligosaccharides were separated on a PGC-column and monitored by their absorbance at 236 nm Spectra from m/z 50–2000 were recorded and those corresponding to PA-oligosaccharide peaks were summarized (A) endoH-PA; (B) PNGaseF-PA; (C) PNGaseA-PA; and (D) Hyd(HFA)-PA Deduced monosaccharide compositions are assigned as in Fig Characterization of endoH-sensitive N-glycans Compound H2-1, the major component of the endoH fraction (Fig 4C), showed pseudomolecular ions [M + Na]+ at m/z 1132.8 in MALDI-TOF-MS consistent with a composition of Hex5HexNAcPA Only mannosyl residues were registered by carbohydrate constituent analysis (Table 1) Methylation analysis demonstrated the presence of terminal and 3,6-disubstituted mannose (Table 2) ESIIT-MS/MS analysis (Table 3) of the permethylated compound revealed protonated fragment ions at m/z 1296 (Hex5HexNAc), 1186 (Hex4HexNAcPA), 968 (Hex3Hex NAcPA), 778 (Hex2HexNAcPA) and 560 (HexHex NAcPA) Treatment with a-mannosidase from jack beans released four mannosyl residues as confirmed by MALDITOF-MS, suggesting the high mannose-type structure shown below MALDI-TOF-MS analyses of fractions H2-2 and H2-3 (Fig 4C) revealed in both cases the presence of two components with pseudomolecular ions [M + Na]+ at m/z 1295.1/1336.3 and 1457.4/1498.5 consistent with compositions of Hex6HexNAcPA/Hex5HexNAc2PA and Hex7HexNAcPA/Hex6HexNAc2PA, respectively Each of these samples was therefore further subfractionated by RPHPLC at pH 6.0 [39] yielding subfractions H2-2-1, H2-2-2, H2-3-1 and H2-3-2 (not shown) For identification of their isomeric structures, high mannose-type compounds H2-2-1 and H2-3-1 were rechromatographed by RP-HPLC together with authentic oligosaccharide standards Although relative elution time values are not available in the literature for high mannose-type PA-glycans with one GlcNAc residue, it may be postulated from their co-elution with the standards used that these oligosaccharides represented the isomers depicted below The presence of terminal, 2-substituted and 3,6-disubstituted mannosyl residues could Ó FEBS 2002 N-Glycans of KLH (Eur J Biochem 269) 5465 Fig Detection of fucose and HexNAc species by methylation analysis of KLH PAoligosaccharide fractions Partially methylated alditol acetates were separated by gas chromatography and registered in the positive-ion mode after chemical (A–H) or electron impact (I) ionization (A–D) Detection of terminal and monosubstituted fucose in total KLH glycopeptides (A), as well as in fractions PNGaseF-PA (B), PNGaseA-PA (C), and Hyd(HFA)-PA (D); (E–H) monitoring of HexNAc-derivatives in KLH glycopeptides (E), as well as in fractions PNGaseF-PA (F), PNGaseA-PA (G), and Hyd(HFA)-PA (H); (I) electron impact mass spectrum of the 1,4,5tri-O-acetyl-2,3-di-O-methylfucitol derivative reflecting a 4-substituted fucose 1, terminal Fuc; 2, 4-substituted Fuc; 3, terminal GlcNAc; 4, 4-substituted GlcNAc; 5, 3-substituted GlcNAc; 6, 3-substituted GalNAc; 7, 3,4-disubstituted GlcNAc; 8, 4,6-disubstituted GlcNAc; *contaminant Fig HPLC fractionation of glycans by sequential use of a PGC column (A, B) and an amino-phase column (C, D) Elution conditions are as described in the Experimental procedures (A) endoH-PA; (B) PNGaseF-PA; and (C) and (D) subfractionation of fraction H2 and fraction F4 by amino-phase HPLC, respectively be additionally confirmed by linkage analysis (data not shown) Methylation analysis of subfraction H2-3-2 (cf Table 2) verified terminal, 2-substituted and 3,6-disubstituted Man as well as terminal Gal and 3-substituted GlcNAc in agreement with the results obtained in the case of total endoH-PA glycans On the basis of these data and the known structural requirements for endoH sensitivity, the indicated structure may be proposed Subfraction H2-22 could not be further analyzed due to small amounts Likewise, compounds with four hexosyl residues (Fig 2A), detected in fraction H1 (Fig 4A) by ESI-MS (Table 1), have not been characterized Ó FEBS 2002 5466 T Kurokawa et al (Eur J Biochem 269) Table Oligosaccharide components from KLH obtained after enzymatic release, pyridylamination and HPLC-fractionation The molecular masses were measured by MALDI-TOF-MS and the monosaccharide constituents were determined by carbohydrate analysis Molar ratios are based on the sum of monosaccharides determined by MALDI-TOF-MS minus the PA-substituted GlcNAc +, presence, –, absence; N.D., not done Molecular mass [M + Na]+ Carbohydrate constituents Fraction Experimental Theoretical Molecular composition H1 H2-1 H2-2-1 H2-2-2 H2-3-1 H2-3-2 F1-5 F2-1 F2-2 F3-1 F4-1 F4-2 F4-3 F4-4 F5-1 F5-2 947.9b 1132.8 1295.1 1336.3 1457.4 1498.5 1539.8 850.0 1012.2 1498.3 995.5 1011.6 1157.9 1174.2 1157.7 1319.8 948.4 1133.0 1295.2 1336.3 1457.3 1498.4 1539.4 849.8 1011.9 1498.4 995.9 1011.9 1158.1 1174.1 1158.1 1320.2 Hex4HexNAcPA Hex5HexNAcPA Hex6HexNAcPA Hex5GlcNAc2PA Hex7HexNAcPA Hex6GlcNAc2PA Hex5HexNAc3PA Hex2HexNAc2PA Hex3HexNAc2PA Hex6HexNAc2PA Hex2HexNAc2dHexPA Hex3HexNAc2PA Hex3HexNAc2dHexPA Hex4HexNAc2PA Hex3HexNAc2dHexPA Hex4HexNAc2dHexPA a Gal Man GlcNAca Fuc Relative amount (%) N.D – – + – + 1.9 – 1.1 – – – 0.9 0.9 – 0.8 N.D + + + + + 3.0 2.2 2.0 5.8 2.0 3.4 1.9 3.2 3.1 2.9 N.D – – + – + 2.1 0.8 0.9 1.2 0.8 0.6 0.9 0.9 0.7 0.9 N.D – – – – – – – – – 