Glycobiology vol. 7 no. 3 pp. 367-372, 1997
Cloning and expression of a Xenopus laevis oocyte lectin and characterization of its
mRNA levels during early development
Jin Kyu Lee 1 , Phillip Buckhaults1, Christopher Wilkes1,
Meredith Teilhet1, Mary Lou King, Kelley W.Moremen1,
and Michael Pierce1"2
Department of Cell Biology and Anatomy, University of Miami School of
Medicine, Miami, FL 33101, USA and 'Department of Biochemistry and
Molecular Biology and the Complex Carbohydrate Research Center,
University of Georgia, Athens, GA 30602, USA
^ o whom correspondence should be addressed
cDNA clones encoding a soluble, calcium-dependent, melibiose-binding lectin from Xenopus laevis oocytes have been
isolated, characterized, and expressed in bacteria. This lectin has been shown by others to be localized in oocyte cortical granules where it ultimately is released and participates in the formation of the fertilization envelope. A lectin
with similar specificity has been purified by others from
blastula and immunolocalized to specific locations in developing embryos, which suggests it may also function after
fertilization in regulating cell adhesion and migration. We
have used melibiose affinity chromatography to isolate the
oocyte lectin (monomer molecular masses of about 45 and
43 kDa) and shown that after exhaustive treatment with
JV-g]ycanase, only one major protein band at 35 kDa was
observed, suggesting that a single polypeptide with variable
TV-linked glycosylation is expressed in the oocyte. After obtaining internal peptide sequences, a PCR-based cloning
approach allowed the isolation of full length cDNAs from
an ovary Xgtll library encoding a protein of 313 amino
acids with three potential jV-linked oligosaccharide sites.
Although this lectin, termed XL35, requires calcium ions
for oligosaccharide binding, its sequence does not contain
the sequence motif defined for "C-type" lectins. A 6-Histagged form of the lectin was expressed in E.coli and purified on a Ni2+-NTA column from bacterial extracts. The
recombinant lectin was active using an agglutination assay,
and this activity was inhibitable by EDTA and melibiose,
properties exhibited by the native lectin. Southern blot
analysis revealed a single hybridizing band, arguing
against the existence of a multigene family. Northern blot
analysis demonstrated that the lectin mRNA is expressed in
oocytes and remains at relatively high levels through late
gastrulae, continuing until tadpole stages. The persistence
of the lectin mRNA, coupled with results from earlier studies, strongly suggests that XL35 is zygotically expressed
and functions during morphogenesis.
Key words: lectin/carbohydrate binding protein/fertilization/
morphogenesis
Introduction
Studies by two laboratories have demonstrated that Xenopus
laevis oocytes and embryos contain soluble, calcium© Oxford University Press
dependent, lectins which form multimers of about 500 kDa
(Roberson and Barondes, 1982; Nishihara et al., 1986). On
reducing SDS-PAGE gels these lectins migrate as overlapping
diffuse bands of about 45 and 43 kDa. (Roberson and Barondes, 1982; Outenreath et al, 1988). These proteins are contained within the cortical granules of the oocytes, as well as in
several other locations, and they are released from the cortical
granules at fertilization (Wyrick et al, 1974; Nishihara et al,
1986). Several lines of evidence suggest that the released multimeric lectins bind to oligosaccharide targets on glycoproteins
in the egg jelly coat where they participate in the formation of
the fertilization envelope which blocks sperm entry (Wyrick et
al., 1974; Roberson and Barondes, 1983; Monk and Hedrick,
1986). Proteins purified from the oocytes by melibiose affinity
chromatography agglutinate trypsinized rabbit erythrocytes in
the presence of Ca + , and this reaction is strongly inhibited by
a-galactosides such as melibiose, suggesting that the lectins
bind to the abundant glycolipids on these erythrocytes terminated by a-galactose residues (Clark et al., 1987).
