Neutral N-glycans of the gastropod
Arion lusitanicus
Martin Gutternigg, Karin Ahrer, Heidi Grabher-Meier, Sabine Bu¨ rgmayr and Erika Staudacher
Department fu
¨
r Chemie, Universita
¨
tfu
¨
r Bodenkultur Wien, Vienna, Austria
The neutral N-glycan structures of Arion lusitanicus (gas-
tropod) skin, viscera and egg glycoproteins were examined
after proteolytic digestion, release of the glycans from the
peptides, fluorescent labelling with 2-aminopyridine and
fractionation by charge, size and hydrophobicity to obtain
pure glycan structures. The positions and linkages of the
sugars in the glycan were analysed by two dimensional
HPLC (size and hydrophobicity) and MALDI-TOF mass
spectrometry before and after digestion with specific
exoglycosidases. The most striking feature in the adult tis-
sues was the high amount of oligomannosidic and small
paucimannosidic glycans terminated with 3-O-methylated
mannoses. The truncated structures often contained modi-
fications of the inner core by b1,2-linked xylose to the
b-mannose residue and/or an a-fucosylation (mainly a1,6-)
of the innermost GlcNAc residue. Skin and viscera showed
predominantly the same glycans, however, in different
amounts. Traces of large structures carrying 3-O-methylated
galactoses were also detected. The egg glycans contained
mainly ( 75%) oligomannosidic structures and some pau-
cimannosidic structures modified by xylose or a1,6-fucose,

species gives an overview on its biosynthetic capacity for
glycosylation. It is the first step for the identification of
glycosylation related target enzymes for inhibition.
So far, N-glycan structures derived from the hemocyanins
of the snails Helix pomaia, Lymnaea stagnalis, Rapana
venosa and the keyhole limpet Megathura crenulata have
been published. The Helix pomatia glycans show complex
structures containing a common core with an a1,6-linked
fucose to the reducing GlcNAc and a b1,2-linked xylose to
the b-mannose residue. One or both a-mannose residues
may be substituted by GalNAcb1,4GlcNAcb1,2 elements
which contain two to four b1,3- or b1,6-linked galactoses
with or without 3- or 4-O-methyl groups [1]. Lymnaea
stagnalis hemocyanin contains low and high molecular mass
biantennary oligosaccharides. They lack the a1,6-linked
fucose to the inner GlcNAc residue, but some antennae
terminate with an a1,2-linked fucose. Similarly to Helix
pomatia, the basic element of the antennae is Galb1,3Gal-
NAcb1,4GlcNAc [2,3]. The two N-glycans of the functional
unit RvH1-a of Rapana venosa hemocyanin are biantennary
nonfucosylated oligosaccharides with 3-O-methylated ter-
minal b1,3-linked galactose residues. One of these residues
also carries a sulfate group on the a1,6-linked core mannose
and a 3-O-methylated GlcNAc residue b1,2-linked to the
b-mannose of the core [4]. Megathura crenulata hemocyanin
is substituted by a novel type of N-glycan with galactoses
directly linked in b1,6-linkage to mannose residues [5].
Recently a core structure terminated with two 3-O-methy-
lated mannose residues linked to the major soluble protein
of the organic shell matrix of Biomphalaria glabrata was

GalNAc-transferase which shows similar characteristics to
mammalian b1,4-galactosyltransferase [8], a b1,3-galacto-
syltransferase and an a1,2-fucosyltransferase [9,10].
Hybridization experiments using a bovine b1,4-galactosyl-
transferase cDNA probe resulted in the isolation of a clone
encoding a b1,4-GlcNAc-transferase which is similar to the
mammalian galactosyltransferase in acceptor specificity but
requires a different nucleotide sugar. It is definitely not
involved in the biosynthesis of the chitobiose core of
N-glycans [11–13]. The function of this enzyme in vivo is
not clear. The prostate glands of these snails also contain a
b1,4-glucosyltransferase forming Glcb1,4GlcNAc units
[14]. Furthermore an a1,3-fucosyltransferase catalysing
the transfer of fucose from GDP-fucose to a Galb1,4Glc-
NAc acceptor forming the Lewis
X
-unit has been found in
the connective tissue of Lymnaea stagnalis [10] and an
a1,3-fucosyltransferase catalysing the transfer of fucose
from GDP-fucose to the asparagine-linked GlcNAc has
been found in the albumin and prostate glands of the same
snail[15].However,noLewis
X
-containing structures, core
a1,3-fucosylated structures, or glucosylated units have been
detected in the glycans of this snail so far. An a1,2-
L
-galactosyltransferase which seems to be involved in the
elongation of the storage polysaccharide of the snail was
found in Helix pomatia [16]. Although in vitro this