1.2 – 1.3 – 1.2 1.4 N.D 16.0 1.8 0.5 1.6 1.1 0.9 3.5 0.7 10.3 7.5 3.3 2.3 1.2 4.0 2.5 The innermost GlcNAc residue was not detected due to reductive amination b [M + H]+ registered by ESI-MS Table Methylation analysis of major PA-oligosaccharides derived from KLH PA-oligosaccharide fractions were permethylated and hydrolyzed The partially methylated alditol acetates obtained after reduction and peracetylation were analyzed by capillary GLC/MS The absence or presence of individual components in indicated by – or +, respectively (+) trace amounts PA-GlcNAc derivatives were not registered Presence in oligosaccharide fraction Alditol acetate H2-1 H2-3-2 F1-5 F1–5a F2-1 F2-2 F2–2a F3-1 F4-1 F4-2 F4-3 F4-4 F5-1 F5-2 F5–2b Linkage 2,3,4-FucOH 2,3,4,6-ManOH 3,4,6-ManOH 2,3,4-ManOH 2,4,6-ManOH 3,4-ManOH 2,4-ManOH 2,3,4,6-GalOH 3,4,6GlcN(Me)AcOH 3,6-GlcN(Me)AcOH 4,6-GlcN(Me)AcOH – + – – – – + – – – – a – + + – – – + + – – + After b-galactosidase treatment – – + + – + + + + + – b – + + – – – + (+) + + – – + – + – – – – – + – – + – – – – + + – + – – + – – + – – – – + – – + – + – – + – – + – + + – + – – – – – + – – + – – – – + – – + – + – – + – – – + – + – – + – + – – + + – + – + + – – – – + – – + – + + – + – – + + – + – + – – + – – – + – + – Fuc(1Man(1-2)Man(1-6)Man(1-3)Man(1-2,6)Man(1-3,6)Man(1Gal(1GlcNAc(1-4)GlcNAc(1-3)GlcNAc(1- After a-mannosidase treatment Characterization of glycans released by PNGase F PNGaseF-released KLH N-glycans can be divided into two groups due to the absence (F2-1, F3-1, F4-1, F4-2 and F5-1) or presence (F1-5, F2-2, F4-3, F4-4 and F5-2) of additional galactosyl residues As the latter species represent novel glycoprotein-N-glycan structures, they are separately discussed MALDI-TOF-MS of compound F2-1 revealed pseudomolecular ions [M + Na]+ at m/z 850.0 consistent with the composition Hex2HexNAc2PA, corresponding to the smallest N-glycan preparatively isolated from KLH in this study Carbohydrate constituent and methylation analyses demonstrated the presence of terminal mannose, 6-substituted mannose and 4-substituted GlcNAc (cf Tables and 2) ESI-IT-MS/MS analysis of the permethylated compound led to protonated fragment ions at m/z 668 (Hex2HexNAc) and 370 (HexNAcPA) (Table 3) Treatment with a-mannosidase resulted in the release of one mannosyl residue, thus indicating the truncated structure depicted below Likewise, compound F4-1 could be demonstrated to represent the fucosylated counterpart of F2-1 The protonated fragment ion at m/z 544 (dHexHexNAcPA), obtained by ESI-IT-MS/ MS analysis of the permethylated compound (Table 3), as well as its sensitivity towards treatment with a-fucosidase from bovine kidney (Table 4) demonstrated fucose to be a-glycosidically linked to the innermost GlcNAc residue The fact that this glycan was sensitive to PNGase F further allowed the conclusion that the fucosyl residue was located at C6 of the respective GlcNAc moiety [36] By the same line of reasoning based on equivalent analytical data, compounds F4-2 and F5-1 could be identified as nonfucosylated and (a1–6)-fucosylated representatives of the standard pentasaccharide core of glycoprotein-N-glycans Ó FEBS 2002 N-Glycans of KLH (Eur J Biochem 269) 5467 In contrast, MALDI-TOF-MS of compound F3-1 revealed pseudomolecular ions [M + Na]+ at m/z 1498.3 consistent with the composition Hex6HexNAc2PA (Table 1) Carbohydrate constituent analysis indicated the presence of Man and GlcNAc residues and methylation analysis provided evidence for the occurrence of terminal, 6-substituted and 3,6-disubstituted mannosyl residues in the ratio of 2.8 : 1.0 : 2.2 as well as 4-substituted GlcNAc (Table 2) ESI-IT-MS/MS (Table 3) revealed sodiated fragment ions at m/z 1335, 1173, 1011, 850, 687 and 525, corresponding to Hex5)0HexNAc2PA in addition to the protonated fragment ion at m/z 300 (HexNAcPA) Treatment with a-mannosidase released five mannosyl residues (Table 4) RP-HPLC analysis according to Hase and Ikenaka [40] disclosed a different relative elution time in the case of F3-1 glycans which did not match with the corresponding values of Man6GlcNAc2-PA isomers published so far, as all of these reference compounds contained an (a1–2)-linked instead of an (a1–6)-linked mannosyl residue The precise linkage position of the additional (a1–6)-bound mannose could not be further assigned Possibly due to the lack of 2-substituted and the presence of 6-substituted mannose, fraction F3-1 glycans represented high mannose-type oligosaccharide isomers which did not fulfill the structural criteria for endoHsensitivity [46] On the basis of the data obtained, the structure of F3-1 glycans may be proposed as follows Fig MALDI-TOF-MS spectra of compound F4-3 after sequential enzymatic digestions (A) Starting material; (B) after b-galactosidase (jack beans) digestion; (C) after treatment with a-mannosidase (jack beans); (D) after degradation with a-fucosidase (bovine kidney) Except for the [M + H]+ ion at m/z 665.4 in (D), signals represent [M + Na]+ ions Fig Nano-ESI-IT-MS/MS spectrum of the doubly charged pseudomolecular ion [M + H + Na]2+ at m/z 579.2 produced by compound F4-3 Possible fragmentation pathways are included in the structure The assignment of fragments is in agreement with the nomenclature introduced by