After fertilization, lectins with very similar physical properties and ligand specificity have been purified from blastulae
(Roberson and Barondes, 1982). Moreover, a polyclonal antibody prepared against the oocyte lectin preparation showed
binding to cleavage furrows and to areas of active cell migration (Roberson and Barondes, 1983), including the blastopore
region and the roof of the blastocoel (Outenreath et al., 1988).
At these locations, it has been suggested that the lectins are
involved in cell adhesion and migration. In order to define in
detail the structure and function of the lectins during fertilization and early development, we have affinity-purified the oocyte lectins on immobilized melibiose (Roberson and Barondes, 1982; Roberson et al, 1985) and shown that after Nglycanase treatment, only one major polypeptide of 35 kDa
could be detected. We obtained peptide sequences from the
purified protein and used PCR techniques to isolate cDNA
clones which encode this polypeptide. Expression of the cDNA
in E.coli yielded lectin, which after purification demonstrated
specific agglutination activity. Southern and Northern blot
analyses demonstrated a single genomic sequence and expression of the lectin mRNA in the embryo up to at least the
tail-bud stage, respectively.
Results and discussion
Characterization of the melibiose-binding oocyte lectin
The lectin was purified by affinity chromatography on melibiose-sepharose (Roberson and Barondes, 1982), revealing a diffuse pair of bands at about 45 and 43 kDa after SDS-PAGE.
Exhaustive treatment of the preparation with N-glycanase,
which cleaves Asn-linked oligosaccharides, and subsequent
SDS-PAGE revealed a single major band at 35 kDa (Figure 1).
These results demonstrate that the oocyte lectin, termed XL35,
367
Fig. 1. SDS-PAGE analysis of the oocyte lectin. Lane A, affinity-purified
lectin; lane B, lectin after exhaustive digestion with N-glycanase. Molecular
weight standards are depicted at left.
is expressed as a single polypeptide and that the diffuse protein
bands observed after affinity purification differ primarily in
their Asn-linked oligosaccharide structures.
Isolation of X U 5 cDNAs
The protein preparation was blotted onto nitrocellulose and
subjected to trypsinization and peptide separation on reversephase HPLC. Several peptides were sequenced, and two of
them (Materials and methods) were used to design degenerate
PCR primers, as depicted in Figure 2. Two degenerate oligonucleotides, 29 and 26 basepairs in length, were synthesized
and utilized in an RT-PCR reaction with total ovary RNA. The
159 bp amplimer generated (Figure 2) was subcloned and sequenced. This amplirner was then used to screen a Xgtll
cDNA library prepared from Xenopus laevis ovary. The insert
from a partial clone was isolated as a 401 bp EcoRI restriction
fragment, subcloned, sequenced, and used as a probe for
Southern and Northern blot analyses. The inserts from the remaining five clones were PCR-amplified using either vector
primers flanking the cloning site or lectin-specific primers
flanking the coding region. The respective coding region amplimers were subsequently subcloned and sequenced. Each of
these clones contained a complete open reading frame, and the
consensus sequence is shown in Figure 2.
