a mammalian sperm lectin related to rat hepatocyte lectin-2/3

Molecular and Cellular Biochemistry 103: 155-161, 1991. (~) 1991 Kluwer Academic Publishers. Printed in the Netherlands.

A mammalian sperm lectin related to rat hepatocyte lectin-2/3 Purification f r o m rabbit testis and identification as a zona binding protein

Munir Abdullah*, Esther E. Widgren and Michael G. O'Rand Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; * Present address: Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA

Received 21 August t990; accepted 4 December 1990

Key words: lectins, zona pellucida, asialogylcoprotein receptor

Abstract

In rat liver the asialoglycoprotein receptor is composed of three polypeptides, RHL-1, RHL-2 and RHL-3 [6]. In rat testis and spermatozoa a galactosyl receptor (RTG-r) which is immunologically related to RHL-2/3 has been described [7]. We now report that in addition to its presence in the rat, an antigenic species of 54 kDa related to RHL-2/3 is present on rabbit, human, pig and mouse spermatozoa. Purified rabbit testis galactosyl receptor (RbTG-r) consists of two major proteins of 54 and 49 kDa, while purified rabbit liver galactose lectin consists of two major proteins of 43 and 40 kDa. In an ELISA the purified rabbit testis galactosyl receptor was shown to bind biotinylated heat solubilized rabbit zonae, while the purified liver galactose lectin did not. We conclude that one of the mammalian sperm's zona binding proteins is a galactose lectin of 54 kDa related to rat liver RHL-2/3.

Introduction

Mammalian hepatic asialoglycoprotein receptor is a membrane glycoprotein which mediates the specific internalization and lysosomal degradation of a wide range of glycoconjugates [for review see 1]. The receptor is specific for terminal galactose or N-acetylgalactosamine residues [2] and was origi- nally detected by the ability of cells to selectively endocytose desialylated glycoproteins which have galactose as their terminal sugar [1]. The receptor has been purified from rabbit, human and rat liver [3-5] and is composed of multiple polypeptide species. In rat liver, the asialoglycoprotein receptor is composed of three polypeptide species; a major form referred to as rat hepatic lectin 1 (RHL-1) which has an apparent molecular mass of 41.5 kDa

and two minor forms referred to as RHL-2 and RHL-3 (rat hepatic lectin 2 and 3) which have molecular masses of 49 and 54 kDa respectively [6]. RHL-2 and RHL-3 posses the same polypeptide backbone but differ in their degree of glycosylation and are distinct from RHL-1 [2].

In rat testis and spermatozoa a galactose binding protein referred to as rat testis galactosyl receptor (RTG-r), has been described which has the same molecular mass and is immunologically related to RHL-2/3 [7]. Although no function has been as- cribed to RTG-r, it is possible that the testis en- codes a protein which is present on spermatozoa and which functions as a lectin to bind the galactose residues of the zona pellucida.

Lectins are considered to play an important role during cellular adhesion processes including inter-

156

action between sperm and egg [for review, see 8, 9]. Indeed in the mouse, galactose is present at the nonreducing terminal of the egg's sperm receptor (ZP3) [10]. On rabbit spermatozoa a lectin-like membrane glycoprotein, RSA (rabbit sperm autoantigen), is present which binds sulfated, complex carbohydrates with high affinity and mono- saccharides such as galactose with relatively lower affinity [11]. RSA functions as a high affinity zona binding protein, but additional zona binding proteins with undetermined affinity are also present on rabbit spermatozoa [12]. Included in this class of zona binding proteins is a 54 kDa protein which had been identified by zona blotting [12].