Domon and Costello [57] The molecular compositions of the respective ions are given in Table Compounds F1-5, F2-2, F4-3, F4-4 and F5-2 were found to represent a novel type of N-glycans as they comprised, at least, one galactose b-glycosidically linked to a mannosyl residue MALDI-TOF-MS of the smallest representative of this class, F2-2, revealed pseudomolecular ions [M + Na]+ at m/z 1012.2 consistent with the composition Hex3HexNAc2PA Carbohydrate constituent and Ó FEBS 2002 5468 T Kurokawa et al (Eur J Biochem 269) Table Fragment ions from native and permethylated (*) pyridylamino-oligosaccharides obtained by positive-ion ESI-IT-MS/MS Mean values of the determined masses, rounded to the next integer, are given +, presence; –, absence; minor signals are given in parentheses Ions (m/z) Ions obtained from PA-oligosaccharide fraction Native Permethy Pseudolated molecular ion 204 300 322 347 366 446 370 544 560 525 615 550 668 672 778 687 713 872 834 968 993 850 874 1133 1186 996 1011 1036 1296 1077 1173 1198 1239 1335 1376 418 425 499 506 570 578 587 607 668 688 760 [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M [M + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + H]+ H]+ Na]+ Na]+ H]+ H]+ H]+ Na]+ H]+ Na]+ H]+ Na]+ H]+ Na]+ Na]+ H]+ Na]+ H]+ H]+ Na]+ Na]+ H]+ H]+ Na]+ Na]+ Na]+ H]+ Na]+ Na]+ Na]+ Na]+ Na]+ Na]+ H + Na]2+ H + Na]2+ H + Na]2+ H + Na]2+ H + Na]2+ H + Na]2+ H + Na]2+ H + Na]2+ H + Na]2+ H + Na]2+ H + Na]2+ H2-1* F1-5 F2-1* F2-2 F3-1 F4-1* F4-2* F4-3 F4-4 F5-1 F5-2 Composition HexNAc HexNAcPA HexNAcPA Hex2 HexHexNAc HexNAcdHexPA HexHexNAcPA HexNAc2PA HexNAc2PA Hex2HexNAc Hex2HexNAc Hex4 Hex2HexNAcPA HexHexNAc2PA Hex3HexNAc Hex3HexNAc HexHexNAc2dHexPA Hex3HexNAcPA HexHexNAc2dHexPA Hex2HexNAc2PA Hex4HexNAc Hex3HexNAc2 Hex4HexNAcPA Hex2HexNAc2dHexPA Hex3HexNAc2PA Hex5HexNAc Hex5HexNAc Hex4HexNAc2 Hex4HexNAc2PA Hex6HexNAc Hex5HexNAc2 Hex5HexNAc2PA Hex4HexNAc3PA HexHexNAc2dHexPA Hex2HexNAc2PA Hex2HexNAc2dHexPA Hex3HexNAc2PA Hex3HexNAc2dHexPA-H2O Hex4HexNAc2PA-H2O Hex4HexNAc2PA Hex3HexNAc3PA Hex5HexNAc2PA Hex4HexNAc3PA Hex5HexNAc3PA-H2O methylation analyses demonstrated the presence of terminal galactose, terminal mannose, 3,6-disubstituted mannose and 4-substituted GlcNAc Sequential treatments with bgalactosidase from jack beans and a-mannosidase released one hexosyl residue in each case Without prior treatment with b-galactosidase, however, the terminal mannosyl residue was insensitive towards a-mannosidase which might indicate that the a-mannosyl residue is linked to C3 of the branching mannose [47] This assumption could be confirmed by methylation analysis of the b-galactosidasetreated compound, demonstrating the presence of terminal – – – – – – + – – – – – + – – – – + – – – – + – – – + – – – – – – – – – – – – – – – – – – + – – + – – (+) – (+) – – – – – – – – – (+) – – – – + – – + + – + + + – – – – – + – + + + + – + – – – – – – – – + – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – + + – – – – (+) – – – – – (+) + – – – – + – – – – – – – – – – – – – (+) + – (+) – – – – – – – – + – – – – – + – – – – – + – – – – – + – – – – + + – – + + – + – – – – + – – + – + – – – – – – – + – – – – + – – – – – – – + – – – – – – – – – – – – – – – – – – – – – – – – – – + – – – – – – + – – – – – – + – – – – – + – – – – – – – – – – – – – – – – – – – – – – + + – – – (+) – – – – – – – – + – (+) – – (+) – – – + + – – – – – – – – + + + + + – – – – – – – + + + – – – + – + – – – + + – – – – + + – – – + – – – – – – – – – + – + – – – – – – – + + (+) – – + – – – – – – – – + – – – – + – – – + + – – – – – – – – (+) + + + + – – – – – – – + + – (+) (+) – – – – – + – + + – (+) – – + + – – – + – – – + – – – – + – (+) + – + + – – – – mannose, 3-substituted mannose and 4-substituted GlcNAc (Table 2) MALDI-TOF-MS of compound F4-3 led to pseudomolecular ions [M + Na]+ at m/z 1157.9 consistent with the composition Hex3HexNAc2dHexPA (Table 1) Carbohydrate constituent and methylation analyses revealed the presence of terminal fucose, terminal galactose, 6-substituted mannose and 4-substituted GlcNAc (Table 2) The Y4a and Y3a ions at m/z 996 and 834 obtained by ESI-ITMS/MS analysis are in agreement with a linear arrangement of hexoses whereas the Y1a and B4a ions at m/z 446 and 713 Ó FEBS 2002 N-Glycans of KLH (Eur J Biochem 269) 5469 Fig Nano-ESI-IT-MS/MS spectrum of the doubly charged pseudomolecular ion [M + H + Na]2+ at m/z 770.2 produced by compound F1-5 Possible fragmentation pathways are delineated together with the structures of the isomers I and II In isomer II only fragments differing from isomer I are marked and given in parentheses As in Fig 5, the nomenclature of Domon and Costello [57] is used Identical fragments may also originate from other fragmentation pathways which are not indicated for the sake of clarity For the type of ions, see Table Table On-target cleavage of PA-oligosaccharides with exoglycosidases Oligosaccharides samples were analyzed in the positive-ion reflectron mode and sequentially or independently digested directly on the MALDI target with the enzymes indicated Given mass values indicate the average masses of the pseudomolecular ions [M + Na]+ Products of incomplete enzymatic cleavage are given in parentheses Sequential enzymatic digestion Independent enzymatic digestion