Isolation and Characterization of Rhamnose-binding Lectins from
Eggs of Steelhead Trout (Oncorhynchus mykiss) Homologous to
Low Density Lipoprotein Receptor Superfamily*
Hiroaki
Tateno
,
Ayako
Saneyoshi
,
Tomohisa
Ogawa
,
Koji
Muramoto
§,
Hisao
Kamiya¶, and
Mineo
Saneyoshi
From the
Department of Biological Resource Sciences,
Graduate School of Agriculture, Tohoku University, Sendai 981-8555, the ¶ School of Fisheries Sciences, Kitasato University, Sanriku,
Iwate 022-0101, and the
Department of Biological Sciences,
Teikyo University of Science and Technology, Uenohara,
Yamanashi 409-0193, Japan
 |
ABSTRACT |
Two L-rhamnose-binding lectins
named STL1 and STL2 were isolated from eggs of steelhead trout
(Oncorhynchus mykiss) by affinity chromatography and ion
exchange chromatography. The apparent molecular masses of purified STL1
and STL2 were estimated to be 84 and 68 kDa, respectively, by gel
filtration chromatography. Sodium dodecyl sulfate polyacrylamide gel
electrophoresis and matrix-assisted laser desorption ionization time of
flight mass spectrometry of these lectins revealed that STL1 was
composed of noncovalently linked trimer of 31.4-kDa subunits, and STL2
was noncovalently linked trimer of 21.5-kDa subunits. The minimum
concentrations of STL1, a major component, and STL2, a minor component,
needed to agglutinate rabbit erythrocytes were 9 and 0.2 µg/ml,
respectively. The most effective saccharide in the hemagglutination
inhibition assay for both STL1 and STL2 was L-rhamnose.
Saccharides possessing the same configuration of hydroxyl groups at C2
and C4 as that in L-rhamnose, such as
L-arabinose and D-galactose, also inhibited. The amino acid sequence of STL2 was determined by analysis of peptides
generated by digestion of the S-carboxamidomethylated protein with Achromobacter protease I or
Staphylococcus aureus V8 protease. The STL2 subunit of 195 amino acid residues proved to have a unique polypeptide architecture;
that is, it was composed of two tandemly repeated homologous domains
(STL2-N and STL2-C) with 52% internal homology. These two domains
showed a sequence homology to the subunit (105 amino acid residues) of
D-galactoside-specific sea urchin (Anthocidaris
crassispina) egg lectin (37% for STL2-N and 46% for STL2-C,
respectively). The N terminus of the STL1 subunit was blocked with an
acetyl group. However, a partial amino acid sequence of the subunit
showed a sequence similarity to STL2. Moreover, STL2 also showed a
sequence homology to the ligand binding domain of the vitellogenin
receptor. We have also employed surface plasmon resonance biosensor
methodology to investigate the interactions between STL2 and major egg
yolk proteins from steelhead trout, lipovitellin, and
'-component, which are known as vitellogenin digests. Interestingly,
STL2 showed distinct interactions with both egg yolk proteins. The
estimated values for the affinity constant (Ka) of
STL2 to lipovitellin and
' component were 3.44 × 106 and 4.99 × 106, respectively. These
results suggest that the fish egg lectins belong to a new family of
animal lectin structurally related to the low density lipoprotein
receptor super- family.
 |
INTRODUCTION |
Lectins are a group of sugar-binding proteins that recognize
specific carbohydrate structures and agglutinate a variety of animal
cells by binding to cell-surface glycoproteins and glycolipids. The
present state of knowledge permits us to organize the known animal
lectins into several categories depending on sequence similarity and
common characteristics such as sugar binding specificity, conserved
carbohydrate recognition domains, and ion requirements, i.e.
C-type, I-type, galectins, pentraxins, and P-type lectin (1-4).
Although a number of egg lectins have been isolated from various fish
families such as Salmonidae (5-9), Clupeidae
(10), and Cyprinidae (11), they have not been classified to
any animal lectin family. The fish egg lectins are characterized by
their binding affinity to L-rhamnose, with a few exceptions
(8, 11). In other species, L-rhamnose-binding lectins have
been found only in American cockroach (Periplaneta
americana) (12) and Streptomyces 27S5 (13).
