3980
Biochemistry 1996, 35, 3980-3985
Characterization of a Mouse ZP3-Derived Glycopeptide, gp55, That Exhibits Sperm Receptor and Acrosome Reaction-Inducing Activity in Vitro Eveline S. Litscher‡,§ and Paul M. Wassarman*,§ Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110 ReceiVed NoVember 15, 1995; ReVised Manuscript ReceiVed January 29, 1996X
ABSTRACT:
During fertilization, free-swimming mouse sperm bind to mZP3 (∼83 000 Mr), one of three zona pellucida glycoproteins, and once bound undergo the acrosome reaction, a type of cellular exocytosis [Wassarman, P. M., & Litscher, E. S. (1995) Curr. Top. DeV. Biol. 30, 1-19]. Sperm recognize and bind to specific serine/threonine-linked oligosaccharides located at the mZP3 combining site for sperm. Here, we examined certain characteristics of gp55, a ∼55 000 Mr glycopeptide derived from the carboxyterminal half of mZP3 polypeptide to which sperm bind [Rosiere, T. K., & Wassarman, P. M. (1992) DeV. Biol. 154, 309-317]. gp55 is heterogeneous with respect to Mr (∼47 000-62 000 Mr) and has a relatively low pI (∼4.3-4.5) compared to the polypeptide portion of the glycopeptide (pI ∼6.5). gp55 inhibits binding of sperm to eggs (i.e., exhibits sperm receptor activity) and induces sperm to undergo the acrosome reaction in Vitro at about the same concentrations required for intact mZP3 (∼50-200 nM). Each of three different size-fractions of gp55, separated by SDS-PAGE, also exhibits bioactivity in Vitro. Removal of asparagine-linked (N-linked) oligosaccharides from gp55, by extensive digestion with N-glycanase, reduces its Mr to ∼21 000 and increases its pI to ∼5.3, but does not significantly affect its ability to inhibit binding of sperm to eggs or to induce sperm to undergo the acrosome reaction. Similarly, digestion of gp55 with either endo-β-galactosidase or neuraminidase alters its Mr and/or pI, but does not significantly affect either of its bioactivities. These observations are consistent with the proposal that neither N-linked oligosaccharides nor sialic acid is an essential element of the mZP3 combining site for sperm. They also indicate that a relatively small mZP3 glycopeptide is able to induce sperm to undergo the acrosome reaction (i.e., cellular exocytosis) in Vitro.
Mammalian eggs are surrounded by a thick, transparent extracellular coat, called the zona pellucida (ZP;1 Gwatkin, 1977; Dietl, 1989; Dunbar & O’Rand, 1991; Yanagimachi, 1994). Generally, the ZP is composed of three glycoproteins, called ZP1-3, that form a loose meshwork of interconnected filaments (Greve & Wassarman, 1985; Wassarman, 1988, 1993; Wassarman & Mortillo, 1991). In mice, hamsters, and many other mammals, including human beings, free-swimming sperm recognize and bind to ZP3 to initiate the fertilization process (Bleil & Wassarman, 1980; Wassarman, 1990, 1993; Wassarman & Litscher, 1995). Mouse ZP3 (mZP3; ∼83 000 Mr) is an acidic glycoprotein (pI ∼4.5) that consists of a ∼44 000 Mr polypeptide (pI ∼6.5), three or four complex-type, asparagine-linked (Nlinked) oligosaccharides, and an undetermined number of serine/threonine-linked (O-linked) oligosaccharides (Salzmann et al., 1983; Wassarman, 1988, 1993). Purified mZP3 inhibits binding of sperm to eggs and induces sperm to undergo the acrosome reaction in Vitro (Bleil & Wassarman,
1980, 1983; Wassarman, 1990). Acrosome-intact sperm apparently recognize and bind to specific O-linked oligosaccharides located at the mZP3 combining site for sperm (Florman & Wassarman, 1985; Bleil & Wassarman, 1988; Kinloch et al., 1995; Wassarman, 1989, 1990, 1992; Litscher & Wassarman, 1993). Previously, we reported that digestion of purified mZP3 by either papain or V8 protease produced an ∼55 000 Mr glycopeptide that was derived from the carboxy-terminal half of the polypeptide and inhibited binding of sperm to eggs in Vitro (Rosiere & Wassarman, 1992). Here, following digestion of mZP3 by papain, we purified the glycopeptide, called gp55, in order to assess the potential role of N-linked oligosaccharides, sialic acid, and other factors in its bioactivities. In addition, we asked whether this portion of mZP3 was sufficient to induce sperm to undergo the acrosome reaction in Vitro. A preliminary report of these results has appeared (Litscher & Wassarman, 1994). MATERIALS AND METHODS
* Author to whom correspondence should be addressed. ‡ E.S.L. was supported in part by a postdoctoral fellowship from the Swiss National Science Foundation. § Present address: Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029-6574. X Abstract published in AdVance ACS Abstracts, March 15, 1996. 1 Abbreviations: ZP, zona pellucida; PBS, phosphate-buffered saline; PVP, polyvinylpyrrolidone; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; N-linked, asparagine-linked; Olinked, serine/threonine-linked; N-GLYase, peptide-N4-(N-acetyl-βglucosaminyl)asparagine amidase; EBGase, endo-β-galactosidase; NEURase, neuraminidase.