PA-oligosaccharide Composition [M + Na]+ b-Gala a-Manb a-Fucc a-Manb a-Fucc b-GlcNAcd H2-1 F1-5 F2-1 F2-2 F3-1 F4-1 F4-2 F4-3 F4-4 F5-1 F5-2 Man5GlcNAcPA Gal2Man3GlcNAc3PA Man2GlcNAc2PA Gal1Man2GlcNAc2PA Man6GlcNAc2PA Man2GlcNAc2FucPA Man3GlcNAc2PA Gal1Man2GlcNAc2FucPA Gal1Man3GlcNAc2PA Man3GlcNAc2FucPA Gal1Man3GlcNAc2FucPA 1133.4 1539.7 850.2 1012.2 1498.3 996.3 1012.1 1158.3 1174.0 1158.4 1320.5 – 1376.9f – 850.2 – – – 995.9 1012.2 – 1158.0 – 1215.3 – 687.3 – 833.9 – 834.0 687.8 833.5 833.5 – – – – – 687.8 – 687.6 – 687.4 687.8 461.9e 1539.0 687.5 1011.8 687.4 833.9 687.5 1157.9 1012.3 833.6 1158.3 – – – – – 850.2 – 1012.2 – 1011.6 1174.0 – 1337.5 (1539.6) – – – – – – – – – Exoglycosidases: a b-galactosidase (jack beans), b a-mannosidase (jack beans), c a-fucosidase (bovine kidney), d b-N-acetylhexosaminidase (jack beans) e [M + H]+ ion f The second galactose could be only liberated by extensive enzymic degradation in a tube demonstrated the presence of dHexHexNAcPA, suggesting that the fucosyl residue is linked to the innermost GlcNAc (cf Figure and Table 3) These results could be corroborated by sequential exoglycosidase treatment (Fig and Table 4) yielding the release of one galactosyl, one mannosyl and one fucosyl residue Hence, it can be concluded that compound F4-3 represented the galactosylated variant of compound F4-1 carrying Gal in (b1–6)-linkage at its outermost mannosyl residue By the same line of evidence, it could be demonstrated that compounds F4-4 and F5-2 represented the Gal(b1–6)-substituted derivatives of F4-2 and F5-1, respectively In the case of F5-2, the linkage position of Gal could be assigned to the (a1–6)-linked mannosyl residue by methylation analysis after preparative treatment with a-mannosidase (see Table 2) Compound F1-5 represented the most complex structure of hemocyanin N-glycans recovered so far MALDI-TOFMS revealed pseudomolecular ions [M + Na]+ at m/z 1539.8 consistent with the composition Hex5HexNAc3PA Carbohydrate constituent analysis further indicated the presence of Gal, Man and GlcNAc residues in a ratio of 1.8 : 2.9 : 2.0 (Table 1) Methylation analysis demonstrated the presence of terminal galactose, 2- and 6-substituted mannose, 2,6- and 3,6-disubstituted mannose as well as terminal and 4-substituted GlcNAc In agreement with methylation data, treatment with a-mannosidase did not lead to a shift in molecular mass, whereas digestion with b-galactosidase or N-acetylhexosaminidase from jack beans resulted in the release of one (40%) and two (60%) galactosyl residues or the partial liberation (about 30%) of GlcNAc, respectively Sequential treatment with b-galactosidase and a-mannosidase further confirmed the subterminal position of one mannosyl residue (Table 4) Consequently, methylation analysis after exhaustive b-galactosidase digestion led to the almost complete disappearance of 6-substituted and 2,6-disubstituted mannosyl residues in conjunction with the considerable removal of terminal Gal and the simultaneous expression of terminal 5470 T Kurokawa et al (Eur J Biochem 269) Man ESI-IT-MS/MS analysis revealed Y4 ions at m/z 1335.0 and 1375.8 indicative for the presence of terminal HexNAc and Hex residues (cf Fig and Table 3) Furthermore, B2a and Y3a fragment ions at m/z 550.6 and 1011.0 indicated the presence of a Hex2HexNAc unit, whereas no evidence has been obtained for a Hex3fragment Instead, the doubly charged Y3b fragment ion at m/z 607.5 indicated a Hex2-antenna Likewise (B2a) and doubly charged [(Y4a)-H2O]2+ fragment ions at m/z 366.0 and 577.9 support the presence of a second structural isomer comprising a N-acetyllactosamine unit (Fig 7) in agreement with the simultaneous presence of 2-substituted and 2,6disubstituted Man shown by methylation analysis Considering the linkage position of b-Gal residues found in the other compounds and the common route of biosynthesis of N-linked glycans, it may be postulated that compound F1-5 represents a mixture of two isomers containing either a Gal(b1–4)GlcNAc(b1–2)Man(a1–3) or a GlcNAc(b1–2)[Gal(b1–6)]Man(a1–3) unit in addition to a Gal(b1–6)Man(a1–6) antenna On the basis of the results obtained, the following structures may be proposed DISCUSSION The main purpose of this study was to give a first, detailed account of carbohydrate structures of KLH To this end, whole hemocyanin comprising both isoforms, KLH1 and KLH2, was employed The carbohydrate content and monosaccharide composition deviated to some extent from literature data [28] This might be due to differences either in the overall carbohydrate structures of the glycoprotein preparations used or in the relative abundance of KLH1 and KLH2 isoforms present in the starting material The latter aspect is important in so far as it has been demonstrated that KLH1 and KLH2 differ in their monosaccharide pattern [29; Wuhrer, unpublished results] Hence, variations in the ratio of KLH isoforms, which are observed, for example, during prolonged captivity of the Ó FEBS 2002 animals [2], may clearly influence the molar proportions of the monosaccharide constituents of this glycoprotein Furthermore, the ratio of KLH1 and KLH2 varies considerably in the pelleted hemocyanins from individual limpets, thus demonstrating that the origin of the starting material