Expression of X U 5 cDNA in E.coli
The sequence encoding XL35 was modified by PCR to introduce a Sun restriction site at the predicted signal sequence
cleavage site, and cloned into the pQE-9 QIAexpress vector
such that when expressed in E.coli, the resulting recombinant
protein contained six histidine residues at the N-terminus (Materials and methods). E.coli transformed with this construct
were induced to express lectin with IPTG, and an expression
time course revealed the appearance of a protein with apparent
molecular mass of 35 kDa (data not shown). After harvesting,
the cells were disrupted in guanidinium hydrochloride, and the
extract was chromatographedon a N ~ ~ + - N Tcolumn,
A
to which
the 35 kDa protein bound. After exchanging the guanidinium
HCl buffer to one with 8 M urea, elution of the recombinant
lectin from the affinity column was accomplished by lowering
the pH to 4.5. These eluent fractions contained a single major
band at 35 m a , whose N-terminal 25 amino acid sequence
demonstrated it to be XL35. The purified recombinant lectin
was assayed for its ability to agglutinate trypsinized, fixed
rabbit erythrocytes. When all urea was removed from the solution containing the recombinant lectin, it slowly precipitated,
perhaps due to its lack of glycosylation. In 2 M urea, however,
as summarized in Table I, the recombinant lectin was active in
agglutinating the erythrocytes, similar to the activity observed
with the affinity-purified oocyte lecrin. Moreover, the recombinant XL35 agglutination activity was completely inhibited by
EDTA and by melibiose, but not by sucrose. These characteristics are similar to those observed for the affinity-purified
oocyte lectin (Table I) (Roberson and Barondes, 1982). The
purified oocyte lectin was also active in 2 M urea with the same
specificity of binding, but with significantly lower specific
activity. That the recombinant, nonglycosylated lectin shows
agglutination specificity similar to that observed for the oocyte
lectin demonstrates that the XL35 cDNA we have isolated and
expressed does indeed encode the oocyte lectin purified by
melibiose chromatography.
Southern and Northern blot analyses
The 401 bp cDNA probe (Figure 2) was used to probe Southern
and Northern blots. Analysis of the Southern blot shows that
the probe hybridized with one major band in five genomic
DNA restriction digestions (Figure 3), although small amounts
of incomplete digestion were observed with EcoRl and PstI.
These results argue against the existence of multiple, homologous genes in Xenopus
To examine the expression patterns of lectin mRNA at fertilization and during embryo development, Northern analysis
was performed on total RNA purified from Stage VI oocytes
and from embryos at various stages of development. Northern
analysis (Figure 4a) shows that relatively high levels of XL35
mRNA were present in the Stage VI oocytes and persisted
through gastrulation, after which it declined. Compared to the
levels of expression in gastrulae, very little XL35 mRNA was
present in hatching tadpoles. The same blot was then probed
with a cDNA encoding a fragment of mitochondrial rRNA in
order to normalize these results to the amount of RNA in each
lane of the blot (Figure 4b), and this normalization c o n f i i e d
the results shown in Figure 4a. Since it is highly unlikely that
maternal mRNAs have persisted until tadpole stages, together
with the observation of a slight increase of RNA levels at
gastrulation, it appears that XL35 mRNA is most likely newly
transcribed at the mid-blastula transition along with many other
zygotic RNAs. The fact that these RNAs are transcribed zygotically, as well as maternally, would strongly support the
hypothesis that XL35 displays multiple functions during fertilization and development, as suggested by the immunolocalization experiments.
Characterization of XU5 and possible functions
in morphogenesis
XL35 activity is assayed by agglutination of trypsinized, fixed
rabbit erythrocytes (Roberson and Barondes, 1982). This ag-
Xenopus oocyte lectin
GGCTTGGAACTTGGTACTAAGCTCCATGAAAG
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GCATTGTTTGGCAACTTTATTAAACCACAATCCTGTCTTGAGATAAGCCGAATTCCAGCACACTGGCG
Fig. 2. Nucleotide and amino acid sequences of XL35. The position in the lectin coding region that was used as a hybridization probe for Southern and
Northern blots is indicated by an underline. The positions in the coding region corresponding to the initial primers designed from peptide sequence are
denoted by a dotted underline. Asterisks below the peptide sequence indicate the three potential A'-glycosylation sites.
glutination activity requires calcium and can therefore be inhibited by EDTA. Many animal lectins have been shown to
require calcium ions for activity ("C-type" lectins) and a sequence motif for the calcium-binding site of these lectins has
been defined (Drickamer, 1993). Interestingly, XL35 does not
contain this sequence motif. The activity of recombinant XL35
does, however, demonstrate that the TV-linked oligosaccharides
on the native lectin are not required for ligand binding. The
precise structures of the glycoconjugates in the egg jelly to
which XL35 binds remain to be determined.
