Báo cáo Y học: Hemocyanin from the keyhole limpet Megathura crenulata (KLH) carries a novel type of N-glycans with Gal(b1–6)Man-motifs doc

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, t

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, Gu¨nter Lochnit2, Hildegard Geyer2, Ju¨rgen Markl3

and Rudolf Geyer2,4

1

Pharmaceutical Discovery Center, Pharmaceutical Research Division, Takeda Chemical Industries, Ltd, Osaka, Japan;

2

Institute of Biochemistry, University of Giessen, Giessen; and3Institute 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

enzymati-cally liberated from tryptic glycopeptides, pyridylaminated

and separated by two-dimensional HPLC Only neutral

oligosaccharides were obtained and characterized by

car-bohydrate 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 molluskglycoprotein is characterized by a unique oligosaccharide pattern compri-sing, in part, novel structural elements

Keywords: keyhole limpet hemocyanin; carbohydrate struc-ture analysis; mass spectrometry; N-glycans

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

immuno-genic 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 vac-cines [8–11] In addition, it has been demonstrated that KLH shares a cross-reacting oligosaccharide epitope with glyco-conjugates 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 toRudolf Geyer, Biochemisches Institut am Klinikum

der Universita¨t 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-acetylglucos-aminidase H from Flavobacterium meningosepticum; Hex, hexose;

HexNAc, N-acetylhexosamine; IT, ion trap; KLH, keyhole limpet

hemocyanin; MS/MS, tandem mass spectrometry; PA,

2-aminopyri-dine; PGC, porous graphitic carbon; PNGase A, peptide-N 4

-(N-ace-tyl-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,

tosyl-L -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-N 4

-(N-acetyl-b-glucosami-nyl)asparagine amidase A (EC 3.5.1.52); peptide-N4

-(N-acetyl-b-glu-cosaminyl)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)

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

carbo-hydrate 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,

N-acetylglucosamine, N-acetylgalactosamine and fucose in

differing amounts [29; Wuhrer, unpublished results]

Fur-thermore, 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

hemo-cyanins 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

E X P E R I M E N T A L P R O C E D U R E S

Materials

KLH (Vacmune) 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 1 : 2 (KLH1/KLH2); the purity of this

material was controlled by nondenaturing gel

electropho-resis [2,34] Purified KLH can be stored at 4C for at least

1 year without detectable proteolytic degradation

Isomalt-osyl oligosaccharides with 2–30 glucose units were prepared

by partial hydrolysis (0.1MHCl, 80C, 2 h) of dextran 4

(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 jackbeans, a-galactosidase from green

coffee beans, endo-b-N-acetylglucosaminidase H from

Streptomyces plicatus (endoH), and peptide-N4

-(N-acetyl-b-glucosaminyl)asparagine amidase F from Flavobacterium

meningosepticum (PNGase F) were obtained from Roche

Diagnostics (Mannheim, Germany)

b-N-Acetylhexosa-minidase from jackbeans and a-fucosidase from bovine

kid-ney 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 jackbeans was obtained

from Glyco (Upper Herford, UK)

Tryptic digestion of KLH

Thirty milligrams of KLH were reduced with 2 mmol of

dithiothreitol (Sigma) in 4 mL of 38 mMTris/HCl buffer,

pH 8.8, containing 6Mguanidinium chloride (Sigma) and

0.38 mMEDTA for 3.5 h at 37C in the dark

Iodoacet-amide (2.2 mmol; Sigma) dissolved in 1 mL of 50 mMTris/

HCl buffer, pH 8.8, containing 8M guanidinium chloride

and 0.5 mMEDTA was added to the reaction mixture and

incubated at 37C for 1 h in the dark After addition of

1 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, Toso-Haas, Stuttgart, Germany) with 25 mMNH4HCO3buffer,

pH 8.5, containing 4M urea (Sigma) as running solvent The carboxymethylated KLH fraction was diluted twice with 25 mM NH4HCO3buffer, pH 8.5, and digested with

1 mg of tosyl-L-phenylalanine-chloromethylketon (TPCK) treated trypsin (Sigma) for 18 h at 37C The tryptic digest was desalted on a reversed-phase cartridge (C18ec; Mache-rey and Nagel, Du¨ren, Germany) and the (glyco)peptides, 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 glycopep-tides by sequential treatment with 4.5 nkat endoH, 0.8 nkat PNGase F and 0.08 nkat PNGase A overnight at 37C 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 mLÆh)1 with 10 mMammonium acetate buffer, pH 6.0, as running solvent Pyridylaminated (PA)-oligosaccharides were moni-tored 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 (k¼ 337 nm) The matrix, 6-aza-2-thiothymine (5 mgÆmL)1; Sigma) and the PA-oligosaccharides (1–20 pmol) were mixed on the stain-less 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;

