From the
We have purified a sperm membrane protein, designated zonadhesin, that binds in a species-specific manner to the extracellular matrix (zona pellucida) of the egg, and cloned its cDNA. The cDNA encodes a novel protein with a single transmembrane segment separating a 36 amino acid, highly basic intracellular C terminus from a 2418-amino acid extracellular region. The extracellular sequence specifies a mosaic protein comprising a unique N-terminal domain, a mucin-like domain, and five tandem domains proximal to the membrane that are homologous to prepro von Willebrand factor. The N-terminal and mucin-like domains were absent from zonadhesin that bound to the egg extracellular matrix, suggesting that processing occurs during sperm maturation and/or capacitation. By Northern blotting and in situ hybridization, zonadhesin mRNA was detected only within the testis, where it was expressed primarily in haploid spermatids. The unique domain structure of zonadhesin suggests multiple functions, one of which is to mediate sperm adhesion to the zona pellucida.
Fertilization in both invertebrates and vertebrates is
species-specific, and events such as motility activation, gamete
adhesion, and induction of the acrosome reaction show absolute or
relative species
specificity(1, 2, 3, 4, 5) .
Thus, sperm membrane proteins that interact with the egg in a
species-specific manner are likely to be primary gamete recognition
and/or signaling components. Efforts to reach a molecular understanding
of mammalian fertilization have been hampered by the apparent
asynchrony of the sperm population; at any given time only a fraction
of spermatozoa possess the ability to fertilize the egg. To overcome
this problem we used the porcine egg extracellular matrix (zona
pellucida) as an affinity medium to isolate proteins from large
quantities of pig sperm membranes(6) . One of these proteins
bound in a species-specific manner to the egg extracellular matrix and
migrated in SDS-PAGE at M 150,000 under
nonreducing conditions and as M
105,000 and 45,000
subunits (p105 and p45) after disulfide bond reduction(6) . We
have now purified this sperm membrane protein (named zonadhesin),
obtained partial amino acid sequence, and isolated its cDNA. Zonadhesin
is expressed by the haploid spermatid and is homologous to both von
Willebrand factor and mucins.
Zonadhesin was purified to apparent homogeneity based on its zona pellucida binding activity (Fig. 1). Approximately 30 µg of p105 and 10-15 µg of p45 were obtained from 880 mg of sperm membrane protein, using 44 mg of native, particulate zona pellucida as the affinity matrix. Amino acid sequences of eight p105 and five p45 tryptic peptides (Table 1) were not present in existing protein sequence data bases, suggesting that zonadhesin was a novel protein.
Figure 1: Purification of zonadhesin. Lanes 1-3, SDS-PAGE/Western blot of sperm biotinylated proteins (disulfides reduced), detected with streptavidin-peroxidase. Lane 1, 0.2 µg of total membrane protein (starting material for purification of zonadhesin). Lanes 2 and 3, proteins from 20 µg of membrane that bound to native porcine and bovine zona pellucida, respectively. After extensive washing with detergent solutions, only zonadhesin remained bound to the porcine zona pellucida (lane 2). Zonadhesin did not bind to the bovine (lane 3) or mouse zona pellucida(6) , nor to the Xenopus laevis egg envelope (6) under these conditions. Lane 4, SDS-PAGE (disulfides reduced) of purified zonadhesin, stained with Coomassie Brilliant Blue R-250.
Degenerate oligonucleotide primers based on the sequences of two p105 tryptic peptides were used to clone cDNAs encompassing the zonadhesin coding sequence by a combination of PCR, cDNA library screening, and 5`-RACE. The 7785-base composite sequence of the cDNAs contains a single major open reading frame, and the 2476-amino acid sequence deduced from it includes the sequences of the eight p105 and five p45 peptides in its C-terminal two-thirds ( Table 1and Fig. 2).
Figure 2:
Deduced amino acid sequence of zonadhesin. a, N-terminal and mucin-like domains. The predicted signal
peptide is underlined. The mucin-like domain begins at
Ser; spaces have been inserted to delineate its
53 heptapeptide imperfect repeats. b, multiple sequence
alignment of the tandem D-domains (designated D0-D4) of
zonadhesin. Domain boundaries are the same as those defined for the
D-domains of prepro-von Willebrand factor(21) . Note the
characteristic alignment of nearly all of the cysteine residues and
presence of the CGLCG motif in D1 and D2. c, C-terminal
sequence. The transmembrane segment is underlined. In b, alignment was by the clustal method using the PAM 250
alignment matrix. Pairwise alignment parameters were: window size
= 5, gap penalty = 3. Multiple alignment parameters were:
gap penalty = 10, gap length penalty =
10.
