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-

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

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].

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

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-

(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

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

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

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

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.

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

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

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

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

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.

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

(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

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

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]

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

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]

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

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

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

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

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-

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].

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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