ThermoHypersil, Kleinostheim, Germany) using an

Ulti-mate 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-IT-MS (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 150C with a nitrogen stream of 8 LÆmin)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 nLÆmin)1

at room temperature After injecting the sample, elution was

performed with 100% eluent A for 2 min, and a linear

gradient to 25% eluent B (H2O/acetonitrile 20 : 80, v/v,

containing 0.1% formic acid) in 28 min followed by a final

wash with 95% solvent B for 5 min 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 : 1) or

permethyl-ated PA-glycans (in methanol) was loaded into a

laborat-ory-made, gold-coated glass capillary and electrosprayed at

a voltage of 700–1000 V The solvent was evaporated at

120 or 80C for native or permethylated

PA-oligosaccha-rides, respectively, with a nitrogen stream of 4 LÆmin)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

per-formed in the positive-ion mode using helium as collision

gas

Anion-exchange HPLC

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 mLÆmin)1 The elution was performed using a

linear gradient from 0 to 12% 0.5M ammonium acetate,

pH 9.0, during the first 3 min, followed by a further increase

up to 40% in the next 28 min, and to 100% in the last

5 min The eluate was monitored by fluorescence with an

excitation wavelength of 310 nm and an emission

wave-length of 380 nm

Preparative separation of PA-oligosaccharides

The PA-oligosaccharides were separated by

prepar-ative HPLC using a PGC column (Hypercarb, 7 lm,

4.6 mm· 100 mm; ThermoHypersil) equilibrated with

elu-ent A (H2O/acetonitrile 95 : 5, v/v, containing 0.1% formic

acid) at a flow rate of 0.8 mLÆmin)1 After injecting the

sample, elution was performed with 100% eluent A for

2 min, followed by a linear gradient to 25% eluent B (H2O/

acetonitrile 20 : 80, v/v, containing 0.1% formic acid) in

28 min and a final wash with 95% solvent B for 5 min 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 (Nucle-osil 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 mLÆmin)1[38] After injecting the sample, elution was performed with 100% eluent A for 5 min, a linear gradient

to 70% eluent B (200 mMacetic acid/triethylamine, pH 7.3/ acetonitrile 60 : 40, v/v) in 35 min and a final wash with 100% solvent B for 5 min 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, Ger-many) 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-oligosac-charides 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 4M aqueous trifluoroacetic acid (Merck, Darmstadt, Germany) at

100C for 4 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 else-where [42]

Methylation analysis 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 micro-techniques described elsewhere [43,44]

Digestion with exoglycosidases PA-oligosaccharides were degraded with a-mannosidase from jackbeans (0.85 nkat), a-galactosidase from green coffee beans (0.85 nkat), b-galactosidase from jackbeans (0.17 nkat), a-fucosidase from bovine kidney (0.07 nkat) and b-N-acetylhexosaminidase from jackbeans (1.1 nkat)

on a stainless steel MALDI-TOF-MS target as described elsewhere [45] All enzymes were dialyzed before use against

25 mMammonium 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 kid-ney), 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

mass by MALDI-TOF-MS, the sample spot was

reconsti-tuted with 2–3 lL of enzyme solution The target was

incubated at 37C over night in a screw-capped jar

containing the respective 25 mMammonium 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

R E S U L T S

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

mono-saccharide constituents

Analysis of total KLH glycans

In order to take an overview of the complexity of KLH

glycosylation, intact glycoprotein was subjected to

automa-ted hydrazinolysis Resulting glycans were pyridylaminaautoma-ted

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

mul-tiple, 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/Fuc¼ 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,

respect-ively Analytical anion-exchange HPLC of the

pyridyl-aminated 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 KLH-derived glycans (Fig 1A) In the case of endoH-PA species, the monosaccharide compositions deduced from the pre-vailing 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 glyco-peptides as well as endoH-PA, PNGaseF-PA,

PNGaseA-PA and Hyd(HFA)-PNGaseA-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 con-tain type-1 (Galß3GlcNAc-) or type-2 (Galß4GlcNAc-) N-acetyllactosamine antennae Oligosaccharides liberated

by PNGaseF disclosed, in addition, terminal fucose, 6-sub-stituted, 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 addi-tion, linkage analyses revealed the presence of increased levels of 3-substituted GalNAc as well as 3,4- and 4,6-disubstituted 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 exclu-sively on the structural elucidation of the major carbohy-drate 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 col-umn was performed, resulting in a large number of PA-oligosaccharide subfractions (referred to as H2-1 for subfraction 1 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

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

con-stituent analysis (Table 1) Monosaccharide linkage

posi-tions 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 5 and Table 3) Monosaccharide sequencing and determination of the anomeric configurations of the corresponding glycosidic linkages were performed by degradation with exoglyco-sidases (Fig 6 and Table 4) All glycans containing galactosyl residues were found to be sensitive towards digestion with b-galactosidase from jackbeans 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 perme-thylation) 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 con-tained (a1–6)-linked fucosyl residues

Fig 1 Positive-ion nano-LC-ESI-IT-MS

analysis of total KLH-derived

PA-oligosac-charides released by hydrazinolysis

PA-oligo-saccharides 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).