The physiological significance of the existence and the structure of
the L-rhamnose-binding proteins in fish eggs have not been
clarified yet. However, they may be involved in a variety of biological
functions, including regulation of carbohydrate metabolism (14),
prevention of polyspermy (15, 16), cross-linking of carbohydrate-rich
proteins of the fertilization envelope (17), bactericidal effects (6,
18), mitogenesis (19), lectin-mediated cellular cytotoxicity (20), and
opsonization of pathogens (7).
In the present paper, we report the isolation and characterization of
L-rhamnose-binding lectins from the egg of steelhead trout
(Oncorhynchus mykiss) and the complete amino acid sequence of the lectin, STL2. Surprisingly, its tandemly repeated domains are
homologous to the ligand-binding domain of the vitellogenin receptor,
which belong to the low density lipoprotein
(LDL)1 receptor superfamily.
Furthermore, the interaction of STL2 with two major egg york proteins,
lipovitellin (Lv) and
' component, which derived from vitellogenin,
was demonstrated by a biosensor based on surface plasmon resonance
(21). Based on their structural properties and kinetic measurements, we
discuss the biological functions of the L-rhamnose-specific
lectins from fish eggs.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The eggs were obtained from mature steelhead
trout (5~6 years old, 2~3 kg weight) cultured in Tomakomai
Experimental Forest Station, Hokkaido University, Hokkaido, Japan.
Achromobacter protease I, Staphylococcus aureus
V8 protease, and
-L-rhamnose monohydrate were purchased
from Wako Pure Chemical Ind. (Osaka, Japan), Epoxy-activated Sepharose
6B, Superdex 200, and HiTrap Q column were purchased from Amersham
Pharmacia Biotech. An L-rhamnose-Sepharose 6B gel was
prepared according to the directions by Amersham. All other reagents
were the purest grade commercially available.
Isolation of Steelhead Trout Egg Lectins--
Eggs (5 kg) was
homogenized and defatted with acetone and filtered through filter
paper, and the filtrate was dried at room temperature to yield 1.7 kg
of the acetone powder. The acetone powder (50 g) was homogenized with
300 ml of ice-cold 0.15 M NaCl containing 0.1 mM phenylmethanesulfonyl fluoride (Sigma) and centrifuged at 15,000 × g for 20 min at 4 °C. The supernatant
was mixed with L-rhamnose-Sepharose 6B gel (~50 ml), and
the suspension was incubated at 4 °C for 10 h with gentle
shaking. Unabsorbed substances were removed by washing the gel with 50 mM sodium acetate buffer (pH 5.5) containing 0.15 M NaCl (ABS buffer). The gel was packed in a column
(3.0 × 10 cm) and washed with ABS, and then the adsorbed substance was eluted with 0.2 M L-rhamnose in
ABS. The fractions with significant absorption at 280 nm were collected
and dialyzed against 20 mM Tris-HCl buffer (pH 7.5). The
lectin fraction was further purified by anion exchange chromatography
on a HiTrap Q column (5 ml, Amersham) pre-equilibrated with 20 mM Tris-HCl buffer (pH 9.0) and eluted with a linear
gradient of 0 to 1 M NaCl in the same buffer. Each peak was
collected, dialyzed against distilled water thoroughly, and then
lyophilized. During the purification steps, protein concentration was
determined by the Bradford dye binding assay (22) using bovine serum
albumin as a standard.
Hemagglutination Assay and Inhibition Assay--
Samples were
diluted with 50 µl of 0.15 M NaCl on microtiter plates
and mixed with 50 µl of 4% rabbit erythrocyte suspension. The
mixture was allowed to stand at room temperature for 30 min, and then
the hemagglutination activity was measured. The hemagglutination activity was defined as the titer value of maximum dilution with positive agglutination of 2% rabbit erythrocytes.
The inhibitory effects of saccharides on hemagglutination were assayed
as follows. The saccharide solutions (25 µl) tested in this study
were diluted 2-fold in series on microtiter plates and incubated with
25 µl of the lectin solution having hemagglutination titer values of
23 for 15 min. The rabbit erythrocytes suspension (4%, 50 µl) was added to the mixture and incubated for 30 min. The inhibitory activities were estimated by the minimum concentration of sugar needed
to cause negative hemagglutination.
