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

Hemocyanin from the keyhole limpet
Megathura crenulata
(KLH)
carries a novel type of N-glycans with Gal(b1–6)Man-motifs
Tomofumi Kurokawa
1,2
, Manfred Wuhrer
2
,Gu¨ nter Lochnit
2
, Hildegard Geyer
2
,Ju¨ rgen Markl
3
and Rudolf Geyer
2,4
1
Pharmaceutical Discovery Center, Pharmaceutical Research Division, Takeda Chemical Industries, Ltd, Osaka, Japan;
2
Institute of Biochemistry, University of Giessen, Giessen; and
3
Institute 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 mollusk glycoprotein 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 to Rudolf 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:
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-N
4
-(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-N
4
-(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 />(Received 25 July 2002, accepted 10 September 2002)
Eur. J. Biochem. 269, 5459–5473 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03244.x
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.
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 1 : 2 (KLH1/KLH2); the purity of this
material was controlled by nondenaturing gel electropho-
resis [2,34]. Purified KLH can be stored at 4 °C for at least
1 year without detectable proteolytic degradation. Isomalt-
osyl oligosaccharides with 2–30 glucose units were prepared
by partial hydrolysis (0.1
M
HCl, 80 °C,2h)ofdextran4
(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-N
4
-(N-acetyl-
b-glucosaminyl)asparagine amidase F from Flavobacterium

meningosepticum (PNGase F) were obtained from Roche
Diagnostics (Mannheim, Germany). b-N-Acetylhexosa-
minidase from jack beans and a-fucosidase from bovine kid-
ney were purchased from Sigma (Deisenhofen, Germany).
Peptide-N
4
-(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 2 mmol of
dithiothreitol (Sigma) in 4 mL of 38 m
M
Tris/HCl buffer,
pH 8.8, containing 6
M
guanidinium chloride (Sigma) and
0.38 m
M
EDTA for 3.5 h at 37 °C in the dark. Iodoacet-
amide (2.2 mmol; Sigma) dissolved in 1 mL of 50 m
M
Tris/
HCl buffer, pH 8.8, containing 8
M
guanidinium chloride
and 0.5 m
M
EDTA was added to the reaction mixture and

incubated at 37 °C 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 m
M
NH
4
HCO
3
buffer,
pH 8.5, containing 4
M
urea (Sigma) as running solvent.
The carboxymethylated KLH fraction was diluted twice
with 25 m
M
NH
4
HCO
3
buffer, pH 8.5, and digested with
1mgoftosyl-
L
-phenylalanine-chloromethylketon (TPCK)
treated trypsin (Sigma) for 18 h at 37 °C. 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 37 °Cas
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 m
M
ammonium 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 (k ¼ 337 nm). The
matrix, 6-aza-2-thiothymine (5 mgÆmL
)1
;Sigma)andthe
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;
5460 T. Kurokawa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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 lmIDwith10lm opening) from New Objective

(Cambridge, MA, USA) were used. The solvent was
evaporated at 150 °C 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 (H
2
O/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 (H
2
O/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 80 °C 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% 0.5
M
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 (H
2
O/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 (H
2
O/
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 m
M
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 m
M
acetic 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 lLof4
M
aqueous
trifluoroacetic acid (Merck, Darmstadt, Germany) at
100 °C 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 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 m
M
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 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 lLofmatrix
solution were mixed on the target and dried in a gentle
stream of cold air. After determination of the molecular
Ó FEBS 2002 N-Glycans of KLH (Eur. J. Biochem. 269) 5461
mass by MALDI-TOF-MS, the sample spot was reconsti-
tutedwith2–3lL of enzyme solution. The target was
incubated at 37 °C over night in a screw-capped jar
containing the respective 25 m
M
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 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 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 mul-
tiple, incompletely resolved oligosaccharides. About 30
signals were assigned to molecular compositions of
Hex
0)7
HexNAc
2)3
dHex
0)3
PA (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, 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 Hex
4)7
HexNAc
1

PA
(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)-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.
Hex
5)6
HexNAc
2
PA 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
recoveredinfractionsPNGaseA-PAandHyd(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
5462 T. Kurokawa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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 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 dHex
1
HexNAc
1
PA 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).
Ó FEBS 2002 N-Glycans of KLH (Eur. J. Biochem. 269) 5463
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 Hex
5
HexNAcPA. 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
(Hex
5
HexNAc), 1186 (Hex
4
HexNAcPA), 968 (Hex
3
Hex
NAcPA), 778 (Hex
2
HexNAcPA) and 560 (HexHex
NAcPA). Treatment with a-mannosidase from jack beans
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 Hex
6
HexNAcPA/Hex
5
HexNAc
2
PA and
Hex
7

