The Xenopus laevis cortical granule lectin: cDNA cloning, developmental expression, and identification of the eglectin family of lectins

Comparative Biochemistry and Physiology Part A 137 (2004) 115–129

The Xenopus laevis cortical granule lectin: cDNA cloning, developmental expression, and identification of the eglectin family of lectins夞 Betty Y. Chang, Thomas R. Peavy, Nathan J. Wardrip, Jerry L. Hedrick* Section of Molecular and Cellular Biology, University of California, One Shields Avenue, Davis, CA 95616, USA Received 10 June 2003; received in revised form 3 September 2003; accepted 5 September 2003

Abstract A Xenopus laevis egg cortical granule, calcium-dependent, galactosyl-specific lectin participates in forming the fertilization layer of the egg envelope and functions in establishing a block to polyspermy. We report the cDNA cloning of the lectin, expression of the cortical granule lectin gene during oogenesis and early development, and identification of a new family of lectins. The translated cDNA for the cortical granule lectin had a signal peptide, a structural sequence of 298 amino acids, a molecular weight of 32.7 K, contained consensus sequence sites for N-glycosylation and a fibrinogen domain. The lectin cDNA was expressed during early stages of oogenesis. Lectin glycoprotein levels were constant during development with 2y3 of the lectin associated with the extracellular perivitelline space and the eggy embryo fertilization envelope. Lectin mRNA levels were from 100- to 1000-fold greater in ovary than in other adult tissues. The lectin had no sequence homology to the previously identified lectin families. The lectin had 41–88% amino acid identity with nine translated cDNA sequences from an ascidian, lamprey, frog, mouse, and human. Based on the conserved carbohydrate binding and structural properties of these glycoproteins, we propose a new family of lectins, the eglectin family. 䊚 2003 Elsevier Inc. All rights reserved. Keywords: Cortical granule lectin; Oocyte lectin; Intelectin; Serum lectin; Eglectin; Block to polyspermy; Calcium-dependent lectin; Fertilization layer

1. Introduction The extracellular matrix of anuran eggs has been studied extensively to understand its role in fertilization and early development (Hedrick and Nishihara, 1991). In particular, ultrastructural and molecular changes in the egg envelope at fertilization produce a block to polyspermy, thereby 夞 Betty Y. Chang and Thomas R. Peavy have contributed equally to this work. *Corresponding author. Tel.: q1-530-752-3192; fax: q1530-752-0175. E-mail address: [email protected] (J.L. Hedrick).

insuring maintenance of the diploid state of the fertilized egg. At fertilization in Xenopus laevis, a fertilization layer is added to the egg envelope formed by the interaction of a cortical granule lectin (CGL) and a jelly coat ligand as proposed by Wyrick et al. (1974). The CGL is released from the egg via sperm induced exocytosis of cortical granules. CGL diffuses through the vitelline envelope and binds to its jelly coat ligand located on the outer surface of the envelope, thereby forming the fertilization layer (Grey et al., 1974; Greve and Hedrick, 1978). The fertilization layer blocks subsequent sperm binding to and

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penetration of the egg envelope (Grey et al., 1976) and protects the developing embryo from its environs (Hedrick and Nishihara, 1991). CGL has been purified from the egg cortical granules and characterized (Nishihara et al., 1986). The lectin’s binding specificity is for galactosides and requires calcium for ligand binding. It is a metalloglycoprotein composed of 12–14 subunits (Chamow and Hedrick, 1986). Deglycosylation of the CGL by either trifluoromethane sulfonic acid or PNGase F generated a protein with an apparent molecular weight of 39 K by SDS-PAGE (Wardrip and Hedrick, 1989). Initial characterization of the CGL glycans established the presence of both acid complex and neutral high mannose N-linked oligosaccharides. Removal of the glycan moiety significantly reduced the lectin’s carbohydrate binding function (Wardrip and Hedrick, 1990; Hedrick et al., 1993). The structure–function relations of the CGL are being investigated to understand CGL’s mechanism and role in establishing a block to polyspermy at fertilization. Towards this goal, we report here: (1) the cDNA cloning of the CGL, (2) experiments which further define its biological function in fertilization and early development (CGL tissue specific gene expression and the lectin’s persistence in the embryo until the hatching stage of development), (3) provide evidence for the existence of a CGL gene in the human and mouse genomes, and (4) reveal the discovery of a new ten-membered glycoprotein family of lectins. We propose the name eglectin for this new lectin family. The eglectins join the other previously described twelve families of lectin proteins and glycoproteins (Kilpatrick, 2002). Some of the results included in this paper were previously published as a preliminary report (Hedrick et al., 1993). 2. Materials and methods 2.1. Procurement of gametes X. laevis females were hormonally-stimulated with 35 IU pregnant mare serum gonadotropin and 500–1000 IU human chorionic gonadotropin as described previously (Hedrick and Hardy, 1991). Ovulated eggs were collected and stored at room temperature in 1.5x OR-2 (125 mM NaCl, 3.8 mM KCl, 1.5 mM CaCl2, 1.5 mM MgCl2, 1.5 mM Na2HPO4, 7.5 mM HEPES, 5.7 mM NaOH). A