highest quality available from Merck or Sigma.
Preparation of N-glycans
Thawed slugs (10 individuals for each preparation) were
washed to remove the extraneous mucous components and
dissected into three fractions; the skin and inner organs
(viscera) were lyophilized separately, while the intestinal
tract was discarded. The dry material (skin, viscera or eggs)
was suspended in 200 mL of 50 m
M
Tris/HCl buffer
pH 7.5, homogenized with an IKA Ultra Turrax T25
(IKA-Labortechnik, Janke and Kunkel GmbH, Staufen,
Germany) at 15 000 r.p.m. for 2· 20 s and centrifuged at
5000 g for 10 min. The supernatant was adjusted to 80%
(w/v) of ammonium sulfate and centrifuged at 27 500 g for
40 min. The precipitate was dialyzed against water, con-
centrated on rotary evaporation and made up to 150 m
M
of
Tris/HCl, 1 m
M
CaCl
2
, pH 7.8. Thermolysin (ICN
Biomedicals, Vienna, Austria) was added at a 40 : 1
(w/w) ratio of protein/enzyme and incubated for 20 h at
50 °C. The digest was dialyzed against 2% (v/v) acetic acid
andappliedtoacolumnof100mLofDowex50W·2
equilibrated in 2% (v/v) acetic acid. The column was
washed with 150 mL of the same solution and the

)1
. Solvent A was 50 m
M
Tris/HCl,
pH 8.5; solvent B was 1
M
NaCl in solvent A. The run
was started with 5 min at 100% solvent A followed by a
linear gradient of 5% per min to 50% solvent B, continued
with 10% per min to 100% solvent B and terminated by
1 min at 100% solvent B. Fluorometric detection was
carried out at excitation and emission wavelengths of 320
and 400 nm, respectively.
Ó FEBS 2004 Neutral N-glycans of Arion lusitanicus (Eur. J. Biochem. 271) 1349
The neutral fraction was further fractionated by a two
dimensional mapping technique starting with separation
according to hydrophobicity on an Hypersil ODS column
(0.4 · 25cm, 5l, Forschungszentrum Seibersdorf, ARC
Seibersdorf research GmbH, Seibersdorf, Austria) [21].
Fluorometric detection was performed at excitation and
emission wavelengths of 320 and 400 nm, respectively, and
peaks were collected and dried prior to subfractionation by
size, in the second dimension. The method was modified
from the procedure of Khoo et al. [23] using a Palpak
type N column (4.6 · 250 mm, Takara, Japan) at a flow
rate of 1 mLÆmin
)1
. Solvent A was 75 : 25 (v/v) acetonitrile/
stock solution [3% (w/v) acetic acid-triethylamine buffer at
pH 7.3 with 10% (v/v) acetonitrile]. Solvent B was 50 : 50

Roche) was used at a concentration of 2 mU in 0.15
M
citrate-phosphate buffer, pH 5.0 containing 0.1
M
NaCl;
a-mannosidase (jack bean, Sigma) at 2 mU in 50 m
M
sodium acetate, pH 4.5 containing 0.2 m
M
ZnCl
2
; a-fuco-
sidase (bovine kidney, Sigma) at 2 mU in 50 m
M
sodium
citrate, pH 4.5; a1,2-fucosidase (recombinant, Sigma) at
0.2 mU in 50 m
M
sodium phosphate pH 5.0; b-galactosi-
dase (bovine testis, Roche) at 1.6 mU in 50 m
M
sodium
citrate, pH 5.0 and b-hexosaminidase (bovine kidney,
Sigma) at 25 mU in 20 lLof0.1
M
sodium citrate,
pH 5.0). Incubations were carried out in 20 lL of appro-
priate buffer at 37 °Covernight.
For chemical release of fucose a1,3-linked to the
inner GlcNAc-residue, the dry sample was incubated for