Experiments which examined the binding of polyclonal anTable L Agglutination properties of native andrecombinantXL35
Source
Agglutination activity*
No additions
EDTA"
Melibiose"
Sucrose"
Oocyte lectin
Recombinant
lectin
'Agglutination was assayed using trypsinized, fixed rabbit erythrocytes as
described in Materials and methods and included 2 M urea to retain
recombinant lectin solubility. The degree of agglutination was observed
under low power using an inverted microscope and scored as: n i l , total
agglutination of erythrocytes; +++, <10% of erythrocytes not agglutinated;
++, half of erythrocytes not agglutinated; +, some agglutination detectable;
—, no agglutination detectable.
""Final concentrations of additions: EDTA, 0.10 M; melibiose, 0.25 M;
sucrose, 0.25 M.
tibodies prepared against the oocyte lectin demonstrated that
populations of the lectin are localized in cortical granules, are
released at fertilization, and participate in formation of the
fertilization envelope to prevent polyspermy (Nishihara et al.,
1986). The conclusion that XL35 mRNA is expressed in developing embryos is consistent with the results of two previous
studies: one that demonstrated significant amounts of a lectin
with similar structure and specificity to the oocyte lectin in
blastulae (Roberson and Barondes, 1982), and another that
used a polyclonal antibody to the oocyte lectin to localize
cross-reacting material to the blastopore region and to extracellular locations on the roof of the blastocoel (Outenreath et
al, 1988). Taken together, these results suggest strongly that
the XL35 functions not only in formation of the fertilization
membrane, but also in cell adhesion and migration during early
morphogenesis. The nature and localized expression of the
glycoconjugate ligands in the embryo are unknown, although
there are recent reports of a blood group-B active determinant
expressed on glycoconjugates from Xenopus blastulae. These
determinants are expressed in areas of cell-cell contact, and it
seems very possible that XL35 or lectins similar to it could be
binding to these glycoconjugates and regulating intercellular
adhesion in the embryo (Nomura et al., 1995). Structural
analyses of the oligosaccharide ligands of the lectin, as well as
in situ hybridization and immunocytochemical experiments to
refine the locations of the lectin biosynthesis and secretion are
in progress to investigate this hypothesis.
Sequence similarity searches using the XL35 DNA and pep369
Fig. 3. Southern blot analysis of XL35. Twelve micrograms of genomic
DNA derived from Xenopus laevis ovaries was digested for 24 h with
BamH1, lane 1; EcoRI, lane 2; HindIII, lane 3; Psrl, lane 4; and XhoI, lane
5. The blot was then subjected to hybridization using the 401 bp lectin
restriction fragment (Materials and methods) as the radiolabeled probe.
tide sequences detected no similarity to any known published
sequences, including plant or animal lectins. A human EST
sequence was detected with 930% DNA sequence similarity to
the XL35 lectin. Using this EST sequence, we have recently
isolated cDNAs from several human and mouse tissues which
show similar sizes to XL35, as well as high degrees of identity
to the Xenopus lectin (60% at the amino acid level; J.-K. Lee,
K.W.Moremen, and M. Pierce, unpublishedobservations). These
results suggest that a family of lectins with similarity to XL35
exists in vertebrates, and that members of this family perform
physiological functions in adult organisms as well as in Xenopus oocytes and embryos.
Materials and methods
Materials
Restriction enzymes, Thennus aquaticus DNA polymerase, agarose, and other
chemicals were purchased from major chemical suppliers. Nitrocellulose fild m were purchased from Amersham.
ters, DNA labeling kits, and (a-32P]
Nick and N a g 5 desalting columns were purchased from Pharmacia. UltrafreeMC Low Binding Durapore Membranes were purchased from Millipore and
used for gel purification of DNA fragments. Zeta-Probe GT nylon membranes
for Southern analysis were purchased from Bio-Rad. N-Glycanase was from
Boehringer. BSA, IPTG, melibiose, and immobilized melibiose were from
Sigma. Sephaglas reagents were from Pharmacia. N~~'-NTAcolumns and
vectors used to construct the 6-His tag were from Qiagen and were used
according to the manufacturer's instructions.