may play an important role N- and possible O-linked oligosaccharides were released from tryptic KLH glycopeptides by sequential treatment with endoH, PNGase F and PNGase A, as well as by hydrazinolysis, converted into their PA-derivatives and analyzed by on-line nano-liquid chromatography-ESI-MS using a nano-PGC-column This technique turned out to be a versatile tool for simultaneous desalting, fractionation and analysis of picomolar amounts of carbohydrates For preparative purposes, PA-glycans, recovered after endoH and PNGase F treatment, were separated by two-dimensional HPLC employing a PGC column in combination with an amino phase column More than 30 different PA-oligosaccharide subfractions were obtained 15 of which, representing about 60% of the totally released N-glycans, were analyzed by MALDI-TOF-MS, carbohydrate con- stituent and methylation analyses, ESI-IT-MS/MS and exoglycosidase digestions The results revealed that KLH carries high mannose-type sugar chains as well as truncated glycans derived thereof carrying, in part, fucose at the innermost GlcNAc As the latter monosaccharide unit had been modified by reductive amination, the linkage positions of respective fucosyl residues could not be assigned by methylation analysis Due to the sensitivity of the respective glycans towards PNGase F and a-fucosidase from bovine kidney, however, fucose residues could be proposed as being (a1–6)-linked [48] As native KLH is characterized by a multimeric, rigid structure [21,22], high concentrations of guanidinium chloride and urea had to be employed during carboxymethylation and tryptic digestion, respectively, in order to achieve sufficient solubility of starting material and reaction Ó FEBS 2002 products as well as fragmentation of the glycoprotein Although the resulting glycopeptides have not been analyzed in detail, proteolytic cleavage appeared to be incomplete Small precipitates occasionally observed, however, did not contain carbohydrates Hence, the observation that the pool of glycans finally released by PNGase A and hydrazinolysis (Fig 2C,D) still comprised, in part, oligosaccharides without Fuc might be the result of an incomplete action of PNGase F due to sterical hindrance and/or diverging oligosaccharide structures The latter assumption is corroborated by the observation that carbohydrate fractions, released after PNGase F-treatment by PNGase A and, finally, by hydrazinolysis, comprised, at least in part, different monosaccharide constituents, such as 4-substituted fucose, 3-substituted GalNAc and 3-substituted or 4,6-disubstituted GlcNAc residues In agreement with this finding, constituent analyses of these oligosaccharide fractions confirmed different monosaccharide compositions (data not shown) Furthermore, glycans obtained by hydrazinolysis might include O-linked oligosaccharides in addition to N-glycans As a striking feature, part of the studied glycans carried Gal(b1–6)-moieties bound to either b- or a-glycosidically linked mannosyl residues So far, terminal and internal Gal(b1–6)Man units have been found, for example, in some O-specific side chains of Salmonella lipopolysaccharides [49], capsular polysaccharides of Klebsiella pneumoniae [50] and glycoglycerolipids from archaebacteria [51,52] In the context of glycoprotein-N-glycans, however, such Gal(b1–6)Man-motifs represent novel structural elements The question as to whether these glycans are able to induce antibodies recognizing this particular carbohydrate epitope awaits further study Nevertheless, this aspect has now to be considered when KLH is used as an immunogen or carrier of low molecular mass, carbohydrate-based haptens Glycosyltransferases that are possibly involved in the biosynthesis of hemocyanin glycans in Gastropoda have been studied extensively in the past [28] It could be demonstrated that the connective tissue of snails contains enzymes which differ, at least in part, in their substrate specificities from corresponding glycosyltransferases characterized so far from other sources From the carbohydrate structures described above, it may be concluded that M crenulata expresses a novel type of b-1,6-galactosyltransferases conveying Gal to mannosyl residues of glycoprotein-N-glycan cores The precise substrate specificity of these enzymes remains to be investigated Gal(b1–6)Man-motifs have neither been detected in the hemocyanin-linked glycans of the pulmonate gastropods Helix pomatia and Lymnaea stagnalis nor in those of the spiny lobster Panulirus interruptus [30–33,53,54] N-linked carbohydrate chains from H pomatia and L stagnalis represent mostly core xylosylated monoantennary and diantennary complex-type glycans with extended, often multiply branched antennae and 3-O-methylated monosaccharide constituents In contrast, the N-glycans from M crenulata characterized so far are mainly high mannose-type or truncated complex-type sugar chains The question as to whether glycans with Gal(b1–6)Man-motifs are limited to KLH or further distributed amongst marine gastropods cannot be answered yet The well-studied hemocyanin from Haliotis tuberculata would provide an excellent model system in this context [22] N-Glycans of KLH (Eur J Biochem 269) 5471 A major goal which can now be addressed is to unravel biological