A 29-amino acid putative signal
peptide (14) is present at the N terminus of the deduced
sequence (Fig. 2a). The sequence also predicts a
2418-amino acid extracellular region, a single membrane-spanning
segment, and a 36-amino acid intracellular C terminus containing
numerous basic residues. Cleavage of the putative signal peptide at
serine 29 would produce a 2447-amino acid mature polypeptide chain with
a calculated molecular molecular mass of 267,000 Da. This predicted
molecular mass is larger than the sum (M 150,000)
of the apparent sizes of the zonadhesin subunits. The cDNA therefore
encodes a precursor protein. Processing of this precursor apparently
involves proteolytic generation of p105 and p45 as well as removal of
approximately the N-terminal one-third of the protein. Spermatozoa
acquire the capacity to fertilize the egg during maturation in the
epididymis (1) and capacitation in the female reproductive
tract(15) ; the changes that occur in either environment could
involve proteolysis of zonadhesin. A requirement for sperm surface
proteolytic activity to facilitate sperm adhesion to the zona pellucida
has been reported(16) .
A region of highly repetitive sequence is present between amino acids 300 and 700 of zonadhesin; it comprises 53 imperfect repeats of the consensus sequence PTE(K/R)(P/T)T(V/I). Repetitive sequences rich in proline and threonine are characteristic of mucins, which are O-glycosylated on numerous serines and threonines and have extended structures owing to their high proline content(17) . These structural properties reflect the functions of mucins in regulating cellular interactions; the large, carbohydrate-rich domains extend beyond most other cell surface glycoproteins and thereby either inhibit or promote cell adhesion(17, 18) . The mucin-like domain of zonadhesin could function similarly during sperm migration through the male or female reproductive tracts, inhibiting inappropriate trapping of spermatozoa or promoting adhesion to the oviductal isthmus, which serves as a sperm reservoir in several species(19, 20) .
Five homologous domains, in
tandem, follow the mucin-like domain (Fig. 2b).
Sequence comparisons (PIR data base) revealed that these domains are
homologous to the D-domains of prepro-von Willebrand factor
(vWF)(21) . Conservation of nearly all of the cysteines within
the five domains is readily apparent, and several other amino acids are
also conserved at various positions in the sequences. The D0 domain of
zonadhesin is about one-fourth the length of the D1-D4 domains,
and it truncates at the same point in its sequence as a partial
D-domain present in prepro-vWF. The sequence CGLCG of prepro-vWF
D1-D3 domains is conserved in the zonadhesin D1 and D2 domains.
The vicinal cysteines in this sequence may mediate covalent
oligomerization of prepro-vWF monomers(22) . Four vWF-like
D-domains were recently identified in the human intestinal mucin
MUC2(23) , which also exists as disulfide-bonded oligomers and
possesses the CGLCG motif in its D1 and D3 domains. We previously
observed high M sperm proteins (SDS-PAGE,
nonreducing conditions) that bound to the porcine zona pellucida and
appeared to be covalent oligomers of zonadhesin(6) . Thus, the
CGLCG motif in the D1 and D2 domains of zonadhesin may mediate covalent
oligomerization, which in turn could be important in sperm adhesion to
the zona pellucida because of the increased binding avidity of
multivalent interactions. The zona pellucida is itself a repetitive
structure, comprising strands of noncovalently associated multimers of
ZP2-ZP3 heterodimers(24) , that could interact
productively with an oligomeric receptor. Oligomerization could also
promote membrane protein aggregation that may be important for
induction of the sperm acrosome reaction(25, 26) .
Northern blots of poly(A) RNAs from several pig
tissues detected expression of a 7.5-8 kb zonadhesin mRNA in
testis, but not in heart, liver, lung, spleen, brain, kidney, or
epididymis (Fig. 3). Because testis is a heterogeneous tissue
containing several different cell types, we localized zonadhesin
expression more precisely by in situ hybridization (Fig. 4). Some but not all of the seminiferous tubules in
testicular sections hybridized strongly with the zonadhesin probe, and
this hybridization was restricted to the germinal epithelia of the
tubules. The apparent expression of zonadhesin mRNA by only a fraction
of the tubules in a given tissue section probably reflects the
asynchrony of spermiogenesis among tubules. Within strongly hybridizing
tubules, expression was detected only in the haploid spermatids.