Lecrrn punficofron and generotinn of PCR primers
The oocyte lectrns were punfied usrng a procedure previously described (Roberson and Barondes. 1982). followed by a C,-reverse phase HPLC step uslng
Fig. 4. (A) Northern blot analysis of total RNA (20 pg/lane) from oocyte or
embryos at various stages: lane I , stage VI oocytes; lane 2, cleavage stage
embryos; lane 3, blastula; lane 4, late gastrula; lane 5, late neurula; lane 6,
hatching embryos; lane 7, tadpole. The blot was hybridized with the 401 bp
lectin restriction fragment (Materials and methods) as the radiolabeled
probe. (B) Densitometer values obtained from the Northern blot were
normalized to the values obtained when the same blot was probed with a
mitochondrial rRNA fragment (Yost et al.. 1995).
acetonitrile-TFA buffers to desalt and concentrate the lectins. An aliquot of the
protein peak from the column was dried, and the sample was sent to the
Harvard Microchemistry Facility (Cambridge, MA), where it was then reduced
with dithiothreitol. alkylated with iodoacetamide, resuspended in urea. and
subjected to exhaustive tryptic digestion. The digested sample was chromatographed on a reverse phase HPLC column and two of the resolved peaks were
subjected to gas phase Edman degradation. The isolated peptides yielded the
following sequences: Peak 43-ESCNAEHVCIGGGGYWEADPR and Peak
Xenopus oocyte lectin
54-SQFTPGYIQFRPINTEK. Two degenerate oligonucleotides were designed
based on the peptide sequences: Peak 54 primer CARTTYAaCCIGGITAYATHCARTT, Peak 43 primer CICCDATRCAIACRTGYTCIGCRTTRCA.
Generation of a PCR product and cDNA library screening
Total RNA from Xenopus laevis ovary was isolated by the guanidinium
isothiocyanate method (Chirgwin et al., 1979), and 15 p.g was subjected to
reverse transcription using MMLV reverse transcriptase and oligo-d(T) primers as described in the Perkin Elmer RT-PCR kit The cDNA preparation was
subjected directly to PCR using 3 (iM of each degenerate primer. The diermal
cycling conditions were 95°C, 1 min; 55°C, 1 min; 72°C, 1 min, for a total of
30 cycles. The amplification products were resolved on a 3% agarose gel, and
a 159 bp PCR amplification product was purified and subcloned into the pCR
II vector using the TA Cloning kit (Invitrogen). The Xenopus laevis ovary
cDNA library in Xgtl 1, originally prepared by D. Melton's laboratory (Rebagliati et al., 1985) was screened by plaque hybridization using the cloned 159
bp PCR product as a radiolabeled probe. The 159 bp amplimer was excised
from pCR II by digestion with EcoRI and isolated using Sephaglas reagents
(Pharmacia). Twenty nanograms of DNA were labeled using [-y-32P] dCTP and
the Mega-Prime labeling kit (Amersham). Unincorporated nucleotides were
removed by desalting over a Sepharose G-50 column (Nick column, Pharmacia). Filter lifts were prepared using nylon membranes (HyBond, Amersham)
and UV crosslinked using a Stratalinker (Stratagene). Prehybridization was
carried out at 42°C in 50% formamide, 1 x Denhardts, 5 x SSC, 0.1% SDS and
100 ng/ml heat-denatured herring sperm DNA. Hybridization was performed
in the same solution with the addition of heat-denatured radiolabeled probe to
a final concentration of 1 x 106 cpm/ml. Positive phage clones identified by
plaque hybridization were purified through four rounds of plaque purification.
Lambda DNA from positive clones was isolated from 100 ml LB cultures
containing Y1090 host cell and 10 raM MgSO4 and 0.2% maltose by the
polyethylene glycol precipitation procedure followed by chromatography over
a Qiagen Tip-100 column as described by the manufacturer (Qiagen Corp.).