sperm suspension was obtained by macerating testes in approximately 2 ml ice cold 1.5x OR-2. Large debris was allowed to settle, and the sperm suspension was stored on ice prior to use for fertilization. 2.2. Fertilization Fertilization conditions were essentially as described previously (Hedrick and Hardy, 1991). Immediately before insemination, eggs were rinsed several times in fertilization medium (0.33 DeBoers solution; 35 mM NaCl, 0.25 mM CaCl2, 10 mM Tris, pH 8.0). An aliquot of sperm was added and the gametes swirled to mix. After initiation of cleavage, excess sperm were removed by rinsing embryos in fertilization medium. Development proceeded at room temperature, and stages were identified using the staging criteria of Nieuwkoop and Faber (1967). 2.3. Affinity purification of CGL, dgCGL and antidgCGL CGL was affinity purified from dejellied egg lysates using mercaptan–solubilized jelly as the coupled ligand (Nishihara et al., 1986). CGL was chemically deglycosylated with trifluoromethane sulfonic acid (Karp et al., 1982). Antibodies specific for deglycosylated CGL (anti-dgCGL) were affinity purified from a goat polyclonal antisera directed against dgCGL prepared by Fabry and Hedrick (1992) by using dgCGL as the coupled ligand. 2.4. Isolation of CGL cDNA from X. laevis ovary cDNA libraries Affinity purified anti-dgCGL was used for expression screening of a X. laevis ovarian cDNA expression library in lgt11 (a kind gift from the late M. Dworkin) as described in Sambrook et al. (1989). Positive clones were further screened, purified, subcloned, and sequenced using Sequenase娃 (USB, Cleveland, OH) (Sanger et al., 1977). Full length CGL cDNA clones were obtained by rescreening the ovarian lgt11 library using w32Pxlabeled cDNA (Bercegeay et al., 1993). CGL cDNAs were also identified from another ovarian cDNA library constructed by Yang and Hedrick (1997) by using a PCR approach with primers

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designed from sequenced cDNAs. PCR products were cloned and commercially sequenced. 2.5. Isolation and sequencing of internal CGL peptides CGL was reduced and S-carboxymethylated with w14Cx-iodoacetic acid by the method of Crestfield et al. (1963). Aliquots of w14Cx-CGL were treated with either 0.2% wyw clostripain or V8 protease in 20 mM Tris–HCl, pH 7.5 at 37 8C for 16 h. Samples were dried, redissolved in 0.1% TFA, and separated by reverse phase HPLC. After analysis by SDS-PAGE, purified peptides were submitted to the UC Davis Molecular Structure Laboratory for sequencing. CGL (4 mg) was deglycosylated with N-glycanase (10 U) at 37 8C for 72 h in 50 mM Tris–HCl, pH 7.5. In the absence of EDTA or protease inhibitors, a proteolytic activity associated with Nglycanase caused fragmentation of CGL. The resulting deglycosylated peptides were ethanol precipitated, S-carboxymethylated, and separated by preparative SDS-PAGE. Peptides were gel eluted as described by Gerton et al. (1982) and submitted for sequencing as above. 2.6. Deblocking of CGL peptide N-terminus with pyroglutamate aminopeptidase After SDS-PAGE, dgCGL was electroblotted onto a polyvinyl difluoride membrane and stained with 0.1% Ponceau S. After excising and destaining the dgCGL band, the membrane was incubated in 100 mM acetic acid, 0.5% wyv polyvinylpyrrolidone-40 at 37 8C for 30 min and then washed extensively in deionized water. Pyroglutamate aminopeptidase digestion of dgCGL was performed in 100 mM sodium phosphate buffer, 10 mM Na2EDTA, 2 mM DTT, 5% glycerol, pH 8.0 for 18 h at 4 8C followed by 4 h at 25 8C (Podell and Abraham, 1978; Hirano, 1991). 2.7. FAB-MS of CGL peptide for amino terminal sequence determination Carboxymethylated CGL was digested with trypsin (Type III, Sigma, St. Louis, MO) before and after pyroglutamate aminopeptidase treatment by using 20 mg trypsin ymg CGL at 37 8C for 20 h in 100 mM NH4HCO3, pH 7.8. After addition of TFA to a final concentration of 0.1%, peptides

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were separated by C18 reverse phase HPLC. The peptide predicted by size to be the N-terminal peptide was submitted for Fast Atom Bombardment-Mass Spectrometry (FAB-MS) analysis by the UC Davis Molecular Structure Facility. 2.8. Northern blot analysis of CGL Ovaries from hormonally-stimulated animals were surgically removed and the oocytes released by collagenase treatment (Smith et al., 1991). Oocytes corresponding to stages I–VI according to Dumont (1972) were hand selected and subjected to either RNA or protein extraction. Northern blots were performed according to Sambrook et al. (1989). Total RNA from oocytes and embryos were electrophoresed using 1.2% formaldehyde gels and transferred to a positively charged nylon membrane (Hybond-Nq, Amersham Life Sciences, Buckinghamshire, England). A randomprimed w32Px-CGL cDNA probe was heat denatured and added to membranes prehybridized at 50 8C in 50% formamide, 6X SSC, 5X Denhardt’s solution, 0.5% SDS, and 100 mgyml of salmon sperm DNA. After 8 h, blots were washed in a low stringency buffer (1X SSC, 0.1% SDS) for 20 min at room temperature, and then in a high stringency buffer (0.2X SSC, 0.1% SDS) three times at 68 8C for 20 min each. Hybridizing bands were visualized by exposure to X-ray film. 2.9. RT-PCR of CGL Total RNA was isolated from the following tissues: the ovaries of a hormonally-induced female; the heart, fat body, intestine and spleen of a female not induced with hormones; and the testes, lung, liver and muscle from a male not induced with hormones. After purification of mRNA by the Oligotex娃 mRNA Kit (Qiagen, Chatsworth, CA), RT-PCR experiments were performed using either 100 ng or 1 mg of mRNA template with primers (forward primer 59-TTGAAGCATATAACCTTGGGGTGTG-39; and reverse primer 59-ACAGCGGCCTCAGTTATCTCTATGC-39) designed to amplify a 499 bp fragment of the CGL cDNA by the Titan娃 One Tube RT-PCR System (Boehringer Mannheim, Indianapolis, IN). Reactions were reverse transcribed at 50 8C for 30 min and then PCR amplified using the following cycling conditions: one denaturing cycle of 94 8C for 2 min; 35 cycles of 94 8C for 40 s, 65 8C for