The thermostability of the lectins was examined by the hemagglutination
assay described above after incubating for various periods at 40, 50, 60, or 70 °C. To test the dependence of divalent cations on
hemagglutination, the lectins were treated in 0.1 M EDTA
for 15 min at room temperature and dialyzed against 0.15 M
NaCl at 4 °C overnight. The lectin solution was tested for
hemagglutination activity in the absence or presence of
Ca2+ or Mg2+ ions in 0.15 M
NaCl.
Molecular Weight Determination of Proteins and Proteolytic
Products--
The molecular weights of STLs and their subunits were
determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), gel filtration and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (Kompact MALDI
I, Shimadzu, Kyoto, Japan). SDS-PAGE was performed according to the
method of Laemmli (49) using 15% separating gel in the presence or
absence of 2-mercaptoethanol, and protein bands were stained by
Coomassie Brilliant Blue R-250. Gel filtration was performed on a
Superdex 200 column (1 × 30 cm) eluted with 20 mM
Tris-HCl buffer (pH 7.5) containing 0.15 M NaCl and 10 mM L-rhamnose.
For MALDI-TOF mass spectrometry, proteins were embedded in a sinapinic
acid (3, 5-dimethoxy-4-hydroxycinnamic acid) matrix, which absorbs
UV-light, for analysis. Desorption and ionization of the mixed sample
and matrix were induced by a nitrogen laser at 337 nm with a pulse
width of 3 ns. 100 single-shot spectra were averaged to improve the
signal-to-noise ratio. A second measurement was performed with insulin
(Mr 5,734.5) (bovine pancreas, Sigma) as an
internal standard. Thus, it was possible to determine the molecular
weight of the samples with an accuracy of 0.1%.
Preparation of S-Carboxamidomethylated (CAM)-STL2--
STL2 was
reduced with 10 mM dithiothreitol in 0.25 M
Tris-HCl buffer (pH 8.6) containing 10 mM EDTA and 6 M guanidine hydrochloride at 37 °C for 2 h and
reacted with 20 mM iodoacetamide for 30 min at room
temperature with shielding from light to convert freshly generated
cysteine residues into S-carboxamidomethyl cysteine. Excess
reagent was removed by gel filtration on a HiTrap desalting column (5 ml, Amersham) pre-equilibrated with 0.1 M
NH4HCO3 (pH 8.0).
Enzymatic Digestion and Separation of Peptides--
CAM-STL2 (50 nmol) was dissolved in 1 ml of 50 mM Tris-HCl (pH 9.0)
containing 1 M urea and digested with
Achromobacter protease I (S/E = 100:1) at 37 °C for
16 h. CAM-STL2 (50 nmol) was also digested with S. aureus V8 protease (S/E = 50:1) in 1 ml of 50 mM
sodium phosphate buffer (pH 7.8) in the presence of 1 M
urea at 37 °C for 12 h. Each digest was separated by
reversed-phase HPLC on a TSKgel ODS 120T column (5 µm, inner diameter
4.6 mm × 250 mm) (Tosoh, Tokyo, Japan) using a gradient of
acetonitrile in 0.1% trifluoroacetic acid.
Amino Acid Sequence Analysis--
Amino acid compositions were
determined by the precolumn labeling method (23, 24) using
4-dimethylaminoazobenzene-4'-sulfonyl chloride (Pierce).
The amino acid sequences of purified proteins and proteolytic peptides
were determined by a gas-phase protein sequencer (Shimadzu PSQ-1).
Molecular mass determination of the peptides was performed by using
MALDI-TOF mass spectrometry as described above. Homologous sequences
were searched by FASTA program (25) accessed by Genome Net
WWW2 . Hydropathy profile analysis and the
secondary structure prediction of STL2 were performed on the basis of
the primary structure according to the methods of Kyte and Doolittle
(26) and Chou and Fasman (27), respectively.
Surface Plasmon Resonance Analysis of the Interaction between
STL2 and Egg York Proteins--
BIAcoreTM (Amersham),
which is based on surface plasmon resonance, was used to determine the
interaction between STL2 and egg yolk proteins from steelhead
trout.