The insert from one of the phage clones was excised by digestion with EcoRI,
resolved on a 1% agarose gel, subcloned into pBluescript SK- (Stratagene),
and sequenced This product represented a 401 bp partial clone due to an
internal £coRI restriction site and was used as a hybridization probe for Southern and Northern analysis. The full length insert from one of the lambda clones
was isolated by PCR amplification using purified lambda DNA as a template,
and Xgtl 1 forward and reverse primers (New England BioLabs), and Taq
polymerase. The -1.2 kb amplification product was subcloned into the pCR II
vector, and the ends were sequenced. Exact match primers were then designed
against the 5' and 3' untranslated regions of the lectin cDNA sequence (5'
primer: GGAACTTGGTACTAAGCTCCATGAAAG; 3' primer: ATCTCAAGACAC-GATTGTGGTTTAATAAAG) and used to amplify the coding
regions from the remaining clones using me purified Xgtl 1 DNA preparations
as templates. The resulting amplimers were subcloned into pCR II and fully
sequenced.
Northern and Southern analyses
A 401 basepair probe (depicted in Figure 2) was generated and radiolabeled
using the Amersham megaprime DNA labeling system according to manufacturer's instructions. A fragment of mitochondria] DNA was also generated and
labelled for control hybridizations. All blots were probed using the same
protocol (Church and Gilbert, 1984) in which the prehybridization buffer consisted of 75 ml of 1 M phosphate buffer pH 7.0, 0.3 ml of 0.5 M EDTA pH 8.0,
52.5 ml of 20% SDS, 1.5 g of fatty acid free BSA, 1.5 ml of a 10 mg/ml
denatured herring sperm DNA, and 20.7 ml of water. The hybridization buffer
was identical, except for the addition of denatured radiolabeled probe. Prehybridization was performed for 2 h at 65°C and hybridization was performed
overnight at 65°C. The blots were washed three times at 65°C for 15 min in 50
ml of 40 mM phosphate buffer pH 7.0, 1% SDS, 1 mM EDTA, and 0.5%
fraction V BSA and an additional three times at 65°C for 15 min in the same
buffer excluding BSA. The blots were then washed in 50 ml of 1 M phosphate
buffer pH 7.0 at room temperature for 10 min and data collected using a
Molecular Dyiuunics phosphoimager.
ing these primers and the XL 1-2 cDNA clone as a template was cloned directly
into the QIAexpress Vector (pQE-9) (Qiagen Corp.) using Sall-Psil restriction
sites. This procedure appended the sequence, MRGS(His)6GS, to the Nterminus. The resulting plasmid was transfected into E.coli host strain
SB13OO9(pREP4). Recombinant protein production was induced with 1 mM
DTG. The purification of recombinant protein was performed by the method
described in the QIA express instructions. Briefly, the cells were harvested and
subjected to one freeze-thaw, after which 10 g of the pellet (wet weight) were
resuspended in 50 ml of 6 M guanidine hydrochloride, 0.1 M NaH2PO4, 0.01
M Tris, pH 8.0. After stirring for 1 h, the solution was centrifuged (10,000 x
g, 20 min, 4°Q and the clear supernatant loaded on a 4 ml Ni-NTA chromatography column which was equilibrated and eluted using the manufacturer's
instructions. The majority of the major protein band which migrated at 35 kDa
on SDS-PAGE bound to die column. After washing the column with 8 M urea,
0.1 M NaH2PO4, 0.01 M Tris, pH 8.0, the column was step-eluted with the
same buffer at pH 6.3, 5.9, and finally the 35 kDa protein band was eluted
using the same buffer at pH 4.5. The yield of purified recombinant protein was
about 250 mg/1 of culture after purification on the nickel column.