0006-2960/96/0435-3980$12.00/0
Enzymes and Chemicals. Immobilized papain (EC 3.4.22.2; Pierce), N-glycanase (N-GLYase; FlaVobacterium meningosepticum; EC 3.5.1.52 and 3.2.2.18; Genzyme), endo-βgalactosidase (EBGase; Bacteroides fragilis; EC 3.2.1.103; Oxford GlycoSystems), and neuraminidase (NEURase; Arthrobacter ureafaciens; EC 3.2.1.18; Oxford GlycoSystems) were purchased from commercial sources. Ampholytes (Bio-Lyte 3/10 and 4/6; Bio-Rad), molecular weight standards (Bio-Rad), and carrier-free Na125I (Amersham) also were purchased from commercial sources. © 1996 American Chemical Society
Characterization of mZP3-Derived gp55 Collection and Culture of Gametes and Embryos. Sperm, ovulated eggs, and fertilized eggs were obtained from randomly bred, Swiss albino mice (CD-1; Charles River Breeding Labs) and cultured in Vitro, essentially as previously described (Bleil & Wassarman, 1980; Florman & Wassarman, 1985; Moller et al., 1990). Purification of Radiolabeled gp55. Production of 125Ilabeled gp55 and purification of the glycopeptide by HPLC were carried out essentially as previously described (Rosiere & Wassarman, 1992). mZP3 was purified by HPLC from ZP that were isolated by Percoll (Sigma) gradient centrifugation of ovarian homogenates (Bleil & Wassarman, 1986; Bleil et al., 1988). Purified mZP3 was radioiodinated in 0.2 M sodium phosphate (pH 6.8)/0.1% SDS in the presence of carrier-free Na125I (specific activity, 14-16 mCi/µg; sufficient to convert all five mZP3 tyrosine residues to either monoiodo- or diiodotyrosine) and mixed with unlabeled mZP3 (1:10 labeled:unlabeled ratio). The mixture was digested with immobilized papain (250 µg/mL gel), at an enzyme-to-mZP3 ratio of 1:2 by weight, in 50 mM Hepes (pH 6.1)/10 mM EDTA/20 mM cysteine/0.1% SDS, at 37 °C for 2 h, with vortexing for a few seconds every 20 min. Protease was separated from mZP3 by centrifugation and gel-filtration (Bio-Gel P-2) into 0.2 sodium phosphate (pH 6.6)/0.1% SDS, followed by HPLC fractionation (Bio-Sil SEC-250 column, 300 × 7.8 mm; flow rate, 0.1 mL/min). Aliquots were analyzed in a gamma counter, to estimate recovery, and then examined by SDS-PAGE and autoradiography. Fractions containing gp55 were pooled and dialyzed extensively, first against 8 M urea and then against distilled water. High-Resolution Two-Dimensional Electrophoresis. 125Igp55 and partially deglycosylated 125I-gp55 were subjected to isoelectric focusing and SDS-PAGE by using a Mini Protean II-2D cell according to procedures described in the supplier’s (Bio-Rad) guide [procedures based on O’Farrell (1975)]. Gels were stained with silver to visualize internal standards (Bio-Rad) and subjected to autoradiography to identify the positions of gp55 and glycosidase-treated gp55. Fractionation of gp55 into Molecular Weight Classes. Purified 125I-gp55 was subjected to SDS-PAGE followed by autoradiography of the wet gel. The region of gel containing 125I-gp55 was excised and sliced into three fractions based on molecular weight (high, ∼60 000 Mr; medium, ∼55 000 Mr; low, ∼49 000 Mr), and each fraction was electroeluted from the gel slice (Centrilutor, Centricon30; Amicon) and dialyzed extensively, first against 8 M urea and then against distilled water. Treatment of gp55 with Glycosidases. Digestions of 125Igp55 with glycosidases were carried out at 37 °C for 48 h. Typically, ∼1 µg of radiolabeled gp55 was incubated with enzyme in a total volume of 10 µL (2 µL of enzyme plus 8 µL of buffer): 0.5 unit of N-GLYase in 20 mM sodium phosphate (pH 7.5); 8 milliunits of