Both RTG-r and the rabbit 54 kDa zona binding protein might share similar galactose binding properties. Consequently, our aim in this study was to determine the presence of a RTG-r equivalent protein on rabbit spermatozoa and subsequently to purify rabbit galactosyl receptor from rabbit testis. We also wanted to compare the rabbit testis lectin with rabbit liver galactose lectin in order to establish any relationship between the two proteins, as reported for RHL-2/3 and RTG-r [7]. We now report that in addition to its presence in the rat, a protein related to RHL-2/3 is present on spermatozoa of other mammalian species. Purified rabbit testis galactosyl receptor (RbTG-r) has the same molecular mass as reported for both RHL-2/3 and RTG-r and it shares common antigenic determi- nants with these proteins. Purified RbTG-r is different from rabbit liver galactose lectin in molecular mass and the two proteins are immunologically unrelated.

Materials and methods

Materials Frozen rabbit testes were purchased from Pel- Freez Biologicals, Rogers, AR. Ejaculated pig spermatozoa were obtained from North Carolina State University, human ejaculated spermatozoa were obtained from the UNC-fertility clinic, rabbit spermatozoa were collected by artificial vagina and mice spermatozoa were collected from the epididy-

mides. Reagent-grade chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

Protein estimation on lysates and purified samples was done using the BCA protein assay (bi- cinchoninic acid; Pierce, Rockford, IL) with bovine serum albumin as a standard. The protein silver staining kit was purchased from Bio-Rad (Rockville Center, NY).

Preparation of tissue and cell lysates Testis and sperm lysates were prepared in 0.05 M Tris-HCl buffer, pH 8.5 containing 50mM NaC1, 5 mM PMSF and 1% Triton X-100. Sperm samples were centrifuged (700 g, 10 rain) and the pellet was washed three times in phosphate buffered saline (PBS; 10 ml, room temperature) before adding the lysis buffer and sonicating on ice for 3 min. The sonicate was centrifuged at 16,500g for 5 min before use.

Purification procedure Galactose lectin was purified from rabbit and rat liver (RHL-2/3) and from rabbit testis on two galactose-Sepharose columns according to the method of Hudgin et al. [3]. Asialoorosomucoid Sepharose was replaced with galactose-Sepharose as the affinity resin [2], which was prepared as described by Fornstedt and Porath [13]. Purification from liver samples was done using 5 g of acetone powder. For purification from testis, 20g of acetone powder (from 100 testes) were used per preparation.

SDS-PA GE electrophoresis and immunoblotting Protein samples were analyzed on one dimensional 10% acrylamide gels [14] under reducing and denaturing conditions, and the gels were stained with either Coomassie blue or silver.

Western blot analysis was carried out according to the method of Towbin et al. [15]. Purified protein samples or cell lysates were resolved on 10% SDS- PAGE and transfered to Immobilon (Millipore, Medford, MA). Reactive sites on the membranes were blocked for 1 h at 37 ° C with 5% BSA in TBS (Tris-buffered saline). Blots were then incubated overnight at 4°C with either rabbit anti-RHL-2/3 (1:250 dilution) or mouse anti-rabbit liver galac-

tose lectin (1:100 dilution) in 5% bovine serum albumin. Immunoreactivity was detected by incu- bating blots for 1 hr at room temperature in peroxidase conjugated goat anti-rabbit Ig or goat anti- mouse Ig as the second antibody (1 : 500 dilution). Blots were developed using diaminobenzidine as substrate.

Production of antisera Antibodies to purified rabbit liver galactose lectin were prepared in Balb/c mice. 10 tzg of protein % in complete Freund's adjuvant were used for initial immunization. Mice received a booster dose with 10 Ixg of protein in incomplete Freund's adjuvant 4 weeks after the initial injection. Two additional booster injections using the same amount of protein were given at two week intervals. Blood was collected from the tail eight days after the last booster. After serum separation, the immunoglobulin fraction was prepared by ammonium sulfate precipitation. Affinity purified rabbit antisera to RHL-1 and RHL-2/3 were kindly provided by Dr. Kurt Drickamer, College of Physicians & Sur- geons, Columbia University, New York.