Digestion by N-glycanase
Lyophilized affinity purified oocyte lectin (6 jig) was brought to 100 p,l with
a solution of 0.1 M p-mercaptoethanol/0.1% SDS. The sample was heated at
100°C for 30 min, after which 25 uJ of 0.5 M Tris-HCl, pH 7.5, was added,
followed by 10 u.1 of 0.1 M phenanthroline, 10 (j.1 of 10% Triton X-100, and
3 |xl of N-glycanase (200 U/ml). The sample was then incubated for 18 hr at
35°C, after which an additional 3 (J.1 of enzyme was added and the incubation
continued for 4 hr. The protein was then precipitated by trichloroacetic acid
and resuspended in reducing sample buffer for SDS-PAGE. The control incubation was handled exactly as described, except no enzyme was added. SDSPAGE was performed using gradient 4-15% polyacrylamide gels (Bio-Rad),
which were stained with Coomassie blue.
Erythrocyte agglutination assay
The assay was performed essentially as described using trypsinized, glutaraldehyde-fixed rabbit erythrocytes (Barondes and Roberson, 1987) in TCS
buffer (10 mM Tris, pH 7.6, 10 mM CaCl2, 150 mM NaCl). To compare the
purified and recombinant lectins, two lectins were assayed under similar conditions. Because the eluted recombinant lectin was in 8 M urea, a solution of
purified nonrecombinant lectin was also brought to 8 M with a urea solution.
The agglutination assay was performed using 10 (xg of either recombinant or
native lectin in 25 u.1 TCS and 8 M urea was added to wells of a 96-well
micromer plate. Next, 25 uJ of TCS alone or TCS containing 0.4 M EDTA or
1.0 M melibiose or 1.0 M sucrose was added to the wells and mixed with the
lectin solution. To this solution, 50 uJ of a 1:1 suspension of trypsinized,
glutaraldehyde-fixed rabbit erythrocytes in TCS was added and the contents of
the wells were mixed by tnturation. The final concentration of urea in the wells
was 2 M, while that of EDTA was 0.10 M and that of melibiose and sucrose
was 0.25 M. Agglutination activity was scored after 1.5 h at room temperature
using an inverted microscope on low power, based on the criteria listed m the
legend to Table I. After 1.5 h in TCS plus 2 M urea, the purified oocyte lectin
retained about 25-50% of the agglutination activity observed when it was
assayed in TCS alone, but the relative abilities of various sugars to inhibit the
agglutination reaction was similar in the presence or absence of urea. The
recombinant lectin precipitated in the absence of urea; therefore, at least 2 M
urea was used in the agglutination assay.
Acknowledgments
We thank Candice Johnson for performing the A'-glycanase experiments, Frank
Comer and Sergio DiVirgilio for assistance with the reverse-phase chromatography, and Gerardo ManiUa-Alverez for assistance with illustrations. This
research has been supported by a grant from the NCRR Resource Center for
Biomedical Complex Carbohydrates to K.M. and M.P.
Expression of lectin cDNA and purification of recombinant protein
The predicted signal peptide cleavage site was determined to be between
amino acids 18 and 19 (von Heijne, 1985). PCR primers were designed to
amplify the coding sequence excluding the signal peptide sequence (amino
acids 19-313). Two restriction enzyme cleavage sites, Sail and Pstl, were
designed into the 5' and 3' primers, respectively: 5' primer CCCGTCGACGAACCTGTTGTAATAGTAGCCTCAAAA; 3' primer CCCCTGCAGTCATAGATAGAAAAGTAATACAGCGGCCTC. The amplimer obtained us-
Abbreviations
XL35, the calcium-dependent melibiose-binding lectin from Xenopus laevis
oocytes; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate;
PAGE, polyacrylamide gel electrophoresis; IPTG, isopropyl-P-D-thiogalactopyranoside; TCS, buffer containing 10 mM Tris, pH 7.6, 10 mM CaCl2, 150
mM NaCl; BSA, bovine serum albumin.
371
JlAseetaL
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Received on August 23, 1996; revised on September 25, 1996; accepted on
September 30, 1996
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