functions of, as well as immunological responses to, the different carbohydrate moieties attached to the giant hemocyanin molecule As a first step, respective glycans have to be individually localized within the polypeptide chains of KLH1 and KLH2 As in H tuberculata hemocyanin [26], most KLH functional units carry one or two potential attachment sites for N-linked glycans (unpublished data) The assignment of distinct oligosaccharide chains to individual glycosylation sites could reveal as to how the two KLH isoforms are differently glycosylated Preliminary data indicate that this is the case [29] Isoform-specific glycosylation, however, could provide signal structures implied in differential regulation processes, as, for example, the selective disappearance of KLH1 from the hemolymph observed under certain environmental conditions [55] KLH has been reported to display considerable immunological cross-reactivity of serodiagnostic value with S mansoni egg antigens due to the presence of common periodate-sensitive epitopes [56] The glycanic nature of the corresponding determinants could be corroborated by the finding that fucose plays an important role in antibody binding [13] In a recent study, evidence could be provided that terminal Fuc(a1–3)GalNAc-epitopes might be prime candidates for the observed cross-reactivity between KLH and S mansoni egg glycosphingolipids [14] Although total KLH glycopeptides have been found to contain significant amounts of 3-substituted GalNAc by monosaccharide linkage analysis (cf Fig 3), none of the major, PNGase F-released sugar chains described in this study comprised this carbohydrate moiety Instead, respective components have been detected in small amounts in glycan pools liberated by PNGase A and hydrazinolysis Possibly, the relevant cross-reacting epitopes are only expressed in minor, PNGase F-resistant N-glycan species and/or in O-linked sugar chains which have been reported to occur, for instance, in the functional unit KLH2-c [7,29] Hence, further studies are required to structurally define the carbohydrate moieties of KLH which are responsible for the serological crossreactivity with schistosomal glycoconjugates ACKNOWLEDGMENTS We thank P Kaese, W Mink, S Kuhnhardt and S Eisenmann ă (Boehringer Ingelheim) for constituent analysis, methylation, GLC/MS and hydrazinolysis as well as R.D Dennis for fruitful discussions We are grateful to the Biosyn Company, Fellbach, Germany for providing the purified KLH used in this study and to W Gebauer (University of Mainz) for purity control The project was supported by the Deutsche Forschungsgemeinschaft (Ge 386/3-1 and Sonderforschungsbereich 535) REFERENCES van Holde, K.E & Miller, K.I (1995) Hemocyanins Adv Protein Chem 47, 1–81 Harris, J.R & Markl, J (1999) Keyhole limpet hemocyanin (KLH): a biomedical review Micron 30, 597–623 Reid, W., Sadowska, M., Denaro, F., Rao, S., Foulke, J Jr, Hayes, N., Jones, O., Doodnauth, D., Davis, H., Sill, A., O’Driscoll, P., Huso, D., Fouts, T., Lewis, G., Hill, M., KaminLewis, R., Wei, C., Ray, P., Gallo, R.C., Reitz, M & Bryant, J (2001) An HIV-1 transgenic rat that develops HIV-related pathology and immunologic dysfunction Proc Natl Acad Sci USA 98, 9271–9276 5472 T Kurokawa et al (Eur J Biochem 269) Tani, K., Murphy, W.J., Chertov, O., Oppenheim, J.J & Wang, J.M (2001) The neutrophil granule protein cathepsin G activates murine T lymphocytes and upregulates antigen-specific IG production in mice Biochem Biophys Res Commun 282, 971–976 Jurincic-Winkler, C.D., Metz, K.A., Beuth, J & Klippel, K.F (2000) Keyhole limpet hemocyanin for carcinoma in situ of the bladder: a long-term follow-up study Eur Urol 37 (Suppl 3), 45–49 Lamm, D.L., Dehaven, J.I & Riggs, D.R (2000) Keyhole limpet hemocyanin immunotherapy of bladder cancer: laboratory and clinical studies Eur Urol 37 (Suppl 3), 41–44 Wirguin, I., Suturkova-Milosevic, L., Briani, C & Latov, N (1995) Keyhole limpet hemocyanin contains Gal(b1–3)-GalNAc determinants that are cross-reactive with the T antigen Cancer Immunol Immunother 40, 307–310 Danishefsky, S.J & Allen, J.R (2000) From the laboratory to the clinic: a retrospective on fully synthetic carbohydrate-based anticancer vaccines Angew Chem Int Ed Engl 39, 836–863 Wang, Z.G., Williams, L.J., Zhang, X.F., Zatorski, A., Kudryashov, V., Ragupathi, G., Spassova, M., Bornmann, W., Slovin, S.F., Scher, H.I., Livingston, P.O., Lloyd, K.O & Danishefsky, S.J (2000) Polyclonal antibodies from patients immunized with a globo H-keyhole limpet hemocyanin vaccine: isolation, quantification, and characterization of immune responses by using totally synthetic immobilized tumor antigens Proc Natl Acad Sci USA 97, 2719–2724 10 Chapman, P.B., Morrisey, D., Panageas, K.S., Williams, L., Lewis, J.J., Israel, R.J., Hamilton, W.B & Livingston, P.O (2000) Vaccination with a bivalent G(M2) and G(D2) ganglioside conjugate vaccine: a trial comparing doses of G(D2)-keyhole limpet hemocyanin Clin Cancer Res 6, 4658–4662 11 Gilewski, T., Adluri, S., Ragupathi, G., Zhang, S., Yao, T.J., Panageas, K., Moynahan, M., Houghton, A., Norton, L & Livingston, P.O (2000) Vaccination of high-risk breast cancer patients with mucin-1 (MUC1) keyhole limpet hemocyanin conjugate plus QS-21 Clin Cancer Res 6, 1693–1701 