Elisa Enzyme-linked immunosorbent assays were per- formed using serial dilutions of protein (testis galactosyl receptor, and two control proteins: liver galactose lectin, and transferrin) from 0.1/xg to 1 gg, air dried overnight in 96 well assay plates (Corning Easy-Wash). The plates were blocked for 1 1/2 hr with 5% BSA/TBST (bovine serum albumin/tris buffered saline + 0.05% Tween-20) and incubated for 1 hr with 200 ~l/well of a 1/800 dilution of biotinylated heat solubilized rabbit zonae pellucidae in BSA: LB (1 : 4; BSA: Loading buffer, 0.01M Tris-C1, pH 7.8, 0.05M CaC12, 1.25M NaC1, 0.5% Triton-X 100). Plates were washed three times in PBS/Tw (phosphate buffered saline + 0.05% Tween-20), incubated for lhr with avidin-peroxidase (1/500) in BSA/LB (1:4), washed two times in PBS/Tw, once with PBS and finally incubated with TMB substrate (3,3', 5,5'- tetramethylbenzidine; 200/xl/well; Kirkegaard- Perry Laboratories, Inc., Gaithersburg, MD). The

157

reaction was stopped with 100 txl 1N HC1 and the plate read at 450 nm. Heat solubilized rabbit zonae pellucidae were biotinylated using 120 ixl of a 4 rag/ ml solution of d-biotin p-nitrophenyl ester in DMSO per ml of protein solution for 4 hr at room temperature followed by extensive dialysis against PBS in a modification of the procedure of Bayer et al. [16]. The proper dilution of biotinlylated zonae to use in the assay was determined experimentally and varied slightly with different zona batches.

Results

RHL-2/3 related antigen in testis and spermatozoa To determine whether a protein species cross reactive with antibodies to RHL 2/3, as reported for rat testis and spermatozoa [7], is also present in spermatozoa and testis of other mammalian species Western blots were prepared. Figure 1 shows that an antigen with a molecular mass of 54 kDa, which on SDS-PAGE migrated to the same position on the gel as RHL 2/3 (lane 1), is recognized by anti- RHL 2/3 in rabbit, human, pig and mouse sperm lysates (lanes 3-6). A similar immunoreactive band was also detected in rabbit testis lysates (lane 2). An additional cross reactive band in testis lysates (small arrowhead in Fig. 1, lane 2), slightly lower in apparent molecular mass, was identified as rabbit IgG (heavy chain) on blots stained with second antibody only (data not shown).

Rabbit liver and testis galacatose lectins are different in size and are immunologically unrelated Since rat testis galactosyl receptor has the same molecular mass and is immunologically related to rat liver RHL-2/3, galactose lectins from rabbit liver and testis were purified to establish any simi- larities in molecular mass and antigenicity. Analy- sis of purified rabbit liver galactose lectin on SDS- PAGE (Fig. 2) showed that the protein was resolved into three bands. The two major bands had apparent molecular masses of 43 kDa and 40 kDa whereas the minor band had an apparent molecular mass of 92 kDa. The 40 kDa band appeared to be the more abundant species based on protein stain-

158

Fig. 1. Western blot analysis of rabbit testis and mammalian sperm lysates: Lysates were prepared in 0.05 M Tris-HC1 buffer, pH 8.5 containing 50mM NaC1, 5mM PMSF and 1% Triton X-100. Samples were resolved on SDS-PAGE under reducing and denaturing conditions and transfered to Immobilon. The blot was probed with anti-RHL 2/3 (1 : 250 dilution). Lane 1 contains 12/zg of purified RHL-2/3. The large arrowhead indicates the 54 kDa band (RHL-3) common to all spermatozoa and testis. Lane 2 contains rabbit testis lysate (100/xg protein). The small arrowhead indicates rabbit IgG which was present in the lysate and identified by second antibody only staining (goat anti-rabbit immunoglobulin). Lanes 3-6 contain lysates from rabbit, human, pig and mouse sperm respectively (100/~g protein each lane). Four separate blots have been aligned.