Two major egg yolk, Lv and
' component, were isolated according to
the procedure of Hara and Hirai (28). Purified Lv and
' component
were diluted to a concentration of 10 and 50 µg/ml, respectively, in
10 mM sodium acetate buffer (pH 5.0 and 4.0, respectively)
and immobilized to the carboxymethylated dextran-modified gold surface
of a CM5 sensor chip via primary amino groups using carbodiimide
chemistry as described in the manufacture's manual. Briefly, the
carboxyl groups on CM5 were activated by 10 mM
N-hydroxysuccinimide and 400 mM
N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide
followed by the addition of Lv or
' component. The remaining
activated groups were blocked by ethanolamine. Employing these
condition, the immobilization of Lv and
' component resulted in
covalent linking of 3300 and 500 resonance units, respectively. For
most proteins, 1000 resonance units corresponds to a surface
concentration of about 1 ng/mm2. The sensor chip surface
was regenerated with 10 mM HCl (40 µl) at the end of each
measurement.
STL2 was dissolved in HBS (0.01 M HEPES buffer (pH 7.4)
containing 0.15 M NaCl, 3 mM EDTA, and 0.005%
(v/v) surfactant P-20) at the concentration of 12.5 µg/ml, and 2-16
serial solutions were injected over the Lv- or
'
component-immobilized surfaces and over unmodified sensor chip CM5 as a
control at a flow rate of 20 µl/min. The effects of
L-rhamnose to the bindings between STL2 and egg yolk
proteins were also analyzed by adding 0.2 M L-rhamnose in STL2 solution. Sensorgrams were analyzed by
nonlinear least squares curve fitting using BIAevaluation software.
A single-site binding model was adopted for analysis of interactions
between STL2 and immobilized Lv and
' component.
 |
RESULTS |
Isolation and Molecular Characterization of STLs--
A fraction
containing lectin was obtained from the acetone powder of the steelhead
trout eggs by means of affinity chromatography on
L-rhamnose-Sepharose 6B (Fig.
1). The fraction obtained from affinity
chromatography gave two peaks on a HiTrap Q anion exchange chromatography (Fig. 2). Since both peaks
contained protein showing strong agglutination activity on rabbit
erythrocytes and gave a single protein band on SDS-PAGE (Fig.
3), proteins isolated from the first and
second peaks were designated as STL1 and STL2, respectively. Table
I summarizes the purification results of STLs. The yields of STL1 and STL2 from 50 g of saline extract of
eggs were 170 and 120 µg, respectively. The low recovery of lectins
from affinity chromatography was mainly due to the precipitation during
dialysis before ion exchange chromatography. Purified STL2 showed more
potent hemagglutinating activity (0.2 µg/ml) than did STL1 (9 µg/ml).

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Fig. 1.
Purification of steelhead trout egg lectins
by affinity chromatography on a L-rhamnose-Sepharose 6B
column. The fish egg lectins were adsorbed on 50 ml of
L-rhamnose-Sepharose 6B gel for 10 h at 4 °C
batchwise, and then the gel was placed in a glass column (3 × 10 cm). STLs were eluted with 0.2 M L-rhamnose in
50 mM sodium acetate buffer (pH 5.5) containing 0.15 M NaCl. Lectin fractions indicated by a bar were
combined and further purified.
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Fig. 2.
Purification of STLs by anion exchange
chromatography on a HiTrap Q column. STLs obtained from affinity
chromatography were further purified by anion exchange chromatography
on a HiTrap Q column (5 ml) equilibrated with 20 mM
Tris-HCl buffer (pH 9.0) and eluted with a linear gradient of 0 to 1 M NaCl in the same buffer. AUFS, absorbance
units at full scale.
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Fig. 3.
SDS-PAGE of STL1 and STL2 before and after
reduction with 2-mercaptoethanol. Lanes 1 and 3 are STL1 and lanes 2 and 4 are STL2,
respectively. Lanes 3 and 4 are reduced with
2-mercaptoethanol. Bovine albumin (66,000), ovalbumin (45,000),
carbonic anhydrase (31,000), and soybean trypsin inhibitor (21,500)
were used as standard markers.
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Gel filtration on Superdex 200 showed that the approximate molecular
masses of STL1 and STL2 were 84 and 68 kDa, respectively (data not
shown). Each STL gave single bands at 28 kDa (STL1) and 22 kDa (STL2)
on SDS-PAGE in the absence of 2-mercaptoethanol and at 35 kDa (STL1)
and 28 kDa (STL2) in the presence of 2-mercaptoethanol (Fig. 3),
suggesting that STL1 and STL2 possess internal disulfide bonds.