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30 s, 68 8C for 45 s; and one final extension cycle of 68 8C for 10 min. A negative control containing all components except template mRNA was also included. For a positive control, the b-tubulin transcript was RT-PCR amplified using primers designed to amplify a 750 bp fragment from 10 ng of mRNA. Image analysis was performed using the NIH Image software program (http:yy rsb.info.nih.govynih-imagey). 2.10. Southern blotting of RT-PCR A CGL cDNA probe was generated by PCR incorporation of digoxigenin (DIG)-dUTP (Boehringer Mannheim, Indianapolis, IN) utilizing the above CGL primers and 20 pg of CGL cDNA for template DNA. Cycling conditions were performed as described above except without the reverse transcription step. A positive control PCR reaction without DIG-dUTP was also performed and 1 ng was loaded into a well of a 1.2% agarose gel next to 10 ml of the CGL RT-PCR reactions. After electrophoresis, the DNA was transferred to a positively charged nylon membrane (GeneScreen娃 Plus Hybridization Membrane, DuPont NEN, Boston, MA) by capillary blotting and UV crosslinked onto the membrane. Heat denatured DIG-labeled CGL probe (15 ngyml) was added to hybridization buffer (5x SSC; 0.1% N-laurylsarcosine; 0.02% SDS; 1% blocking reagent) and incubated at 65 8C for 10 h. After high stringency buffer (0.5x SSC; 0.1% SDS) washes at 65 8C, hybridized probe was immunodetected using antiDIG-alkaline phosphatase Fab fragments and chemiluminescence substrate (CSPD䉸 Ready-toUse, Boehringer Mannheim). Luminescence was visualized by exposure to X-ray film. 2.11. Qualitative analysis of CGL distribution Protein and glycoprotein components in samples were separated by SDS-PAGE as described by Laemmli (1970) and transferred to nitrocellulose membranes according to Towbin et al. (1979). These membranes were probed with anti-dgCGL serum prepared by Fabry and Hedrick (1992). 2.12. Quantitative analysis of CGL distribution Eggs and embryos were carefully dissected into three components; the egg envelope, the perivitelline space, and the eggyembryo. Components were

quantitatively collected. The outer jelly layers, J3 and J2, were removed manually from embryos (Yurewicz et al., 1975). The inner jelly coat layer J1 was carefully dissolved in a mercaptan solution, avoiding lysis of the egg envelope. The dejellied eggs and embryos were washed in DeBoers solution. The egg envelope was then dissected and removed using watchmakers forceps. The denuded eggs and embryos were collected with a Pasteur pipette. The medium remaining contained the contents of the perivitelline space. These three components were stored frozen until analysis. Eggs and embryos from five different clutches of eggs were used for these experiments. For analysis of CGL content, isolated components were boiled in 1 mM EDTA, 1 mM mercaptoethanol, 50 mM D-galactose, 10 mM Tris, pH 9.5 to disrupt the lectin’s binding with its ligand in the fertilization envelope and disrupt any interaction with other cellular components. Purified CGL was treated similarly and used as an analytical standard for quantification. Samples were serially diluted in 100 mM sodium carbonate and coated overnight onto an ELISA plate at 4 8C. Sample wells were then probed with anti-dgCGL serum. Antibody binding to the absorbed CGL in the sample wells was quantified using ELISA procedures described by Fabry and Hedrick (1992). Preimmune serum was used as a control for antibodies non-specifically binding to egg and embryo samples; control values were subtracted from experimental values. Average values from 5 eggs or embryos were determined at various times after fertilization. 3. Results 3.1. Isolation and characterization of CGL cDNA clones Anti-dgCGL identified six CGL cDNA clones in the X. laevis ovary cDNA library. The largest clone contained an approximately 800 bp insert. After rescreening with w32Px-CGL cDNA, full length cDNA clones were identified with 1.l kb inserts. In addition, the 59 and 39 cDNA ends of CGL were PCR amplified and sequenced from a different ovary cDNA library (Yang and Hedrick, 1997) for sequence confirmation. 3.2. CGL cDNA sequence and peptide sequences The CGL cDNA sequence was 1082 bp in length (Fig. 1). Only one open-reading frame was

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Fig. 1. X. laevis CGL cDNA sequence. (Genbank accession no. X82626). Solid underlining denotes the signal peptide sequence; dotted underlining indicates peptide regions sequenced; gray highlighting specifies N-linked consensus sites; and the asterisk represents the stop codon.

identified for the cDNA sequence starting with an ATG initiation site at base positions 1–3 and a stop codon TGA at base position 940–942. The open reading frame was translated into a protein with a molecular mass of 34 304 Da containing 313 amino acids. The CGL cDNA sequence and translated amino acid sequence were submitted to GenBank (accession number X82626). Sequencing of five internal peptides and the deblocked Nterminal peptide confirmed the amino acid sequence derived from the CGL cDNA. However, a serine residue was notably absent from the deblocked N-terminal peptide sequence (QCEPVVIVA) when compared to the deduced sequence (QSCEPVVIVA). In order to resolve the conflict, FAB-MS was used to analyze the deblocked N-terminal peptide and the resulting spectrum confirmed the existence of the serine residue.

