Materials

Ascidians, Halocynthia roretzi, were collected from the Gulf of Onagawa, Japan.

Restriction enzymes and modified enzymes were obtained from Takara and Toyobo.

Isolation of HrFuc'ase cDNA

Total RNA was prepared from growing testes by the AGPC (acid guanidine phenol chloroform) method (Sambrook et al., 1989) and poly(A)-rich RNA was purified with oligoTex beads (Roche Japan). First-strand cDNA synthesis was performed at 37°C for 1 h using Superscript II (GIBCO-BRL) with a random hexamer (Takara). The degenerate primers deduced from conserved region in α-l-fucosidase of Dictiosterium, rat and human were sense primer F1 (5′-GGNGCNAARTAYGTNGTNYTNAC-3′) and anti-sense primer F2 (5′-CCCCANCKRTCATTNACNAYNAC-3′) (Ficher and Aronson, 1989, White et al., 1985, Muller-Taubenbergrt et al., 1989). The PCR conditions were 30 cycles of 94°C for 30 sec., 48°C for 30 sec., 74°C for 30 sec.

The full-length of HrFuc'ase was amplified with the design of 5′ RACE (Rapid amplification of 5′-cDNA ends) and 3′RACE (Rapid Amplification of 3′-cDNA Ends) primers, as well as appropriate nested primers. The primers were: 5′RACE primer; 5′-TGTCGACTGCGTTCCAGTTC-3′, 5′ nested primer, 5′-CAATTAGTAAATCCTTCGTGA-3′. On 3′RACE, poly(A)⁺ RNA was made from growing testes and used to synthesize single strand cDNA with the dT17-adaptor primer, 5′GACTCGAGTCGACATCGAT173′. The synthesis of the second strand and the further PCR reactions were carried out with the adapter primer; 5′GACTCGAGTCGACATCGA3′ and 3′RACE primer F3; 5′GATTACTTCAGTTCTCGTGC-3′, 3′RACE nested primer F4; 5′GCTTGGTTATACAATGATAGC-3′. On 5′RACE, poly (A)⁺ RNA was made from growing testes and used to synthesize single strand cDNA with the 5′RACE primer; F5 (5′CAATTAGTAAATCCTTCGTGA-3′). This cDNA was then used as a template in a PCR reaction using the 5′primer homopolymeric dA tails added to the 3′end of the ss-cDNA using terminal deoxyribonucleotide transferase at 37°C, for 30 min. The synthesis of the second strand and additional PCR experiments were carried out with the dT17-adapter primer, the adapter primer and F5.

DNA sequencing

The PCR products were cloned into pGEM-T vector (Promega) and sequenced with M13 forward and reverse primers using SequiTherm DNA Polymerase (Epicentre Technologies) and the automated DNA sequencer, LICOR model 4000L (ALOKA). The internal sequence was obtained by nested deletion using a Takara deletion Kit.

Southern Hybridization

Genomic DNA prepared from H. roretzi muscle using standard procedures was digested for 6 hr with restriction enzymes (EcoRI, BamHI and SalI) and then loaded into 0.6% agarose gel and electrophoresed. Southern hybridization was carried out using a 450 bp PCR fragment with F1 and F2.

Preparation of fusion protein of HrFuc'ase and Antibody preparation

The 330 bp fragment consisting of 152 to 262 amino acid of HrFuc'ase was subcloned in frame into pUR292 expression vector (Rüter and Muller-Hill, 1983) with the correct orientation. E. coli F”11recA expressing β-galactosidase (β-gal) fusion protein after IPTG induction was solubilized in SDS-PAGE lysis buffer and extracts were electrophoresed on 5% SDS-PAGE (Laemmli, 1970). After staining with Coomasie Brilliant Blue (CBB; Sigma, St. Louis, MO), a high molecular weight fusion protein was cut from the gels and eluted electrophoretically. Eluted samples were dialyzed against distilled water and freeze dried.