Spermatid-specific expression of zonadhesin supports the hypothesis
that this protein mediates sperm-specific function(s).
Figure 3:
Tissue-specific expression of zonadhesin.
Poly(A) RNAs from various pig tissues were hybridized
with a
P-labeled 900-bp zonadhesin probe. H, 4.5
µg of heart RNA; B, 4.5 µg of brain RNA; E,
3.4 µg of epididymis RNA; K, 3.3 µg of kidney RNA; Li, 2.9 µg of liver RNA; T, 3.0 µg of testis
RNA. Migration of RNA standards (Life Technologies, Inc.) is indicated
on the left (47-h exposure).
Figure 4: Localization of zonadhesin mRNA expression in pig testis by in situ hybridization. a, a hematoxylin- and eosin-stained section of pig testis viewed with bright field illumination (bar = 1000 µm). Note that the field comprises primarily seminiferous tubules, with three blood vessels and some connective tissue. b, hybridization of an antisense RNA probe to a parallel section (same field as a), viewed with dark field illumination. c, hybridization of a sense RNA probe to a parallel section (same field, illumination, and exposure as b). Comparing a-c, specific hybridization of the antisense RNA probe is confined to the seminiferous tubules (positions of two blood vessels are marked for orientation). Only a subset of seminiferous tubules express high levels of the zonadhesin mRNA. d, phase contrast view (no counterstain) of two adjacent seminiferous tubules (boxed region of b). Note the difference in the level of zonadhesin expression between the two tubules. Expression in the strongly stained tubule is high in haploid spermatids. The clear zones appearing as halos surrounding the large round nuclei of diploid spermatocytes (arrowheads) indicate an absence of expression in the cytoplasm of these cells.
The domain structures of zonadhesin, prepro-vWF, and MUC2 are illustrated schematically in Fig. 5. Each is a mosaic protein (27) and among them zonadhesin is smallest and is unique in having a putative transmembrane segment and its D-domains in tandem. The mucin-like domain of zonadhesin is also a substantially smaller fraction of the total protein than is the mucin part of MUC2, which is a prototype for secreted mucins.
Figure 5: Schematic depiction of the domain structures of zonadhesin, prepro-vWF, and MUC2. D-domains are stippled, and mucin domains are hatched. Locations of the N-domain of zonadhesin and the A-, B-, and C-domains of prepro-vWF are labeled. The MUC2 sequence (23) specifies a partial fifth D-domain spanning amino acids 757-858 similar to the D0- and D`-domains of zonadhesin and prepro-vWF, respectively, that was previously unidentified.
In addition to their functions in oligomerization, vWF D-domains bind heparin(28) . This property is of potential interest, because heparin and/or other glycosaminoglycans promote capacitation and the acrosome reaction of bovine and hamster spermatozoa in vitro(29) ; however, the molecular target(s) for these agents have not been identified. It is possible that the D-domains of zonadhesin mediate these heparin/glycosaminoglycan effects. Like heparin, zona pellucida glycoproteins contain sulfated carbohydrate(30) ; this further suggests that zonadhesin's binding activity may derive in part from interaction of its D-domains with sulfated carbohydrates in the egg extracellular matrix.
Zonadhesin differs from other potential
gamete recognition proteins of spermatozoa for which cDNAs have been
cloned(31, 32, 33, 34) . The
substrate specificity of sperm surface galactosyltransferase, a M 60,000 plasma membrane-associated variant of the
Golgi enzyme, is consistent with its hypothesized function in gamete
adhesion(35) . Monoclonal and polyclonal antibodies to PH-20, a M
56,000-64,000 sperm surface hyaluronidase,
inhibit adhesion of guinea pig spermatozoa to the zona
pellucida(32, 36) . Recently, two different proteins
potentially involved in mouse (33) and human (34) sperm-egg adhesion (sp56 and ZRK, respectively) were also
described. Bookbinder et al.(33) reported a
correlation between detectability of sp56 in spermatozoa from various
species and ability of the cells to adhere to mouse eggs; species
specificity has not been addressed for the other molecules. Thus,
zonadhesin is presently the only protein known to bind in a
species-specific manner to the egg extracellular matrix.