EBGase in 50 mM sodium acetate (pH 5.8)/250 µg/mL BSA/1 mM NaCl; 40 milliunits of NEURase in 0.1 M sodium acetate (pH 5.0). Digestions were terminated by addition of 40 µL of distilled water and boiling the sample for 5 min, followed by extensive dialysis against distilled water. BioactiVity Assays. gp55 and its derivatives were tested for their ability to bind to sperm and, thereby, prevent sperm from binding to ovulated eggs in Vitro (“competition assay”), essentially according to the procedure described previously (Bleil & Wassarman, 1980; Florman & Wassarman, 1985;
Biochemistry, Vol. 35, No. 13, 1996 3981 Moller et al., 1990; Litscher et al., 1995). The concentration of gp55 used in the experiments was estimated by gammacounting and autoradiography of gels. Briefly, papain digests of 125I-mZP3 were subjected to one-dimensional SDS-PAGE followed by autoradiography, and the cpm associated with the entire digest as well as the cpm associated with gp55 were determined. Such measurements permitted an estimate to be made of the amount of gp55 present (25-30% of total digest). Capacitated sperm (10 µL) were incubated with gp55, or derivatives of gp55 (dissolved in 2 µL of water and 8 µL of M199-M), in a 20 µL drop, under oil, at 37 °C for 15 min. Control experiments included incubating sperm in the presence of intact mZP3, BSA, or distilled water under identical conditions. Following the 15 min incubation, 12 ovulated eggs and 2 two-cell embryos were added to the drop, and the incubation was continued an additional 4045 min. Eggs and embryos were then washed by mouthpipetting, fixed in 2% formaldehyde/PBS/PVP (pH 7.2) and M199-M (1:1), and the number of sperm bound per egg was determined by light microscopy. gp55 and its derivatives also were tested for their ability to induce sperm to undergo the acrosome reaction in Vitro, essentially according to the procedure described previously (Moller et al., 1990; Litscher et al., 1995). Briefly, sperm collected from the 20 µL drops described above (“competition assay”) were fixed and stained with Coomassie blue, and the status of the acrosome was assessed by light microscopy. RESULTS Experimental Rationale. Previously, we reported that a ∼55 000 Mr glycopeptide, excised from the carboxy-terminal half of mZP3 by either papain or V8 protease, inhibited binding of sperm to eggs (“sperm receptor activity”) in Vitro (Rosiere & Wassarman, 1992). However, we did not determine whether or not the glycopeptide, like intact mZP3, could induce sperm to undergo the acrosome reaction subsequent to binding. Furthermore, we did not characterize the glycopeptide with respect to the potential role of N-linked oligosaccharides and/or other moieties in its bioactivities. Experiments that address these issues are presented here. It should be noted that all molecular weights reported here should be considered as “apparent molecular weights,” since it is well known that glycoproteins and glycopeptides migrate anomalously on SDS-PAGE due, in part, to a low chargeto-mass ratio for the molecules (Leach et al., 1980). Production and Electrophoretic Analysis of gp55. Papain digests of purified, radiolabeled mZP3 were fractionated by HPLC on a size-exclusion column, and aliquots of fractions were analyzed by SDS-PAGE and autoradiography, as described under Materials and Methods. As seen in Figure 1, an mZP3 glycopeptide with an average Mr of ∼55 000 (gp55) was a major digestion product. In addition to gp55, the digests contained mZP3 glycopeptides with average Mrs of ∼26 000, ∼13 000 and