Thus, the CGL mRNA codes for a 15 amino acid signal sequence as predicted by the Webbased software program SignalP (Nielsen et al., 1997). A polypeptide of 32 743 Da remains after signal sequence cleavage with a theoretical pI of 5.29. Two putative N-glycosylation sites were found at residues 154–156 (NLT) and 163–165 (NSS). These two sites are located in hydrophilic regions, predicted to be on the protein surface, and located at reverse turns or coils, consistent with the location of other N-linked oligosaccharides (Struck and Lennarz, 1980). 3.3. Sequence comparisons of related genes Significant sequence identities were found when a BLASTP search (Altschul et al., 1997) was performed using the translated X. laevis CGL

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Table 1 Eglectin conserved structures Structure Cys

b

N-Glycosyl sites Fibrinogen domain Conserved sequencesc Signal peptided Number residues % Identity

Sequence

xlCGLa

xtCGL

mmINTL

hsINTL1

hsINTL2

xlXEEL

xlSer1

xlSer2

ljSer

hrPlas

18 35 41, 70, 94 199*251, 259 265*, 280 Total cys. 154–156

q q q

q – q

0 – q

– 0 q

0 0 q

0 – q

0 – q

0 – q

0 0 q

0 – q

10 q

9 q

9 q

10 q

13 q

10 q

11 q

9 q

17 q

8 q

163–165 39–82

q q

q q

– q

q q

– q

q q

q q

– q

– q

– q

76–88 97–102

q

q

q

q

q

q

q

q

q

q

15Q

18Q

19A

18T

27A

21G

20C

19E

19N

21Q

313

320

313

313

325

342

338

315

333

348

100

88.2

63.2

62.7

59.7

65.2

61.9

59.6

50.5

41.0

a

xlCGL, X. laevis cortical granule lectin; xtCGL, X. tropicalis cortical granule lectin; mmINTEL, mouse intelectin; hsINTL1, human intelectin 1; hsINTL2, human intelectin 2; xlXEEL, X. laevis embryonic epidermal lectin; xlSer1, X. laevis serum lectin 1; xlSer2, X. laevis serum lectin 2, ljSer, lamprey serum lectin; hrPlas, ascidian plasma lectin. b Sequence position using the X. laevis CGL numbering system; present (q); absent (–); present but not aligned (0). cConserved residues in all ten eglectin glycoproteins were 76y313s 24.2%; in 8y10 eglectin glycoproteins were 160y313s51.1%. d Total residues in the signal peptide and the resulting N-terminal amino acid after cleavage. *Missing in the ascidian plasma lectin.

amino acid sequence. Lee et al. (1997) reported the sequence of a X. laevis oocyte lectin (accession no. XLU86699), which was deposited into the database 3 years after our CGL sequence was deposited. The two lectin sequences have 99.2% DNA identity and 98.4% amino acid identity with 5y8 of the DNA differences resulting in amino acid substitutions (i.e. 19E™D, 40S™N, 61S™P, 217 K™N, 226D™G). Most notably, a third potential N-linked consensus sequence is created by the change from 217K™N in the oocyte lectin. We resequenced this region of the cDNA using PCR methods and confirmed the CGL sequence reported in Fig. 1. This oocyte lectin cDNA is likely a genetic variant of CGL found at the same gene locus since we were unable to PCR amplify the oocyte lectin cDNA from our libraries. Nine other glycoproteins have high amino acid identity to CGL ranging from 41.0 to 88.2% (Fig. 2 and Table 1). The ovarian cDNA corresponding to CGL from the closely related frog X.(Silurana) tropicalis had the highest percent identity with CGL as expected (Lindsay et al., 2003). Several cDNAs with the name intelectin share high amino acid identity with CGL (G60%). Mouse intelectin, mmINTL, was cloned from small intestine tissue

(Komiya et al., 1998), but the protein was not characterized or purified. The human sequence referred to as intelectin 1, hsINTL1, was cloned from different tissues and named independently by three groups, intelectin 1 (Tsuji et al., 2001), endothelial lectin-1 (Lee et al., 2001), and lactoferrin receptor (Suzuki et al., 2001); but are the same gene product (accession no. NM_017625). The other human gene, intelectin 2 or hsINTL2 (previously named endothelial lectin-2) (Lee et al., 2001), is 85% identical to hsINTL1 and is located in the same chromosomal region. Serum lectin cDNAs from X. laevis, lamprey Lampetra japonica, and ascidian Halocynthia roretzi were related to CGL but only the ascidian plasma lectin was further characterized as a glycoprotein (Abe et al., 1999). The two X. laevis serum lectin sequences were distinctly different from CGL and one of these may be the same galactoside-binding serum lectin described by Roberson et al. (1985). Also, in X. laevis, an embryonic epidermal lectin cDNA named XEEL was found to be related to CGL (Nagata et al., 2003). In addition, three peptide sequences derived from a X. laevis blood group B-active GPI-anchored membrane glycoprotein (Nomura et al., 1998) had high identity to X.

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Fig. 2.