HexNAcPA/Hex
6
HexNAc
2
PA, 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.
5464 T. Kurokawa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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-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 H2-2-
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
andfractionF4byamino-phaseHPLC,
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-substitutedFuc;3,terminalGlcNAc;
4, 4-substituted GlcNAc; 5, 3-substituted
GlcNAc;6,3-substitutedGalNAc;7,3,4-di-
substituted GlcNAc; 8, 4,6-disubstituted Glc-
NAc; *contaminant.
Ó FEBS 2002 N-Glycans of KLH (Eur. J. Biochem. 269) 5465
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 Hex
2
HexNAc
2

PA, 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 (Hex
2
HexNAc)
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
atC6oftherespectiveGlcNAcmoiety[36].Bythesameline
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 (%)Experimental Theoretical Gal Man GlcNAc
a
Fuc
H1 947.9
b
948.4 Hex
4
HexNAcPA N.D. N.D. N.D. N.D. N.D.
H2-1 1132.8 1133.0 Hex
5
HexNAcPA – + – – 16.0
H2-2-1 1295.1 1295.2 Hex
6
HexNAcPA – + – – 1.8
H2-2-2 1336.3 1336.3 Hex
5
GlcNAc
2
PA + + + – 0.5
H2-3-1 1457.4 1457.3 Hex
7
HexNAcPA – + – – 1.6
H2-3-2 1498.5 1498.4 Hex
6

GlcNAc
2
PA + + + – 1.1
F1-5 1539.8 1539.4 Hex
5
HexNAc
3
PA 1.9 3.0 2.1 – 0.9
F2-1 850.0 849.8 Hex
2
HexNAc
2
PA – 2.2 0.8 – 3.5
F2-2 1012.2 1011.9 Hex
3
HexNAc
2
PA 1.1 2.0 0.9 – 0.7
F3-1 1498.3 1498.4 Hex
6
HexNAc
2
PA – 5.8 1.2 – 10.3
F4-1 995.5 995.9 Hex
2
HexNAc
2
dHexPA – 2.0 0.8 1.2 7.5
F4-2 1011.6 1011.9 Hex
3

HexNAc
2
PA – 3.4 0.6 – 3.3
F4-3 1157.9 1158.1 Hex
3
HexNAc
2
dHexPA 0.9 1.9 0.9 1.3 2.3
F4-4 1174.2 1174.1 Hex
4
HexNAc
2
PA 0.9 3.2 0.9 – 1.2
F5-1 1157.7 1158.1 Hex
3
HexNAc
2
dHexPA – 3.1 0.7 1.2 4.0
F5-2 1319.8 1320.2 Hex
4
HexNAc
2
dHexPA 0.8 2.9 0.9 1.4 2.5
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
LinkageH2-1 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
2,3,4-FucOH – – – – – – – – + – + – + + + Fuc(1-
2,3,4,6-ManOH + + – + + + + + + + – + + + – Man(1-
3,4,6-ManOH – + + + – – – – – – – – – – – -2)Man(1-
2,3,4-ManOH – – + – + – – + + – + + – + + -6)Man(1-
2,4,6-ManOH – – – – – – + – – – – – – – – -3)Man(1-
3,4-ManOH – – + – – – – – – – – – – – – -2,6)Man(1-
2,4-ManOH + + + + – + – + – + – + + + – -3,6)Man(1-
2,3,4,6-GalOH – + + (+) – + – – – – + + – + + Gal(1-
3,4,6GlcN(Me)AcOH – – + + – – – – – – – – – – – GlcNAc(1-
3,6-GlcN(Me)AcOH – – + + + + + + + + + + + + + -4)GlcNAc(1-
4,6-GlcN(Me)AcOH – + – – – – – – – – – – – – – -3)GlcNAc(1-
a
After b-galactosidase treatment.
b
After a-mannosidase treatment.
5466 T. Kurokawa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
In contrast, MALDI-TOF-MS of compound F3-1
revealed pseudomolecular ions [M + Na]
+
at m/z

1498.3 consistent with the composition Hex
6
HexNAc
2
PA
(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 Hex
5)0
HexNAc
2
PA 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 Man
6
GlcNAc
2
-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 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 Hex
3
HexNAc
2
PA. 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