Furthermore, the MALDI-TOF mass spectrometry of STL1 gave a predominant
peak at 31.4 kDa and minor peaks at 63 and 94 kDa, and STL2 gave a
predominant peak at 21.5 kDa and minor peaks at 43 and 64.5 kDa (Fig.
4). These results indicate that STL1 is a
noncovalently linked trimer consisting of the 31.4-kDa subunits and
that STL2 is a noncovalently linked trimer consisting of 21.5-kDa subunits. The isoelectric points of STL1 and STL2 were measured to be
6.75 and 4.70, respectively, by isoelectric focusing gel electrophoresis (data not shown).

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Fig. 4.
Thermostability of STL1 and STL2.
Hemagglutinating activities were measured after preincubation of STLs
in 20 mM Tris-HCl (pH 7.0) containing 0.15 M
NaCl for 0 to 120 min at different temperatures. , 40 °C; ,
50 °C; , 60 °C; , 70 °C.
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STL1 completely lost its hemagglutinating activity by heating at
50 °C for 90 min, whereas STL2 retained half of the activity under
the same conditions (Fig. 5). However,
STL2 was inactivated by heating at 70 °C for 10 min. STLs maintained
their hemagglutination activities between pH 4 and 7 (data not shown).
No appreciable change was seen in the hemagglutinating activities of
STL 1 and STL2 after treatment with 0.1 M EDTA, whereas no
enhanced activity was observed by the addition of Ca2+ ion
or Mg2+ ion (data not shown).

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Fig. 5.
Determination of the molecular masses of STLs
by matrix-assisted laser desorption/ionization time of flight mass
spectrometry. Int., intensity.
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Sugar Specificity--
In the hemagglutination inhibition assay
using rabbit erythrocytes, L-rhamnose was the most potent
monosaccharide inhibitor for both STL1 and STL2 (Table
II). Melibiose, L-arabinose.
and D-galactose, which possess the same hydroxyl group
orientation at C2 and C4 of the pyranose ring structure of
L-rhamnose, also showed inhibitory effects. On the other
hand, other monosaccharides tested (Table II) showed no inhibitory
activity even at concentrations of 0.2 M. Glycoproteins
such as mucine type 1, asialomucine type 1, and fetuin also did not
show any inhibition at concentrations up to 0.1%.
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Table II
Inhibition of hemagglutination activity of STLs by saccharides
2% intact rabbit erythrocytes and 8 hemagglutinating units of STLs
were used in each well.
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Amino Acid Sequence Analysis--
The N-terminal amino acid
sequence of CAM-STL2 identified the first 38 residues including three
CAM-cysteine. The enzymatic digest of CAM-STL2 (50 nmol) with
Achromobacter protease I and S. aureus V8
protease was separated by reversed-phase HPLC as shown in Fig.
6. Each generated peptide, designated
L1~L8 for Achromobacter protease I and V1~V9 for
S. aureus V8 protease, was isolated and subjected to amino
acid analysis and MALDI-TOF mass spectrometry (Table
III). The N-terminal sequence of L8 was identical to that of native protein (Fig. 6). The successive amino acid
sequences of L1~L6, V1~V7, and V9 were determined up to their C
terminus by a gas phase protein sequencer (Fig. 6). L5 was a part of
L8. The peptides necessary for overlaps were obtained from digestion of
CAM-STL2 with V8 protease. The sequences of peptides V3, V9, and V7
established the connections between peptides L8/L5 and L6, between L6
and L7, and between L4 and L3, respectively. The sequence determination
of V8 established the connections between peptides L7 and L2 and
between L2 and L1. Thus, the complete amino acid sequence of CAM-STL2
was determined as shown at the top of Fig.
7. STL2 was composed of 195 amino acid
residues with a molecular mass calculated to be 21,349 Da, which is in
good agreement with the value (21,500 Da) obtained from MALDI-TOF mass
spectrometry. STL2 contained 16 half-Cys and 22 Thr residues. On the
other hand, no His, Met, or Trp residues were present. Sugar analysis
of CAM-STL2 showed no evidence for the existence of sugar chains in the
molecule as predicted from the absence of a potential
N-linked glycosylation site (Asn-Xaa-Ser/Thr).