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laevis CGL, as well as strictly conserved residues, and were included in the sequence alignment figure. This GPI-anchored glycoprotein was proposed to be involved in Ca2q-dependent cell–cell adhesion and recognized a cell surface bloodgroup-B trisaccharide, Gal a1-3(Fuc a1-2)Gal b13-. Although all of the above glycoproteins that have been examined functionally are Ca2qdependent galactose-binding lectins, they do not belong to any of the twelve previously identified lectin families (Drickamer and Taylor, 1993; Dodd and Drickamer, 2001). Based on sequence identity and the conservation of two cysteine residues, CGL and the other related glycoproteins have a fibrinogen-like domain that is 44 amino acids in length (Fig. 2 and Table 1). The N-terminal domains (23 to 51 residues) of the glycoproteins preceding the conserved fibrinogen-like domain exhibited the greatest variability in sequence. The positions of eight cysteines were conserved in all glycoproteins except the ascidian plasma lectin that retained six of the eight residues. One Nlinked consensus site and two sequence motifs were strictly conserved for all glycoproteins. About 24% of the residues were invariant in the alignment, 35% were invariant when the ascidian sequence was excluded, and 51% were invariant when 8 of the 10 sequences were considered. The sequence alignment in Fig. 2 was used to generate a gene tree by maximum parsimony analysis to infer the evolutionary relationships among the ten glycoproteins (Fig. 3). Similar tree topologies were obtained using other phylogenetic analysis methodologies (i.e. maximum likelihood and neighborjoining). 3.4. ESTs similar to CGL When a BLASTP search of the EST database was performed using the translated CGL sequence, 21 human and 23 mouse cDNA sequences were

found to align with significant identity to CGL (probability values ranging from 10y10 to 10y50; Genbank accession numbers: AA552031, AA552527, R06009, W03754, HHEA56Z, AI660866, AA483813, AA551795, AA573809, AA573898, AA573925, AA588114, AA588124, AI318094, AI948957, T31354, AI076155, AI248965, N79237, AI187143, AI193160, AA871147, AA473748, AA871186, AI019082, AA871200, AI504654, AV065110, AV070107, AV062845, AV063110, AV064749, AV065050, AV066202, AV066421, AV066556, AV066662, AV069467, AV069671, AV070936, AV071161, AV049929, AV075887, AI019082). The majority of the EST sequences were from intestinal tissues (13y21 for human; 19y23 for mouse). The other human ESTs originated from a fetal liveryspleen cDNA library (5 clones), a fetal lung cDNA library (2 clones), and an adult atrium cDNA library (1 clone), and the non-intestinal mouse ESTs were from a thymus, stomach, pancreas and an embryo (7-day) pooled organ cDNA libraries (1 clone each). In order to assess whether these were the same gene products being expressed in different tissues, we completely sequenced the following EST clones that we obtained from the American Type Culture Collection (Manassas, VA) and submitted their full length sequences to GenBank: human colon IMAGE clone 1012588 (access. no. AY157361), human fetal liveryspleen IMAGE clone 124878 (AY157362), mouse bowel IMAGE clone 1095883 (AY157363), and mouse thymus IMAGE clone 1378158 (AY157364). The human colon and liveryspleen cDNA sequences were 99.2 and 100% identical to the intelectin 1 sequence, respectively, resulting in 2 non-synonymous substitutions for the colon EST cDNA (hsINT1 109D™V, 313R™P). The mouse EST cDNAs were )99% identical to mouse intelectin but with no amino acid substitutions.

Fig. 2. Amino acid sequence alignment of the eglectin family. The following translated cDNA sequences were aligned using Pileup (GCG, Wisconsin package version 10, GCG, Madison, WI): X. laevis CGL, xlCGL (Genbank accession no. X82626); X. tropicalis CGL, xtCGL (AY079196); human intelectin 1, hsINTL1 (NM_017625); human intelectin 2, hsINTL2 (NM_080878) mouse intelectin, mmINTL (AB016496); X. laevis embryonic epidermal lectin, xlXEEL (AB105372); X. laevis serum lectin 1, xlSerum1 (AB061238); X. laevis serum lectin 2, xlSerum (AB061239); lamprey serum lectin, ljSerum (AB055981); ascidian plasma lectin, hrPlasma (no GenBank entry, manually entered). Also, aligned below the sequence blocks was a domain from the human fibrinogen-b polypeptide, hsFibrin b, (AAK62470) and three peptides from X. laevis blood group B-active GPI-anchored membrane glycoprotein (Nomura et al., 1998). Sequences shaded in black or grey denote position in which 7y10 sequences were either identical or conservative substitutions, respectively. Arrowpoints refer to conserved cysteine positions and asterisks denote a putative N-link consensus site.

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Fig. 3. Eglectin gene tree. The maximum parsimony method for protein sequences within the MEGA2 software package (v2.1) was used to examine the relationship of eglectin family members based on the sequence alignment in Fig. 2. Multiple data sets were produced by resampling the alignment data 1000 times to generate the confidence levels presented next to branch bifurcations.

3.5. Expression of CGL mRNA in X. laevis tissues Initially, a Northern blot analysis was performed using w32Px-labeled CGL cDNA with mRNA purified from ovary, liver, brain, kidney, lung, heart, skin and testes. A hybridizing band, 1.2 kb, was found only in the ovarian RNA (data not shown). In order to increase sensitivity, we used a RT-PCR approach followed by Southern blotting to detect CGL transcripts in mRNA from ovary, liver, fat body, spleen, intestines, lung, heart, muscle and testes (Fig. 4). RT-PCR using either 100 ng or 1000 ng of ovarian mRNA with CGL-specific

primers yielded the expected 500 bp product with an appropriate 5- to 10-fold increase for the 1000 ng over the 100 ng sample. A lower 450 bp band was observed to hybridize in the control CGL plasmid and ovary reactions, indicating that one of the primers had a lower affinity for a different annealing site, and another larger 640 bp band was observed only in the 1000 ng ovary sample. We sequenced these amplimers and confirmed that they were indeed CGL. Hybridizing 500 bp bands were also detected in fat body, intestines and testes samples only when 1000 ng of mRNA was used. In addition, faint bands at 500 bp were observed

Fig. 4. Tissue expression of CGL mRNA. (a) Ethidium bromide stained gel of CGL control plasmid PCR reaction (1 ng loaded) and RT-PCR reactions of tissue mRNA preparations (1 mg of template used plus a 0.1 mg ovary mRNA reaction) using CGL primers. (b) Southern blot of the above gel using a DIG-labeled CGL probe. (c) Ethidium bromide stained gel of RT-PCR reactions using tubulin primers.