ing of the gel. On silver stained gels the 40 kDa protein band f rom purified rabbit liver was resolved into two closely migrating bands with apparent molecular masses of 40 kDa and 38 kDa (Fig. 3, lane t). The abundance of the 40 kDa band may not have allowed the detection of the 38 k D a band on Commassie stained gels, or the 38 kDa may repre- sent a b reakdown product of the 4 0 k D a band. Purified rabbit testis galactosyl receptor on SDS-

P A G E exhibited predominant ly two bands having apparent molecular masses of 54 kDa and 49 kDa (Fig. 3, lane 2). Western blot analysis of purified rabbit liver galactose lectin with anti-liver galactose lectin antiserum showed strong reactivity but rabbit testis galactosyl receptor was not recognized by this same antisera (Fig. 4).

Fig. 2. SDS-PAGE analysis of purified rabbit liver galactose lectin (Commassie blue stained gel). Lane 1 contains 25 tzg of a purified sample of the rabbit liver protein run under reducing and denaturing conditions. Two major proteins of 43 kDa and 40kDa (large arrowheads) and one minor protein of 92kDa (small arrowhead) are seen. Lane 2 contains the molecular weight marker proteins: phosphorylase b (94kDa), albumin (67kDa), ovalbumin (43kDa), carbonic anhydrase (30kDa) and trypsin inhibitor (20.1 kDa) at the dye front.

Rabbit testis galactosyl receptor is antigenically related to R H L 2/3 Western blot analysis of purified rabbit testis galactosyl receptor with anti-RHL-2/3, showed that the 54 kDa protein band was recognized by this antiserum (Fig. 5). Ant i -RHL-1 did not recognize testis galactosyl receptor. Consequently, it appears that purified R b T G - r and the 54 kDa proteins in spermatozoa and testis lysates are all recognized by an t i -RHL 2/3 and therefore share antigenic deter-

minants.

Rabbit testis galactosyl receptor is a zona binding protein E L I S A of the purified liver and testis lectins using

159

Fig. 3. SDS-PAGE analysis of rabbit liver galactose lectin and rabbit testis galactosyl receptor. Samples were run under reducing and denaturing conditions and the gel was stained with silver. Lane 1 contains purified galactose lectin from rabbit liver (12 tzg)- Two major protein bands are seen at 43 kDa, 40 kDa (see Fig. 2). A 38 kDa and a minor band at 92 kDa are also seen (arrowheads). Lane 2 contains purified galactosyl receptor from rabbit testis (5 ~g; RbTG-r). Two major protein bands are seen at 54 kDa and 49 kDa (arrowheads).

biot inylated heat solubilized zona as a p robe shows that the zona binds to the rabbit testis lectin but not

to the l iver lect in or to an unre la ted protein, transferrin (Fig. 6). Similarly, when purif ied R b T G - r , resolved on S D S - P A G E and t ransfered to Immobi -

lon, was p robed with biot inylated zona, bo th the

54 k D a and 49 k D a prote in bands bound zonae (da-

ta no t shown).

Discussion

This repor t has demons t r a t ed that a prote in related to the as ia loglycoprotein receptor found in rat testis and spe rma tozoa is present in rabbit testis and

Fig. 4. Western blot analysis of purified rabbit liver galactose lectin and rabbit testis galactosyl receptor (RbTG-r). Samples were resolved on SDS-PAGE under reducing and denaturing conditions, transfered to Immobilon and immunoreacted with antibodies to purified rabbit liver galactose lectin (1 : 100 dilution). Lane 1 contains RbTG-r (10/xg). RbTG-r does not react with anti-rabbit liver galactose lectin. Lane 2 contains rabbit liver galactose lectin (20/zg). Two major bands at 43 kDa and 40kDa and a minor band at 92kDa are seen (arrowheads).

rabbit, human, pig and mouse spermatozoa . The

purified rabbit testis galactosyl receptor (RbTG-r )

is immunological ly distinct f rom rabbit liver hepa-

tocyte lectin and is immunological ly cross reactive with rat liver RHL-2/3 but not with RHL-1 . Previ-

ous work [7] demons t ra ted that R T G - r was present on rat spe rmatozoa and speculated that it might be

involved in sperm-zona interaction. W e have ex-

t ended that repor t with the demons t ra t ion that a

R b T G - r exists in several species of mammal ian sperm and is a zona binding prote in identical in

molecular mass to the 54 k D a zona binding prote in previously identified on zona blots [12].