The hydropathy profile showed that STL2 is rather hydrophilic protein
except for a hydrophobic segment existing in the N-terminal region
(Fig. 8). STL2 was predicted to form
-structures all over the molecule (data not shown). Amino acid
sequence analysis revealed a unique polypeptide architecture of STL2;
it was composed of tandemly repeated homologous domains, each of which
consisted of 99 and 96 amino acid residues (Fig. 9). Fifty-two percent of amino acids were
identical between the N-terminal domain (STL2-N) and the C-terminal
domain (STL2-C). All half-Cys residues were conserved between two
tandem repeats. The hydrophobicity profiles were also similar to each
other (Fig. 8).

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Fig. 6.
Separation of peptides generated by digestion
of the CAM-STL2 with Achromobacter protease I (A) and
S. aureus V8 protease (B). Peptides were separated by
reversed-phase HPLC on a TSKgel ODS 120T column (5 µm, inner diameter
4.6 mm × 250 mm) using a gradient of acetonitrile in 0.1%
trifluoroacetic acid. The flow rate was 1 ml/min. AUFS,
absorbance units at full scale.
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Table III
Amino acid compositions of STL2 and peptides used for sequence analysis
Values are expressed as residues/molecule by analysis or, in
parentheses, from the sequence in FIG. 7.
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Fig. 7.
Detailed summary of sequence determination of
STL2. The proven sequences of specific peptides are given in
one-letter code (Table III) below the summary sequence. L
and V demonstrate the peptides generated by cleavage of the
CAM protein with Achromobacter protease I and S. aureus V8 protease, respectively. Peptides sequences in
uppercase letters were proven by Edman degradation, and
those in lowercase letters indicate tentative identification
or that deduced from amino acid compositions and MALDI-TOF mass
spectrometry.
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Fig. 9.
Comparison of amino acid sequences of STL2
and other egg lectins. Two repeated domains of STL2 (STL2-N and
STL2-C) are separately aligned for comparison. Amino acid residues
identical to those of both STL2-N and STL2-C are indicated by
boxes. Hatched box shows the conserved
half-cystine residues. SUEL, sea urchin (A. crassispina) egg lectin; Coho salmon, coho salmon
(Oncorhynchus kisutch) egg lectin; SAL, cat fish
(Silurus asotus) egg lectin; OLL, shishamo smelt
(Osmerus lanceolatus) roe lectin; OML1~4, olive
rainbow smelt (Osmerus eperlanus mordax) roe lectins.
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To explore the sequence homology between STL1 and STL2, STL1 was
subjected to a gas-phase protein sequencer after
S-carboxamidomethylation. Initial attempts at amino acid
sequencing revealed that the N terminus of STL1 is blocked. Therefore,
CAM-STL1 was unblocked by treating with trifluoroacetic acid according
to the method of Gheorghe et al. (29) to remove a possible
N-acetyl group, repurified, and sequenced successfully
through the first 34 residues. The N-acetyl group of STL1
was confirmed by MALDI-TOF mass spectrometry of the N-terminal peptide
of CAM-STL1, prepared by digesting with Achromobacter
protease I (data not shown).
Interaction of STL2 with Immobilized Egg Yolk
Proteins--
Specific dose-dependent sensorgrams of STL2
were observed in both cases of the Lv- and
' component-immobilized
surfaces. The representative sensorgrams (7.8-62.5 µg/ml) are shown
in Fig. 10. The binding parameters were
estimated by nonlinear least squares curve fitting on the bases of the
subunit monomer (21.5 kDa). The association rate constant
(kass) of STL2 with Lv and
' component were
1.10 × 104 M
1
s
1 and 8.04 × 103
M
1 s
1, respectively. The
affinity constants, Ka, for the STL2-Lv and
STL2-
' component interactions were calculated to be 4.99 × 106 M and 3.44 × 106
M, respectively. These interactions were completely
inhibited by 0.2 M L-rhamnose (Fig.
11). When 0.2 M glucose was
co-injected with STL2, however, no significant decrease in surface
plasmon resonance was observed (data not shown). These results
suggested that the interactions between STL2 and egg yolk proteins, Lv
and
' component, were L-rhamnose-specific.