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Fig. 5. Northern and Western blot analysis of CGL during oogenesis and early development. (a) Total RNA isolated from X. laevis oocyte stages I–VI and the 32-cell embryo was northern blotted and probed using w32Px-labeled CGL cDNA. Each lane was loaded with total RNA equivalent to 5 oocytesyembryos. (b) Western blots of X. laevis oocyte stages (I–VI) and embryo stages (4-cell, gastrula, neurula and hatched) probed with anti-dgCGL serum. Each lane was loaded with total protein equivalent to 0.5–1 oocyteyembryo.

for liver and lung samples when the film was overexposed. Thus, the relative expression level of CGL measured by density in fat body, intestines and testes was approximately 100- to 500-fold less than in hCG stimulated ovary (mostly stage V– VI oocytes), whereas expression in liver and lung was more than 1000-fold less than in ovary. 3.6. CGL expression during oogenesis and early development The developmental profile of CGL expression during oogenesis was examined by extracting total

RNA from six stages of oocytes and gastrula staged embryos. RNA from 5 oocytesyembryos were loaded onto each lane so that stage-specific expression could be directly compared by Northern blot analysis using a w32Px-labeled CGL cDNA probe (Fig. 5a). CGL message was detected in Stage I oocytes, peaked at stage III, and gradually returned to the stage I level by stage VI. The message persisted after eggs were ovulated; reduced levels of message were detected after fertilization. As a control, the Northern blots were reprobed with b-actin cDNA resulting in the detec-

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tion of approximately equivalent amounts of actin transcripts (data not shown). Western blots of stages I–VI oocyte extracts using anti-dgCGL revealed very small amounts of CGL at the early oocyte stages I and II (Fig. 5b). A strong signal appeared at stage III and the amount of lectin present in oocytes peaked at stage IV. This steady state amount of CGL remained constant from stage IV to VI. CGL in eggsyembryos was analyzed at five time points from 0 to 48 h using ELISA methods. Within the limits of analyses (S.D. of 13%), the total amount of CGL and its distribution did not significantly change as a function of development time (data not shown). Based on a standard curve, an embryo (egg) contained on average 0.22 mg of CGL with a distribution of 37% in the embryo, 43% in the perivitelline space, and 19% associated with the fertilization envelope. Thus, most of the CGL, 62%, was associated with the extracellular matrix of the embryo. We did not detect the presence of CGL mRNA in 32-cell stage embryos by Northern blotting experiments (Fig. 5a). Thus, we did not detect significant gene expression during embryogenesis, a result consistent with the relatively constant amounts of CGL during development. 4. Discussion 4.1. Properties of CGL and the cloned cDNA Based on the sequence identity of peptides derived from the isolated CGL protein and the translated cDNA sequence, we cloned the cDNA for the X. laevis cortical granule lectin. This is the first macromolecule to be cDNA cloned from X. laevis cortical granules with a defined biological function; e.g. establishing a block to polyspermy at fertilization. Only a few other egg cortical granule components have been cloned from other species (fish, mammals, sea urchins) but their biological functions in fertilization and early development are largely speculative (for review see Wessel et al., 2001). The CGL glycoprotein possesses some notable if not unusual properties. The N-terminal amino acid residue was established to be a pyroglutamate produced by cyclization of glutamine. Of particular interest, the ascidian plasma lectin related to CGL also had an N-terminal pyroglutamate residue (Abe et al., 1999). The glutamine cyclization process is

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known to be enzyme catalyzed, as well as occurring non-enzymatically (Fischer and Spiess, 1987). In some proteins, an amino terminal pyroglutamyl structure has functional correlations. For instance, a fertilization-promoting tripeptide found in seminal plasma has an amino terminal pyroglutamyl residue and is a possible regulator of sperm function (Fraser and Adeoya-Osiguwa, 2001); the human monocyte chemotactic protein-2 requires an amino terminal pyroglutamyl residue for chemotactic activity (Van Coillie et al., 1998); an RNase from frog eggs, onconase, has decreased cytotoxic and enzyme activities if an amino terminal methionine was substituted for the naturally occurring amino terminal pyroglutamyl residue (Boix et al., 1996). It is also known that blocked amino terminal residues in proteins protect against amino peptidase proteolysis. Since CGL functions external to the cell, resistance to protease action might be an advantage for structural stability, and hence functioning of the molecule. However, the structural importance of the amino terminal pyroglutamyl residue to the carbohydrate binding function of CGL and its cellular function needs to be experimentally determined. CGL is known to be a glycoprotein from chemical analysis (Nishihara et al., 1986). From the data presented here, CGL has two consensus sequence sites for N-linked glycosylation, 154N and 163 N. These sequons were analyzed as to their potential for glycosylation using the neural network method available on the NetNGlyc 1.0 website -http:yywww.cbs.dtu.dkyservicesyNetNGlycy). The 154N site had a potential for Nglycosylation of 0.13 while the 163N site a potential of 0.57. Potentials of )0.5 are considered positive in this type of analysis. It is possible that the sites are also irregularly glycosylated; e.g. CGL molecules may have a single site glycosylated (154 or 163) as well as both sites glycosylated. Glycosylation microheterogeneity is observed for CGL as demonstrated by SDS-PAGE separations of CGL resulting in broad diffuse bands (Fig. 5b). This heterogeneity seems to be due to variation in Nlinked glycosylation since PNGaseF treatment results in a single sharp band with no further reduction in size when chemical means of deglycosylation were used (Hedrick et al., 1993). The molecular weight of the polypeptide chain after deglycosylation (enzymatic or chemical) by SDSPAGE was 39 K as compared to 32.7 K predicted from the translated cDNA sequence. Expression of