Consistent with our finding of a 54 k D a molecule in mouse spe rmatozoa (Fig. 1, lane 6) is a recent repor t [17] that the mouse zona pellucida glycoprotein, ZP-3, preferential ly binds a mouse sperm pro-

160

Fig. 5. Western blot analysis of purified RbTG-r. RbTG-r (12/xg) was run on SDS-PAGE under reducing and denaturing conditions and transfered to Immobilon. Blot was probed with anti-RHL-2/3 antibodies (1 : 250 dilution) and reactivity detected using peroxidase conjugated goat anti-rabbit Ig (1 : 500 dilution) as the second antibody. The 54 kDa band is recognized by the antiserum (arrowhead).

tein of approximately 56 kDa. In the rat both RHL-1, the major form and

RHL-2/3, the minor form, are thought to be required in order for incorporation of desialylated glycoproteins into lysosomes to occur [18]. Simi- larly, in the human both H1 and H2 are required for internalization of desialylated glycoproteins [19]. Consequently, since only the 54 kDa form is present in spermatozoa, RbTG-r may serve only as a lectin to bind galactose and/or N-acetylgalactosamine of the zona pellucida and may not be capable of functioning as a receptor for internalization of ligands.

Both human [19] and rat [2] receptors have been reported to be in oligomeric complexes when present in membranes, although RHL-1 and RHL-2/3

1.2 i

1.0

0.8

0.6

0.4 ~

0.2 011 0.01

ug ANTIGEN PLATED

Fig, 6. ELISA of rabbit testis RbTG-r, rabbit liver galactose lectin and rabbit transferrin binding biotinylated rabbit zonae pellucidae. RbTG-r, rabbit liver galactose lectin and transferrin were plated on a 96 well assay plate and probed with biotin labeled heat solubilized whole rabbit zonae. ( V ) = RbTG-r; (e) = liver galactose lectin, (O) = transferrin.

appear only to self-associate into homooligomers [2]. In an analogous fashion, the sperm lectin-like protein RSA has been shown to be present in large aggregates in the plasma membrane following the acrosome reaction [20]. Moreover, it is the aggre- gated form of RSA which demonstrates high affinity binding for zona pellucida [11]. It might be expected, therefore, that the sperm RbTG-r would aggregate upon interaction with the zona ligand. RHL-2/3 is known to be a transmembrane protein with its C-terminal and carbohydrate binding do- main external to the plasma membrane and its N-terminal in the cytoplasm [2]. It has been sug- gested that RHL-2/3 is topologically prominent in the hepatocyte membrane [21] and that clustering of galactose within a large ligand, as might occur on the zona pellucida, would lead to specific changes in receptor organization [21]. We would postulate that this might be the case on the sperm surface for RbTG-r.

Acknowledgements

The authors thank Dr. K. Drickamer, Columbia University for his kind gift of antisera and Dr. R. Richardson for reading the manuscript and help with the figures. Supported by NIH grant HD 14232 and HD 23755 to M.O'R.