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Fig. 10.
Sensorgrams showing the interactions of STL2
with immobilized Lv (A) and ' component (B). STL2 (50 µl) at
concentrations ranging from 7.8 to 62.5 µg/ml in HBS was injected
onto sensor chip.
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Fig. 11.
Sensorgrams showing the inhibitory effect of
L-rhamnose on the interactions of STL2 with immobilized Lv
(A) and ' component (B). STL2 was injected onto the sensor chip
at the concentration of 62.5 µg/ml in the presence or absence of 0.2 M L-rhamnose.
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 |
DISCUSSION |
Two L-rhamnose-binding lectins, STL1 and STL2, were
isolated from eggs of steelhead trout. The complete amino acid sequence of STL2 has been determined (Fig. 7). STL2 was found to consist of 195 amino acid residues consisting of two tandem repeat domains with 52%
amino acid homology (Fig. 9). These domains (STL2-N and STL2-C) showed
a significant sequence homology to the
D-galactoside-specific lectin (SUEL) isolated from the sea
urchin (Anthocidaris crassispina) egg (30) with 37 and 46%
amino acid identity, respectively. Therefore, STL2 is most probably
composed of the subunit having tandemly repeated functional domains.
Such structures are considered to be important for increasing the
specificity and maintaining both multivalency and hetero
bifunctionality and in fact, are frequently observed in a number of
lectins; e.g. the macrophage mannose receptor of the C-type
lectin family (31) and tandem-repeat type of galectins (32-37).
Fish egg lectins have been found in more than 25 fish species, and the
N-terminal amino acid sequences of some lectins have been reported
(38-40). Fig. 9 shows the alignment of the N-terminal sequences of
fish egg lectins including STL1 with the amino acid sequences of the
two tandem domains of STL2 and SUEL. The N-terminal sequence homologies
among the fish egg lectins were not so high, with only 21 to 46%
identities. However, the segment Tyr-Gly-Arg is conserved throughout
the lectins. Fish egg lectins have very characteristic properties
compared with other animal lectins, because most of them have specific
binding affinity to L-rhamnose. Some exceptions include
D-glucose/L-fucose-binding lectins from Perca fluviatilis and Dicentrarchus labrax and
lectin from Petromyzon marinus, which is specific to
sialoglycoconjugate (11). Furthermore, it has been reported that fish
eggs or serum contains multiple L-rhamnose-binding
isolectins that widely vary in molecular weight and other properties
(38, 39). In the present study, it was found that steelhead trout eggs
contain two distinct isolectins with subunits of different molecular
sizes, different hemagglutinating activities, and different binding
specificities for some saccharides (Table II). STL2 showed greater
affinity for melibiose than did STL1, whereas STL1 showed greater
affinity for L-arabinose than did STL2. In addition, STL1
was blocked by an N-acetyl residue. These observations
suggest that fish eggs possess multiple isolectins with diverse
molecular properties.
Further studies are needed to determine the physiological significance
of multiple lectins as well as their physiological function. The
hemagglutination activity of STLs was most effectively inhibited by
L-rhamnose and weakly inhibited by D-galactose
and L-arabinose, which have the same hydroxyl group
orientation at C2 and C4 as L-rhamnose. On the other hand,
SUEL is exclusively specific to D-galactose. Therefore,
valuable information concerning the relationship between the structure
and sugar binding specificity must be obtained by investigating the
carbohydrate recognition domains of STLs and SUEL. Since
L-rhamnose is not a naturally occurring sugar in
vertebrates, the physiological significance of the
L-rhamnose-binding lectin is puzzling. However, the ligands for L-rhamnose-binding lectins in animals are not necessary
to have a L-rhamnose moiety in the molecules if their
binding activities to unknown glycoconjugates are taken into account.
One of the physiological roles of fish egg lectins is as a defense
mechanism against pathogenic bacteria. The fish egg lectins from
Oncorhynchus tschawytscha and Rutilus rutilus
showed antibacterial activity (6, 8), those from Oncorhynchus
rhodurus showed an opsonic effect (7), and those from
Oncorhynchus keta showed bacterial agglutination activity
(5). We have not obtained any evidence that STLs play a role in defense
mechanisms, as STLs did not show an antibacterial activity against
Escherichia coli or Bacillus subtilis (data not
shown).