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the CGL cDNA in an E. coli system produced a CGL polypeptide with a molecular weight of 40 K (data not shown). Thus, CGL anomalous behavior on SDS-PAGE was likely due to abnormal SDS binding to the polypeptide moiety. Of particular interest, human intelectin 1 also exhibits anomalous behavior on SDS-PAGE (Tsuji et al., 2001). This aberrant SDS binding behavior of CGL was probably associated with the N-terminal domain of the polypeptide since an N-terminal polypeptide produced by hydroxylamine cleavage predicted to have a molecular weight of 6 K had an observed molecular weight of 14 K on SDSPAGE. 4.2. Tissue expression of CGL The presence of CGL mRNA in tissues other than the ovary could only be detected using sensitive PCR methods compared with Northern blotting methods. The ovary (oocyte) contained more than 1000-fold greater CGL mRNA levels than testes, intestine, and fat body and 100- to 500-fold greater CGL mRNA levels than liver and lung. CGL may be expressed at higher levels in these non-ovarian tissues at other developmental times, but low levels of expression do not eliminate a role for CGL in these somatic tissues. Interestingly, human and mouse intelectins are expressed in many different tissues. For example, human intelectin has been identified by Northern blot analysis in ovary, heart, colon, small intestine, thymus, testes, spleen, lymph node, stomach, and uterus (Lee et al., 1997; Tsuji et al., 2001); and additional tissue expression was detected by RT-PCR in salivary gland, skeletal muscle, pancreas, thyroid, liver, kidney, lung, brain, tonsil, prostate, mammary gland, adrenal gland, bone marrow, and peripheral blood leukocytes (Suzuki et al., 2001). Expression of CGL and their homologues in many adult tissues argues that these lectins may participate in a wide variety of functions during organogenesis and in the fully differentiated organism. 4.3. Role of CGL in fertilization and development During oogenesis, CGL gene expression started at Stage I and reached a maximum by Stage III as determined by Northern blot analysis. Western blot analysis detecting the presence of the CGL polypeptide gave parallel results. Expression of egg envelope genes during oogenesis was also

observed to start at Stage II of oogenesis, the stage at which the egg envelope is first observable as a cytological entity (Yamaguchi et al., 1989). The morphological appearance of cortical granules within the developing oocyte was earlier shown by Dumont to occur during early Stage II (Dumont, 1972). This morphological observation is consistent with our CGL gene expression observations. The presence of CGL within the developing embryo is likely due to the continued presence of cortical granules that did not undergo exocytosis in the cortical reaction (Kotani et al., 1973; Grey et al., 1974; Ikenishi, 1980). Others have reported that CGL mRNA persisted until hatching, Stage 35y36 (Lee et al., 1997) with a sharp spike of gene activity at gastrulation, Stage 10. However, we observed by Northern blot analysis that CGL mRNA levels decreased after fertilization and no CGL mRNA could be detected in Stage 6 (32-cell) embryos. Consistent with our results, Nagata et al. observed by RT-PCR that CGL mRNA expression decreased gradually and was undetectable by stage 20 (Nagata et al., 2003). It is possible that the mRNA detected by Lee et al. in embryos was residual oocyte mRNA or new synthesis from a CGL-related gene such as xlXEEL which begins expressing at gastrulation (Nagata et al., 2003). In addition, others have reported a decrease in the relative amount of CGL glycoprotein present in the developing embryo starting at Stage 1 and undetectable by Stage 24 (Outenreath et al., 1988). However, we observed that the amount of CGL associated with the embryo was approximately constant during prehatching development. In our experiments we used antibodies prepared against the protein moiety of CGL, whereas in the paper by Outenreath et al. (1988), they used antibodies prepared against the CGL glycoprotein. As previously shown, the primary immunogenic response to native CGL is against the carbohydrate moiety (Fabry and Hedrick, 1992). Thus, a possible explanation for the differences is the epitope specificity of the antibodies used for CGL quantitation. We found the majority of the CGL was associated with the extracellular matrix, consistent with its functions in blocking sperm penetration and modifying the permeability of the egg envelope (Bakos et al., 1990). From the results presented here and elsewhere (Nishihara et al., 1986), it can be estimated that the concentration of CGL in the perivitelline space of activated eggs is 1–4 mgy