Characterization of endoH-sensitive N-glycans

Compound H2-1, the major component of the endoH

frac-tion (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)

ESI-IT-MS/MS analysis (Table 3) of the permethylated

com-pound 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 jackbeans

released four mannosyl residues as confirmed by

MALDI-TOF-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 RP-HPLC 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 to-gether 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

Fig 2 Positive-ion nano-LC-ESI-IT-MS analysis of separate KLH-derived PA-oligo-saccharide 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 absorb-ance 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 1.

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-disubsti-tuted Man as well as terminal Gal and 3-substi3,6-disubsti-tuted

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

H2-2-2 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

Fig 4 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

proce-dures (A) endoH-PA; (B) PNGaseF-PA; and

(C) and (D) subfractionation of fraction H2

and fraction F4 by amino-phase HPLC,

respectively.

Fig 3 Detection of fucose and HexNAc

spe-cies by methylation analysis of KLH

PA-oligosaccharide fractions Partially methylated

alditol acetates were separated by gas

chro-matography 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

1,4,5-tri-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-di-substituted GlcNAc; 8, 4,6-di3,4-di-substituted

Glc-NAc; *contaminant.

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

dis-cussed

MALDI-TOF-MS of compound F2-1 revealed

pseudo-molecular ions [M + Na]+at m/z 850.0 consistent with the

composition Hex2HexNAc2PA, corresponding to the

small-est N-glycan preparatively isolated from KLH in this study

Carbohydrate constituent and methylation analyses

dem-onstrated the presence of terminal mannose, 6-substituted

mannose and 4-substituted GlcNAc (cf Tables 1 and 2)

ESI-IT-MS/MS analysis of the permethylated compound

led to protonated fragment ions at m/z 668 (HexHexNAc)

and 370 (HexNAcPA) (Table 3) Treatment with a-man-nosidase 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, com-pounds F4-2 and F5-1 could be identified as nonfucosylated and (a1–6)-fucosylated representatives of the standard pentasaccharide core of glycoprotein-N-glycans

Table 1 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.

Fraction

Molecular mass [M + Na] +

Molecular composition

Carbohydrate constituents

Relative amount (%)

a

The innermost GlcNAc residue was not detected due to reductive amination.b[M + H]+registered by ESI-MS.

Table 2 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.

Alditol acetate

Presence in oligosaccharide fraction

Linkage H2-1 H2-3-2 F1-5 F1–5 a F2-1 F2-2 F2–2 a F3-1 F4-1 F4-2 F4-3 F4-4 F5-1 F5-2 F5–2 b

-3)GlcNAc(1-a After b-galactosidase treatment b After a-mannosidase treatment.

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 lackof 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

endoH-sensitivity [46] On the basis of the data obtained, the

structure of F3-1 glycans may be proposed as follows

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 manno-syl residue MALDI-TOF-MS of the smallest representa-tive of this class, F2-2, revealed pseudomolecular ions [M + Na]+ at m/z 1012.2 consistent with the composi-tion HexHexNAcPA Carbohydrate constituent and

Fig 5 Nano-ESI-IT-MS/MS spectrum of the doubly charged

pseudo-molecular 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 3.

Fig 6 MALDI-TOF-MS spectra of compound F4-3 after sequential enzymatic digestions (A) Starting material; (B) after b-galactosidase (jackbeans) 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.

methylation analyses demonstrated the presence of terminal

galactose, terminal mannose, 3,6-disubstituted mannose

and 4-substituted GlcNAc Sequential treatments with

b-galactosidase from jackbeans 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

con-firmed by methylation analysis of the

b-galactosidase-treated 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 pseudomo-lecular ions [M + Na]+at m/z 1157.9 consistent with the composition Hex3HexNAc2dHexPA (Table 1) Carbohy-drate constituent and methylation analyses revealed the presence of terminal fucose, terminal galactose, 6-substi-tuted mannose and 4-substi6-substi-tuted GlcNAc (Table 2) The

Y4aand Y3aions at m/z 996 and 834 obtained by ESI-IT-MS/MS analysis are in agreement with a linear arrangement

of hexoses whereas the Y and B ions at m/z 446 and 713

Table 3 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)

Pseudo-molecular ion Composition

Ions obtained from PA-oligosaccharide fraction Native Permethy

lated

H2-1* F1-5 F2-1* F2-2 F3-1 F4-1* F4-2* F4-3 F4-4 F5-1 F5-2