References

1. Ashwell G, Harford J: Carbohydrate-specific receptors of the liver. Ann Rev Biochem 51: 531-554, 1982

2. Halberg DL, Wager RE, Farrell DC, Hildreth J, Quesen- berry MS, Loeb JA, Holland EC, Drickamer K: Major and minor forms of the rat liver asialoglycoprotein receptor are independent galactose-binding proteins. J Biol Chem 262: 9828-9838, 1987

3. Hudgin RL, Pricer WE, Ashwell G, Stockert RJ, Morell AG: The isolation and properties of a rabbit liver binding protein specific for asilaoglycoprotein. J Biol Chem 249: 5536-5543, 1974

4. Tanabe T, Pricer WE, Ashwell G: Subcellular membrane topology and turnover of a rat hepatic binding protein specific for asialoglycoproteins. J Biol Chem 254: 1038- 1043, 1979

5. Baenziger JU, Maynard Y: Human hepatic lecfin: Phy- siochemical properties and specificity. J Biol Chem 255: 4607-4613, 1980

6. Drickamer K, Mamon IF, Binns G, Leung JO: Primary structure of rat asialoglycoprotein receptor. Structural evi- dence for multiple polypeptide species. J Biol Chem 259: 770-778, 1984

7. Abdullah M, Kierszenbaum AL: Identification of rat testis galactosyl receptor using antibodies to liver asialoglycoprotein receptor: Purification and localization on surfaces of spermatogenic ceils and sperm. J Cell Biol 108: 367-375, 1989

8. Lis H, Sharon N: Lectins as molecules and as tools. Ann Rev Biochem 55: 35-67, 1986

9. O'Rand MG: Sperm-egg recognition and barriers to in- terspecies fertilization. Gamete Res 19: 315-328, 1988

10. Bleil JD, Wassarman PM: Galactose at the nonreducing

161

terminus of O-linked oligosaccharides of mouse egg zona pellucida glycoprotein ZP3 is essential for the glycoprotein's sperm receptor activity. Proc Natl Acad Sci USA 85: 6778-6782, 1988

11. O'Rand MG, Widgren EE, Fisher SJ: Characterization of the rabbit sperm membrane autoantigen, RSA, as a lectin- like zona binding protein. Dev Biol 129: 231-240, 1988

12. O'Rand MG, Matthews JE, Welch JE, Fisher SJ: Identifi- cation of zona binding proteins of rabbit, pig, human and mouse spermatozoa on nitrocellulose blots. J Exp Zoo1235: 423-428, 1985

13. Fornstedt N, Porath J: Characterization of a new lectin found in seeds of Vicia ervilia. FEBS Lett 57: 187-191, 1975

14. Laemmli UK: Cleavage of structural proteins during as- sembly of the head of bacteriophage T-4. Nature (Lond.) 227: 680-685, 1970

15. Towbin HT, Staehlin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350-4354, 1979

16. Bayer EA, Skutelsky E, Wilchek M: The avidin-biotin complex in affinity cytochemistry. Methods Enzymol 62: 308-315, 1979

17. Bleil JD, Wassarman PM: Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking. Proc Natl Acad Sci USA 87: 5563--5567, 1990

18. McPhaul M, Berg P: Formation of functional asialoglycoprotein receptor after transfection with cDNAs encoding the receptor proteins. Proc Natl Acad Sci 83: 8863-8867, 1986

19. Bischoff J, Libresco S, Shia MA, Lodish HF: The H1 and H2 polypeptides associate to form the asialoglycoprotein receptor in human hepatoma cells. J Cell Biol 106: 1067- 1074, 1988

20. Esaguy N, Welch JE, O'Rand MG: Ultrastructural map- ping of a sperm plasma membrane autoantigen before and after the acrosome reaction. Gamete Res 19: 387-399, 1988

21. Lee RT, Lee YC: Affinity labeling of the galactose/N- acetylgalactosamine-specific receptor of rat hepatocytes: preferential labeling of one of the subunits. Biochem 26: 6320-6329, 1986

Address for offprints: M.G. O'Rand, Dept. Cell Biology and Anatomy, CB # 7090, 210 Taylor Hall, University of North Carolina, Chapel Hill, N.C. 27599, USA

a mammalian sperm lectin related to rat hepatocyte lectin-2/3

Documents