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ml. CGL turnover in an extracellular location is unlikely since observations supporting the hydrolysisyremoval from and secretion of CGL into the amphibian perivitelline space during development have not been reported. It was suggested by others that CGL might play some intracellular role during development (Roberson and Barondes, 1983; Outenreath et al., 1988). However, supporting experimental evidence for this suggestion is lacking. The presence of CGL in vesicles associated with embryonic cells they reported are likely contained within residual cortical granules not released in the cortical reaction. The ‘amorphous vesicles’ they observed are membrane bound, located in the cortex of cells next to the plasma membrane, and have the staining properties and size of cortical granules. Thus, from the observations reported here and analysis of other observations, we believe the primary function of CGL in fertilization and early development is associated with the extracellular matrix of the egg and embryo. CGL participates in forming the fertilization layer, thereby providing a block to polyspermy at fertilization. The fertilization layer (CGL ligand complex), in cooperation with the egg envelope, regulates the embryo’s environment (chemical, physical, and microbiological) and provides a protective and optimal internal environment for the developing embryo. 4.4. The Eglectin gene family X. laevis CGL has no sequence relationship to the recognized calcium-dependent galactose-specific lectin families (i.e. C-type lectins and galectins), but is related to nine other cDNA sequences in the database (X. tropicalis CGL, mouse intelectin, human intelectins 1 and 2, X. laevis embryonic epidermal lectin, X. laevis serum lectins 1 and 2, lamprey serum lectin, ascidian plasma lectin) and to peptide sequences from a X. laevis blood group B-active GPI-anchored membrane glycoprotein. These sequences have significant conservation of amino acid sequence and structural domains as presented in Fig. 2 and Table 1. Of those that have been isolated and characterized wCGLs (Nishihara et al., 1986; Lindsay et al., 2003); ascidian plasma lectin (Abe et al., 1999); human intelectin (Tsuji et al., 2001); human lactoferrin receptor (Suzuki et al., 2001); blood group B-active membrane glycoprotein (Nomura et al., 1998)x, all are glycoproteins, form oligomers, require Caq2 for bind-

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ing, and are specific for galactose-containing sugars. We believe the conserved structural, functional, and biological roles of these ten glycoproteins provide convincing evidence that they constitute a family of related glycoproteins. We, therefore, propose that they be called the eglectin ¯ (e-glec-tin) family of lectins, recognizing that one of the first observed animal lectin (agglutinin) activities came from egg extracts (Prokop et al., 1967; Uhlenbruck and Prokop, 1967) and the first discovered lectin of the eglectin family was present in eggs (Wyrick et al., 1974). Multiple eglectin genes exist in X. laevis and humans indicating that gene duplication events have occurred in these lineages (Fig. 3). All four of the X. laevis genes (CGL, XEEL, serum lectins 1 and 2) are more closely related to each other than to other species’ genes suggesting that these genes duplicated within the amphibian lineage after their divergence from the common ancestor leading to mammals. Similarly, human intelectins 1 and 2 are more closely related to each other than either is to mouse intelectin. This relationship suggests a gene duplication event occurred in the lineage leading to primates after their separation from rodents. It seems likely that there are many more eglectin gene members yet to be discovered. Database mining of genome projects will facilitate a more complete evolutionary analysis of the eglectin gene family. In addition, to being found in many different tissues, eglectins can be found both as soluble lectins and as GPI-anchored surface receptors. Human intelectin exists in a soluble form and also membrane bound as a GPI-anchored lactoferrin receptor (Suzuki et al., 2001). In addition, the peptide sequences from the X. laevis blood group B-active GPI-anchored membrane glycoprotein nearly match X. laevis CGL suggesting that the glycoprotein is either a product of the same gene or closely related. However, neither of these glycoproteins, nor any of the other eglectin family members were identified as being GPI-anchored proteins from sequence analysis using GPI prediction algorithms (http:yymendel.imp.univie.ac.aty gpiygpi_prediction.html). Nonetheless, there is convincing biochemical evidence that the GPIanchored lactoferrin receptor and the X. laevis blood group B-active GPI-anchored membrane glycoprotein are GPI-anchored glycoproteins. Regarding biological roles, eglectin glycoproteins have diverse functions such as preventing

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cell-cell interactions (e.g. block to polyspermy), as phagocytosis stimulatingycell defense molecules, and as GPI-anchored cell surface receptors (binding of ligands or cells). In general, eglectins apparently function in cell-cell or cell surface processes. Further studies are necessary to determine the structure, function and evolution of the eglectin family of glycoproteins. We anticipate that eglectins will prove to be a lectin family of fundamental and widespread biological importance. Acknowledgments The authors are indebted to Dr Heather Harris, Ms Katie Kreimborg, and Mr Darrin Ngo for technical assistance with the ELISA and DNA sequencing experiments. BYC is indebted to the late NJW for his constant support in the lab (Deceased April 29, 1994). Support for Dr Peavy was provided by NIH NSRA fellowship HD0837901 and by NIGMS IRACDA GM00679. This work was also supported in part by USPHS Research Grant HD04906. References Abe, Y., Tokuda, M., Ishimoto, R., Azumi, K., Yokosawa, H., 1999. A unique primary structure, cDNA cloning and function of a galactose-specific lectin from ascidian plasma. Eur. J. Biochem. 261, 33–39. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., et al., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Bakos, M.A., Kurosky, A., Hedrick, J.L., 1990. Enzymatic and envelope-converting activities of pars recta oviductal fluid from Xenopus laevis. Dev. Biol. 138, 169–176. Bercegeay, S., Allaire, F., Jean, M., L’Hermite, A., Bruyas, J.F., Renard, N., et al., 1993. The bovine zona pellucida: differences in macromolecular composition between oocytes, pre-treated with A-23187 or not, and embryos. Reprod. Nutr. Dev. 33, 567–576. Boix, E., Wu, Y., Vasandani, V.M., Saxena, S.K., Ardelt, W., Ladner, J., et al., 1996. Role of the N terminus in RNase A homologues: differences in catalytic activity, ribonuclease inhibitor interaction and cytotoxicity. J. Mol. Biol. 257, 992–1007. Chamow, S.M., Hedrick, J.L., 1986. Subunit structure of a cortical granule lectin involved in the block to polyspermy in Xenopus laevis eggs. FEBS. Lett. 206, 353–357. Crestfield, A.M., Moore, S., Stein, W.H., 1963. The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated protein. J. Biol. Chem. 238, 622–627. Dodd, R.B., Drickamer, K., 2001. Lectin-like proteins in model organisms: implications for evolution of carbohydrate-binding activity. Glycobiology 11, 71R–79R.

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