A new family of animal lectins homologous to STL2 are present not only
in fish but also in other animals such as sea urchin. SUEL has been
suggested to be involved in the fertilization and development of the
embryo (41). SUEL was first accumulated in the cortical cytoplasm of
embryos and then incorporated into the extracellular hyaline layer at
least until the mid gastrula stage. For fish egg lectins, their
involvement in the fertilization and development of the embryo has not
been demonstrated yet. An immunofluorescence study showed that some
fish egg lectins were associated exclusively with the content and
surrounding membrane of cortical vesicles situated within the cytoplasm
of maturing oocytes (16). We observed that the hemagglutinating
activity of chum salmon egg homogenate varied with the stages of
development (5). The activity was reduced markedly after the eyed stage
and disappeared just before hatching.
It should be noted that STL2 showed sequence similarity to the ligand
binding domains of the mammalian LDL receptor and very low density
lipoprotein (VLDL) receptor (Fig. 12).
The LDL receptor superfamily has a ligand binding domain composed of
cysteine-rich repeats, epidermal growth factor precursor-type repeats,
an O-linked sugar domain, a transmembrane domain, and a
cytoplasmic domain (42). Although the sequence homology is less than
30%, it is apparent that the sequence of STL2 can be matched to the
region between repeat 3 and repeat 6 in the ligand binding domain of the LDL receptor (Fig. 12). Recently, it was discovered that the receptor for yolk lipoprotein, VLDL, and vitellogenin (VTG) in chicks
belongs to the LDL receptor superfamily (43, 44). VTG is a
lipophosphoglycoprotein that is produced under female hormonal control
in liver and is transported in the circulation to the female gonads as
a precursor of egg proteins. Recently, it was reported that the VTG in
fish is immediately processed into the Lv,
' component, and
phosvitin by cathepsin D like protease in eggs (45). The present study
showed that STL2 interacted to the major egg yolk proteins, Lv and
'
component, with Ka of 106 M
levels. Although the VTG receptor has been isolated from the egg of
rainbow trout (46, 47), we do not know the relationship between the VTG
receptor and STL2 because of the lack of sequence information of the
fish VTG receptor. However, it is not probable that STL2 is derived
from the VTG receptor, since STL does not have the consensus sequences
for the modular repeats characteristic of LDL receptor superfamily
members as revealed with the mosquito VTG receptor (48). In mammals,
VTG receptors localized in coated pits on the surface of
growth-component oocytes are able to accumulate the VTG and other
ligands in the yolk with high concentrations (42). Nosek et
al. (16) confirmed that fish egg lectins are associated with the
cortical vesicles of maturing oocytes in immunofluorescence studies.
The most attractive hypothesis regarding the biological functions of
fish egg lectins is that they could decode the carbohydrate moieties of
glycoproteins and shuttle them into the proper compartment (14). Our
observations that STL2 interacts with egg yolk proteins supports
the hypothesis.

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|
Fig. 12.
Comparison of amino acid sequences of STL2
and mammalian VLDL receptors. Partial amino acid sequences of
mammalian VLDL and VTG/LDL receptors corresponding to ligand binding
domain (Glu-103~Pro-274, repeat 3~6) are aligned. Boxes
show identical residues, and the stippled box shows the
conserved half-cystine residues.
|
|
 |
FOOTNOTES |
*
This work was supported in part by Grants-in-aid for
Scientific Research 08306011 from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Biological
Resource Sciences, Graduate School of Agriculture, Tohoku University,
Sendai 981-8555, Japan. Tel. and Fax.: 81-22-717-8807; E-mail:
muramoto{at}biochem.tohoku.ac.jp.
1
The abbreviations used are: LDL, low density
lipoprotein; CAM, S-carboxamidomethyl; Lv, lipovitellin;
MALDI-TOF, matrix-assisted laser desorption ionization time of flight;
PAGE, polyacrylamide gel electrophoresis; VLDL, very low density
lipoprotein; VTG, vitellogenin; HPLC, high performance liquid
chromatography; STLs, steelhead trout lectins; SUEL, sea urchin egg
lectin.
2
Internet address: http://www.genome.ad.jp.
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