(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.
Ó FEBS 2002 N-Glycans of KLH (Eur. J. Biochem. 269) 5467
methylation analyses demonstrated the presence of terminal
galactose, terminal mannose, 3,6-disubstituted mannose
and 4-substituted GlcNAc. Sequential treatments with b-
galactosidase 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 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 Hex
3
HexNAc
2

dHexPA (Table 1). Carbohy-
drate constituent and methylation analyses revealed the
presence of terminal fucose, terminal galactose, 6-substi-
tuted mannose and 4-substituted GlcNAc (Table 2). The
Y
4a
and Y
3a
ions 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
1a
and B
4a
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
204 [M + H]
+
HexNAc – – – – – – – + – + –
300 370 [M + H]
+
HexNAcPA – + + + + – + ++++

322 [M + Na]
+
HexNAcPA – – – + – – – – + (+) +
347 [M + Na]
+
Hex
2
– –––––––+––
366 [M + H]
+
HexHexNAc – + – – – – – –––(+)
446 544 [M + H]
+
HexNAcdHexPA – – – – – + – (+) – + (+)
560 [M + H]
+
HexHexNAcPA + – – – – – – ––––
525 [M + Na]
+
HexNAc
2
PA – (+) – (+) + – – – + – –
615 [M + H]
+
HexNAc
2
PA – –– ––– +––––
550 [M + Na]
+
Hex

2
HexNAc – (+) – – – – – – + – –
668 [M + H]
+
Hex
2
HexNAc – – + – – + – ––––
672 [M + Na]
+
Hex
4
– –– ––– – –––+
778 [M + H]
+
Hex
2
HexNAcPA + – – – – – – ––––
687 [M + Na]
+
HexHexNAc
2
PA – – – (+) + – – – + – +
713 [M + Na]
+
Hex
3
HexNAc – – – + – – – ++++
872 [M + H]
+
Hex

3
HexNAc – – – – – – + ––––
834 [M + Na]
+
HexHexNAc
2
dHexPA – – – – – – – (+) – – (+)
968 [M + H]
+
Hex
3
HexNAcPA + – – – – – – ––––
993 [M + H]
+
HexHexNAc
2
dHexPA – – – – – + – ––––
850 [M + Na]
+
Hex
2
HexNAc
2
PA – (+) – + + – – (+) + + +
874 [M + Na]
+
Hex
4
HexNAc – – – – – – – – + – +
1133 [M + H]

+
Hex
3
HexNAc
2
– –– ––– +––––
1186 [M + H]
+
Hex
4
HexNAcPA + – – – – – – ––––
996 [M + Na]
+
Hex
2
HexNAc
2
dHexPA – – – – – – – + – + –
1011 [M + Na]
+
Hex
3
HexNAc
2
PA – +– – +– – ++++
1036 [M + Na]
+
Hex
5
HexNAc – – – – + – – ––––

1296 [M + H]
+
Hex
5
HexNAc + – – – – – – ––––
1077 [M + Na]
+
Hex
4
HexNAc
2
– +– ––– – ––––
1173 [M + Na]
+
Hex
4
HexNAc
2
PA – +– –+– – –––+
1198 [M + Na]
+
Hex
6
HexNAc – – – – + – – ––––
1239 [M + Na]
+
Hex
5
HexNAc
2

– +– ––– – ––––
1335 [M + Na]
+
Hex
5
HexNAc
2
PA – +– –+– – ––––
1376 [M + Na]
+
Hex
4
HexNAc
3
PA – +– ––– – ––––
418 [M + H + Na]
2+
HexHexNAc
2
dHexPA – – – (+) – – – + – (+) +
425 [M + H + Na]
2+
Hex
2
HexNAc
2
PA – – – +– – – +++–
499 [M + H + Na]
2+
Hex

2
HexNAc
2
dHexPA – – – – – – – + – + (+)
506 [M + H + Na]
2+
Hex
3
HexNAc
2
PA – – – (+)+– – ++++
570 [M + H + Na]
2+
Hex
3
HexNAc
2
dHexPA-H
2
O– –– ––– – +–+–
578 [M + H + Na]
2+
Hex
4
HexNAc
2
PA-H
2
O – +– ––– – –––+
587 [M + H + Na]

2+
Hex
4
HexNAc
2
PA – –– –+– – –––+
607 [M + H + Na]
2+
Hex
3
HexNAc
3
PA – +– ––– – ––––
668 [M + H + Na]
2+
Hex
5
HexNAc
2
PA – +– –+– – ––––
688 [M + H + Na]
2+
Hex
4
HexNAc
3
PA – +– ––– – ––––
760 [M + H + Na]
2+
Hex