were identified by their elution behaviour on
reverse-phase.
Methylated oligomannosidic structures. Region II of the
reverse-phase pattern (Fig. 1) contained, in the preparations
of the adult snails, methylated mannosidic structures with
mainly five to seven mannose residues and two or more, often
three, methyl groups (abbreviations of glycan structures are
given in Fig. 2). Methylated M
4
,M
8
and M
9
structures were
also found, however,in very low amounts (Table 1). All these
structures were sensitive to endoglycosidase H (Fig. 3). To
confirm the presence of 3-O-methylmannose residues,
we performed carbohydrate composition analysis by gas
chromatography/mass spectrometry. Incomplete methy-
Fig. 1. HPLC analysis of pyridylaminated neutral N-glycans of Arion
lusitanicus on a reverse-phase column. (A) Isomaltose standard, 4–14
glucose units, (B) skin, (C) viscera and (D) eggs. Regions I–IV are
indicated with arrows. I, oligomannosidic structures; II, methylated
oligomannisidic structures; III, a1,6-fucosylated structures; IV, large
galactose containing structures.
1350 M. Gutternigg et al.(Eur. J. Biochem. 271) Ó FEBS 2004
lated structures were subjected to an a-mannosidase digest
which made it possible to identify the position of the
unmethylated mannose in most cases. For example, if the
terminal mannose of the a1,3-arm of a M

earlier elution times on reversed phase chromatography
confirmed the loss of a fucose linked a1,6 to the inner core.
The main compound was dimethylated Me
2
MMF
6
in skin
and viscera (abbreviations of glycan structures are given in
Fig. 4). However, in viscera the monomethylated variant
MeMMF
6
, and in skin a xylosylated variant Me
2
MMXF
6
were also detected. No a1,6-fucosylated glycans lacking the
methyl groups could be determined in adult tissues
(Table 1).
Fig. 2. Structures of paucimannosidic (four
mannose residues or less) and oligomannosidic
glycans. The abbreviation system applied
herein (according to [18]) names the terminal
residues, starting with the residue on the
6-linked antenna and proceeding counter
clockwise.
Ó FEBS 2004 Neutral N-glycans of Arion lusitanicus (Eur. J. Biochem. 271) 1351
Paucimannosidic structures. Examination of regions I and
II of the reverse-phase pattern suggested the presence of
small paucimannosidic structures. Therefore a further
preparation removing the oligomannosidic structures by

earlier at the positions found for the paucimannosidic
Table 1. Neutral N-glycan profiles of Arion lusitanicus. Wherever the
detected traces are less then 0.2% an exact quantitation is not possible.
Therefore the amount is considered to be 0.1%.
Structure Skin (%) Viscera (%) Eggs (%)
Mannosidic structures
MU  0.1 1.6 1.5
MM – – 5.1
M
4
1.5 2.5 1.0
M
5
1.0 21.6 19.6
M
6
2.1 8.2 16.9
M
7
1.5 3.7 14.8
M
8
1.5 4.2 14.9
M
9
1.0 5.2 1.5
GlcM
9
 0.1 – –
Sum 8.8 47.0 75.3

6.6 9.4 –
Me
1-2
M
7
0.7 2.9 –
Me
3
M
7
0.5  0.1 –
Me
1-2
M
8
 0.1 0.4 –
Me
3
M
8
0.8  0.1 –
Me
1-2
M
9
0.6  0.1 –
Me
3
M
9

Me
2
MMXF
6
1.2 – –
Sum 9.9 4.7 –
Other paucimannosidic structures
MUX – – 1.2
MMX 0.4 0.7 11.5
M
4
X  0.1 – –
MMXF
3
1.7 0.4 –
GnGnXF
3
0.7  0.1 –
Sum 2.9 1.2 12.7
Other methylated paucimannosidic structures
MeMMX 0.4 2.2 –
Me
2
MMX 3.5 1.2 –
MeM
4
X 0.6 0.8 –
Sum 4.5 4.2 –
Complex type structures with methylated galactoses
Sum 3.9 2.0 –