12 Dissous, C., Grzych, J.M & Capron, A (1986) Schistosoma mansoni shares a protective oligosaccharide epitope with freshwater and marine snails Nature 323, 443–445 13 Wuhrer, M., Dennis, R.D., Doenhoff, M.J & Geyer, R (2000) A fucose-containing epitope is shared by keyhole limpet haemocyanin and Schistosoma mansoni glycosphingolipids Mol Biochem Parasitol 110, 237–246 14 Kantelhardt, S.R., Wuhrer, M., Dennis, R.D., Doenhoff, M.J., Bickle, Q & Geyer, R (2002) Fuc(a1 fi 3)GalNAc-: major antigenic motif of Schistosoma mansoni glycolipids implicated in infection sera and keyhole limpet hemocyanin cross-reactivity Biochem J 366, 217–223 15 Mansour, M.A., Ali, P.O., Farid, Z., Simpson, A.J.G & Woody, J.W (1989) Serological differentiation of acute and chronic schistosomiasis mansoni by antibody responses to keyhole limpet hemocyanin Am J Trop Med Hyg 41, 338–344 16 Alves-Brito, C.F., Simpson, A.J.G., Bahia-Oliveira, L.M.G., Rabello, A.L.T., Rocha, R.S., Lambertucci, J.R., Gazzinelli, G., Katz, N & Correa-Oliveira, R (1992) Analysis of anti-keyhole limpet haemocyanin antibody in Brazilians supports its use for the diagnosis of acute schistosomiasis mansoni Trans R Soc Trop Med Hyg 86, 53–56 17 Markl, J., Nour el Din, M., Winter-Simanowski, S & Simanowski, U.A (1991) Specific IgG activity of sera from Egyptian schistosomiasis patients to keyhole limpet hemocyanin (KLH) Naturwissenschaften 78, 30–31 18 Xue, C.G., Taylor, M.G., Bickle, Q.D., Sacioli, L & Renaganthan, E.A (1993) Diagnosis of Schistosoma haematobium infection: evaluation of ELISA using keyhole limpet haemocyanin or soluble egg antigen in comparison with detection of eggs or haematuria Trans R Soc Trop Med Hyg 87, 654–658 Ó FEBS 2002 19 Taylor, M.G., Huggins, M.C., Shi, F., Lin, J., Tian, E.YeP., Shen, W., Qian, C.G., Lin, B.F & Bickle, Q.D (1998) Production and testing of Schistosoma japonicum candidate vaccine antigens in the natural ovine host Vaccine 16, 1290–1298 20 Bashir, M., Bushara, H., Cook, L., Fuhui, S., He, D., Huggins, M., Jiaojiao, L., Malik, K., Moloney, A., Mukhtar, M., Ping, Y., Shoutai, X., Taylor, M & Yaochuan, S (1994) Evaluation of defined antigen vaccines against Schistosoma bovis and S japonicum in bovines Trop Geogr Med 46, 255–258 21 Harris, J.R & Markl, J (2000) Keyhole limpet hemocyanIn molecular structure of a potent marine immunoactivator a review Eur Urol 37 (Suppl 3), 24–33 22 Markl, J., Lieb, B., Gebauer, W., Altenhein, B., Meissner, U & Harris, J.R (2001) Marine tumor vaccine carriers: structure of the molluscan hemocyanins KLH and htH J Cancer Res Clin Oncol 127 (Suppl 2), R3–R9 23 Cuff, M.E., Miller, K.I., van Holde, K.E & Hendrickson, W.A (1998) Crystal structure of a functional unit from Octopus hemocyanin J Mol Biol 278, 855–870 24 Meissner, U., Dube, P., Harris, J.R., Stark, H & Markl, J (2000) Structure of a molluscan hemocyanin didecamer (HtH1 from ˚ Haliotis tuberculata) at 12 A resolution by cryoelectron microscopy J Mol Biol 298, 21–34 25 Lieb, B., Altenhein, B., Markl, J., Vincent, A., van Olden, E., van Holde, K.E & Miller, K.I (2001) Structures of two molluscan hemocyanin genes: significance for gene evolution Proc Natl Acad Sci USA 98, 4546–4551 26 Lieb, B., Altenhein, B & Markl, J (2000) The sequence of a gastropod hemocyanin (HtH1 from Haliotis tuberculata) J Biol Chem 275, 5675–5681 27 Miller, K.I., Cuff, M.E., Lang, W.F., Varga-Weisz, P., Field, K.G & van Holde, K.E (1998) Sequence of the Octopus dofleini hemocyanin subunit: structural and evolutionary implications J Mol Biol 278, 827–842 28 Kamerling, J.P & Vliegenthart, J.F.G (1997) Hemocyanins In Glycoproteins (Montreuil, J., Vliegenthart, J.F.G & Schachter, H., eds), pp 123–142 Elsevier Science, Amsterdam 29 Stoeva, S., Schutz, J., Gebauer, W., Hundsdorfer, T., Manz, C., Markl, J & Voelter, W (1999) Primary structure and unusual carbohydrate moiety of functional unit 2-c of keyhole limpet hemocyanin (KLH) Biochim Biophys Acta 1435, 94–109 30 van Kuik, J.A., van Halbeek, H., Kamerling, J.P & Vliegenthart, J.F.G (1985) Primary structure of the low-molecular-weight carbohydrate chains of Helix pomatia a-hemocyanin J Biol Chem 260, 13984–13988 31 Lommerse, J.P., Thomas-Oates, J.E., Gielens, C., Preaux, G., Kamerling, J.P & Vliegenthart, J.F.G (1997) Primary structure of 21 novel monoantennary and diantennary N-linked carbohydrate chains from alpha D-hemocyanin of Helix pomatia Eur J Biochem 249, 195–222 32 van Kuik, J.A., Sijbesma, R.P., Kamerling, J.P., Vliegenthart, J.F.G & Wood, E.J (1986) Primary structure of a low-molecularmass N-linked oligosaccharide from hemocyanin of Lymnaea stagnalis 3-O-methyl-D-mannose as a constituent of the xylosecontaining core structure in an animal glycoprotein Eur J Biochem 160, 621–625 33 van Kuik, J.A., Breg, J., Kolsteeg, C.E.M., Kamerling, J.P & Vliegenthart, J.F.G (1987) Primary structure of the acidic carbohydrate chain of hemocyanin from Panulirus interruptus FEBS Lett 221, 150–154 34 Sohngen, S.M., Stahlmann, A., Harris, J.R., Muller, S.A., Engel, ă A & Markl, J (1997) Mass determination, subunit organization and control of oligomerization states of keyhole limpet hemocyanin (KLH) Eur J Biochem 248, 602–614 35 Geyer, R & Geyer, H (1993) Isolation and fractionation of glycoprotein glycans