5
HexNAc
3
PA-H
2
O – +– ––– – ––––
5468 T. Kurokawa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
demonstrated the presence of dHexHexNAcPA, suggesting
that the fucosyl residue is linked to the innermost GlcNAc
(cf. Figure 5 and Table 3). These results could be corro-
borated by sequential exoglycosidase treatment (Fig. 6 and
Table 4) yielding the release of one galactosyl, one man-
nosyl 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-TOF-
MS revealed pseudomolecular ions [M + Na]
+
at m/z
1539.8 consistent with the composition Hex
5
HexNAc

3
PA.
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 disap-
pearance of 6-substituted and 2,6-disubstituted mannosyl
residues in conjunction with the considerable removal of
terminal Gal and the simultaneous expression of terminal
Fig. 7. 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 path-
ways are delineated together with the struc-
tures 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 3.
Table 4. 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.
PA-oligosaccharide Composition [M + Na]
+
Sequential enzymatic
digestion Independent enzymatic digestion
b-Gal
a
a-Man
b
a-Fuc
c
a-Man
b
a-Fuc
c
b-GlcNAc
d
H2-1 Man
5
GlcNAcPA 1133.4 – – – 461.9

e
––
F1-5 Gal
2
Man
3
GlcNAc
3
PA 1539.7 1376.9
f
1215.3 – 1539.0 – 1337.5 (1539.6)
F2-1 Man
2
GlcNAc
2
PA 850.2 – – – 687.5 – –
F2-2 Gal
1
Man
2
GlcNAc
2
PA 1012.2 850.2 687.3 – 1011.8 – –
F3-1 Man
6
GlcNAc
2
PA 1498.3 – – – 687.4 – –
F4-1 Man
2

GlcNAc
2
FucPA 996.3 – 833.9 687.8 833.9 850.2 –
F4-2 Man
3
GlcNAc
2
PA 1012.1 – – – 687.5 – –
F4-3 Gal
1
Man
2
GlcNAc
2
FucPA 1158.3 995.9 834.0 687.6 1157.9 1012.2 –
F4-4 Gal
1
Man
3
GlcNAc
2
PA 1174.0 1012.2 687.8 – 1012.3 – –
F5-1 Man
3
GlcNAc
2
FucPA 1158.4 – 833.5 687.4 833.6 1011.6 –
F5-2 Gal
1
Man

3
GlcNAc
2
FucPA 1320.5 1158.0 833.5 687.8 1158.3 1174.0 –
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.
Ó FEBS 2002 N-Glycans of KLH (Eur. J. Biochem. 269) 5469
Man. ESI-IT-MS/MS analysis revealed Y
4
ions at m/z
1335.0 and 1375.8 indicative for the presence of terminal
HexNAc and Hex residues (cf. Fig. 7 and Table 3).
Furthermore, B
2a
and Y
3a

fragment ions at m/z 550.6 and
1011.0 indicated the presence of a Hex
2
HexNAc unit,
whereas no evidence has been obtained for a Hex
3
-
fragment. Instead, the doubly charged Y
3b
fragment ion
at m/z 607.5 indicated a Hex
2
-antenna. Likewise (B
2a
)and
doubly charged [(Y
4a
)-H
2
O]
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 2,6-
disubstituted Man shown by methylation analysis. Consid-
ering 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
animals [2], may clearly influence the molar proportions of
the monosaccharide constituents of this glycoprotein.
Furthermore, the ratio of KLH1 and KLH2 varies consid-
erably 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-dimen-
sional 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 carboxy-
methylation and tryptic digestion, respectively, in order to
achieve sufficient solubility of starting material and reaction
5470 T. Kurokawa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
products as well as fragmentation of the glycoprotein.
Although the resulting glycopeptides have not been ana-
lyzed in detail, proteolytic cleavage appeared to be incom-

plete. Small precipitates occasionally observed, however, did
not contain carbohydrates. Hence, the observation that the
pool of glycans finally released by PNGase A and hydraz-
inolysis (Fig. 2C,D) still comprised, in part, oligosaccha-
rides 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-substi-
tuted fucose, 3-substituted GalNAc and 3-substituted or
4,6-disubstituted GlcNAc residues. In agreement with this
finding, constituent analyses of these oligosaccharide frac-
tions 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-ora-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 lipopolysaccha-
rides [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 char-
acterized 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-galactosyl-
transferases conveying Gal to mannosyl residues of glyco-
protein-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 monosac-
charide constituents. In contrast, the N-glycans from
M. crenulata characterized so far are mainly high man-
nose-type or truncated complex-type sugar chains. The
questionastowhetherglycanswithGal(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].

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 immu-
nological 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 cross-
reactivity with schistosomal glycoconjugates.
ACKNOWLEDGMENTS
We thank P. Kaese, W. Mink, S. Ku
¨
hnhardt and S. Eisenmann
(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).
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