the case of the core fucose, which occurs in
more than one type of linkage, the linkage is
depicted as a superscript.
Fig. 5. HPLC analysis of pyridylaminated MMXF
3
on a reverse-phase
column. (A) Isomaltose standard, 3–11 glucose units, (B) MMXF
3
,
(C) MMXF
3
after incubation with a-fucosidase from bovine kidney
and (D) MMXF
3
after incubation with hydrofluoric acid.
Ó FEBS 2004 Neutral N-glycans of Arion lusitanicus (Eur. J. Biochem. 271) 1353
et al.[1]forHelix pomatia a
D
-hemocyanin, where one or
both antennae of biantennary xylosylated glycans termin-
ate with a varying number of methylated galactose
residues.
Eggs
The egg glycans differed from those derived from adult
tissues (Table 1). While in preparations of adult slugs the
unmethylated, oligomannosidic structures were restricted to
8.8% and 47% in skin and viscera, respectively, in the eggs
 75% of the total N-glycans were oligomannosidic struc-
tures, dominated by M
5

standard oligosaccharides, in combination with the mass
information from MALDI-TOF, led to the conclusions
about the structure which were confirmed by digestion with
specific exoglycosidases. For relative quantitation of the
structures see Table 1.
In the course of our work we found that the percentages
of structures vary slightly with the area where the slugs had
been collected (due to nutritional conditions), the age (size)
of the individuals and their physiological status (carrying
eggs or not). However, skin and viscera preparations
contained the same spectrum of N-glycans. Therefore it
can be ruled out that unusual structures are due to food or
environmental contaminants.
The most obvious structural feature of these slug adult
tissues is the high degree of structures with terminal 3-O-
methylated mannose residues (>80% in skin and  50%
in viscera) and traces of structures with 3-O-methylated
galactoses (Table 1). Methylated sugars were first described
in the early 1970s in the polysaccharides of procaryotes,
lower eucaryotes, algae and fungi with soil habitat. In
gastropod hemocyanin 3-O-methylated mannose and
3-O-methylated galactose were found in 1977 [31]. Since
that time a number of methylated sugars have been found in
polysaccharides from plants and procaryotes. In molluscs
3-O-methylated mannose and/or 3-O-methylated galactose
were found in some hemocyanins [32], 6-O-methylation of
mannose was found in the giant clam Hippopus hippoppus
[33] and 3-O-methyl galactose and 3-O-methyl GlcNAc in
Rapana venosa [4]. In nematodes 2-O-methylated fucose was
found in Toxocara [34] and Caenorhabditis elegans [35].

removes the GlcNAc residue from the Mana1,3-antenna
after fucosylation and xylosylation; such an enzyme has
already been described in insects and C. elegans [27,28].
Due to the small size of the glycans, heterogeneity is
mainly caused by modification of the core. A remarkable
amount of xylose linked b1,2 to the b-mannose and/or
fucosylation of the reducing GlcNAc, was detected. Mainly
a1,6-linked fucose was observed. a1,3-linked fucose, like
that typical for plants, occurred only in trace amounts. A
corresponding a1,3-fucosyltransferase has been detected in
Lymnaea stagnalis [15], but here it is the first time that one of
its products has been found in a snail. It can be speculated
that this structural feature is limited to some very specialized
cells and does not occur randomly in the organism.
There was no evidence for the presence of difucosylation
of the inner GlcNAc-residue found in lepidopteran insects
[29] and squid rhodopsin [38], or of difucosylation in
combination with a core xylose as is present in Schistosoma
japonicum eggs [39]. Terminal fucosylation such as the a1,2-
fucosylation seen in another gastropodian source (Lymnaea
stagnalis)[3]orLe
X
determinants were also not found.
Arion lusitanicus contains an enormous potential for
generating a large set of structural elements commonly
found in eukaryotic N-glycosylation: they sialylate [40], they
carry a1,6-linked as well as a1,3-linked fucose as shown for
some insects, nematodes and trematodes, and b1,2-linked
xylose, as found in plants and trematodes, and they are able
to methylate terminal sugars (mannose and galactose) as

for support on the MALDI-TOF and Dr Iain Wilson for reading the
manuscript. The technical help of Thomas Dalik, Susanna Eglseer and
Denise Kerner is highly appreciated.
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