in small amounts Methods Mol Biol 14, 131–142 Ó FEBS 2002 36 Tretter, V., Altmann, F & Marz, L (1991) Peptide-N4-(N-acetylă beta-glucosaminyl) asparagine amidase F cannot release glycans with fucose attached a1–3 to the asparagine- linked N-acetylglucosamine residue Eur J Biochem 199, 647–652 37 Kuraya, N & Hase, S (1992) Release of O-linked sugar chains from glycoproteins with anhydrous hydrazine and pyridylamination of the sugar chains with improved reaction conditions J Biochem 112, 122–126 38 Hase, S (1994) High-performance liquid chromatography of pyridylaminated saccharides Methods Enzymol 230, 225–236 39 Ohashi, S., Iwai, K., Mega, T & Hase, S (1999) Quantitation and isomeric structure analysis of free oligosaccharides present in the cytosol fraction of mouse liver: detection of a free disialobiantennary oligosaccharide and glucosylated oligomannosides J Biochem 126, 852–858 40 Hase, S & Ikenaka, T (1990) Estimation of elution times on reverse-phase high-performance liquid chromatography of pyridylamino derivatives of sugar chains from glycoproteins Anal Biochem 184, 135–138 41 Anumula, K.R (1994) Quantitative determination of monosaccharides in glycoproteins by high-performance liquid chromatography with highly sensitive fluorescence detection Anal Biochem 220, 275–283 42 Wuhrer, M., Dennis, R.D., Doenhoff, M.J., Bickle, Q., Lochnit, G & Geyer, R (1999) Immunochemical characterisation of Schistosoma mansoni glycolipid antigens Mol Biochem Parasitol 103, 155–169 43 Geyer, R., Geyer, H., Kuhnhardt, S., Mink, W & Stirm, S (1983) ă Methylation analysis of complex carbohydrates in small amounts: capillary gas chromatography – mass fragmentography of methylalditol acetates obtained from N-glycosidically linked glycoprotein oligosaccharides Anal Biochem 133, 197–207 44 Geyer, R & Geyer, H (1994) Saccharide linkage analysis using methylation and other techniques Methods Enzymol 230, 86–107 45 Geyer, H., Schmitt, S., Wuhrer, M & Geyer, R (1999) Structural analysis of glycoconjugates by on-target enzymatic digestion and MALDI-TOF-MS Anal Chem 71, 476–482 46 Kobata, A (1979) Use of endo- and exoglycosidases for structural studies of glycoconjugates Anal Biochem 100, 1–14 47 Beeley, J.G (1985) Structural analysis In Glycoprotein and Proteoglycan Techniques (Burdon, R.H & van Knippenberg, P.H., eds), pp 153–300 Elsevier, Amsterdam N-Glycans of KLH (Eur J Biochem 269) 5473 48 Takahashi, N., Hitotsuya, H., Hanzawa, H., Arata, Y & Kurihara, Y (1990) Structural study of asparagine-linked oligosaccharide moiety of taste-modifying protein, miraculin J Biol Chem 265, 7793–7798 49 Luderitz, O., Jann, K & Wheat, R (1968) Somatic and capsular ă antigens of gram-negative bacteria In Comprehensive Biochemistry (Florkin, M & Stotz, E.H., eds), pp 105–226 Elsevier, Amsterdam 50 Edebrink, P., Jansson, P.E & Widmalm, G (1993) Structural studies of the capsular polysaccharide (S-21) from Klebsiella pneumoniae ATCC 31314 Carbohydr Res 245, 311–321 51 Matsubara, T., Iida-Tanaka, N., Kamekura, M., Moldoveanu, N., Ishizuka, I., Onishi, H., Hayashi, A & Kates, M (1994) Polar lipids of a non-alkaliphilic extremely halophilic archaebacterium strain 172: a novel bis-sulfated glycolipid Biochim Biophys Acta 1214, 97–108 52 Upasani, V.N., Desai, S.G., Moldoveanu, N & Kates, M (1994) Lipids of extremely halophilic archaeobacteria from saline environments in India: a novel glycolipid in Natronobacterium strains Microbiology 140, 1959–1966 53 van Kuik, J.A., van Halbeek, H., Kamerling, J.P & Vliegenthart, J.F (1986) Primary structure of the neutral carbohydrate chains of hemocyanin from Panulirus interruptus Eur J Biochem 159, 297– 301 54 van Kuik, J.A., Sijbesma, R.P., Kamerling, J.P., Vliegenthart, J.F & Wood, E.J (1987) Primary structure determination of seven novel N-linked carbohydrate chains derived from hemocyanin of Lymnaea stagnalis 3-O-methyl-D-galactose and N-acetylD-galactosamine as constituents of xylose-containing N-linked oligosaccharides in an animal glycoprotein Eur J Biochem 169, 399–411 55 Markl, J., Savel-Niemann, A., Wegener-Strake, A., Suling, M., ă Schneider, A., Gebauer, W & Harris, J.R (1991) The role of two distinct subunit types in the architecture of keyhole limpet hemocyanin (KLH) Naturwissenschaften 78, 512–514 56 Hamilton, J.V., Chiodini, P.L., Fallon, P.G & Doenhoff, M.J (1999) Periodate-sensitive immunological cross-reactivity between keyhole limpet haemocyanin (KLH) and serodiagnostic Schistosoma mansoni egg antigens Parasitology 118, 83–90 57 Domon, B & Costello, C (1988) A systematic nomenclature for carbohydrate fragmentation in FAB-MS/MS spectra of glycoconjugates Glycoconj J 5, 397–409  ... automated hydrazinolysis in analytical scale The four oligosaccharide fractions were separately pyridylaminated and designated endoH-PA, PNGaseF-PA, PNGaseA-PA and Hyd(HFA)-PA, respectively Analytical... may also originate from other fragmentation pathways which are not indicated for the sake of clarity For the type of ions, see Table Table On-target cleavage of PA-oligosaccharides with exoglycosidases... N-linked carbohydrate chains from H pomatia and L stagnalis represent mostly core xylosylated monoantennary and diantennary complex -type glycans with extended, often multiply branched antennae and