Purified protein was emulsified in Freund's complete adjuvant (Difco, Detroit, MI), and injected subcutaneously into rabbits following a four-fold booster injection of the purified protein in Freund's incomplete adjuvant. The antiserum was characterized by Western blots of the fusion protein.

Preparation of sperm protein

Dry sperm was diluted with sonication buffer containing 10 mM citrate buffer (pH 4.5) and 10 mM NaCl. The sperm suspension was subjected to sonication for 2 min. The resultant suspension was centrifuged at 15,000 g for 20 min and then 100,000 g for 60 min, and the supernatant was collected.

Western blotting

After SDS-PAGE, the gels were stained with CBB to visualize separated proteins. For immunochemical detection of antigens, proteins were electrophoretically transferred using a semi-dry blotting system to Immobilon-P (Millipore Corp. Bedford, MA) PVDF membrane (Towbin et al., 1979). Blotted membranes were blocked for 2 hr in Tris-buffered saline (TBS, pH 7.2) containting 5% nonfat dry milk (Difco, Detroit, MI), and incubated overnight with the antiserum at 1/1,000 dilution. After rinsing with TBS containing 0.5% Tween 20, the blots were incubated for 1 hr with an Avidin-Biotin-Complex (ABC) conjugated goat anti-rabbit antibody (Vector Laboratories) at 1/2,000 dilution and positive reactions were detected with Konica Immunostaining HRP-1000 (Konica, Tokyo).

Fertilization assay

Aliquots of sperm suspension (10⁶ cells/100 μl) were incubated with antiserum against HrFuc'ase or preimmune serum for 15 min at room temperature. Various dilutions of antiserum and sperm were examined. Then, approximately 100 unfertilized eggs were added and incubated for another 10 min. For inactivation of the sperm, a final concentration of 0.04% SDS was added, incubated for 10 min and washed with sea water. After 1 hr incubation, fertilized eggs were scored.

Identification and sequence of the ascidian α-l-fucosidase cDNA clone

Using one pair of degenerate primers encoding the conserved region of the fucosidase domain [GAKYVVLT and VVVNDRW] (Ficher and Aronson, 1989; White et al. 1985; Muller-Taubenbergrt et al. 1989), a 450 bp fragment of the ascidian fucosidase homologue, named HrFuc'ase, was isolated with RT-PCR. 5′RACE was used to obtain the 5′ portion of the encoding region, and 3′RACE was used to obtain the 3′ portion of the encoding. The entire coding sequence based on the 5′ fragments obtained with RT-PCR with the conserved fucosidase domain of 150 amino acids, 5′RACE and 3′RACE suggested a protein of 466 amino acids. This open reading frame has three putative initiation codons, but we have assigned the initiation codon to the ATG at nucleotide position 261, because the 21-amino acid sequence following this ATG possessed the features characteristic of signal sequences and this ATG was flanked by sequences that fit Kozak's criteria for translation initiation codon (Kozak, 1981). The initiation was followed by an open reading frame of 1398 bp. An inframe stop codon occurred at nucleotide position 1659 and the 3′-untranslated region composed of 377 bp included polyadenylation sites (AATAAA). The deduced amino acid sequences suggested that cleavage of the signal peptide would yield a protein of 445 amino acid with a calculated molecular weight of 52.4 kDa. This signal peptide-like sequence was a typical of the membrane-spanning domain of many membrane proteins. The protein contained one α-l-fucosidase motif composed of 12 amino acids at residues 283-294. The α-l-fucosidase clone was named as HrFuc'ase.

To confirm that the cloned HrFuc'ase can bind to the fucose residue, the molecular modeling was performed using with Quanta/Charmm force files and the Protein Data Base (data not shown). The molecular model shows that it has a putative space for interaction of fucose residue thus HrFuc'ase may bind a fucose residue.

Sequence analysis of H. roretzi α-l-fucosidase

Fig. 1 shows a comparison of the amino acid sequence of HrFuc'ase with other α-l-fucosidases. HrFuc'ase shows amino acid identities related to the human (47.7%), rat (47.4%) and Dictiosterium (37.6%) with fucosidase of motifs (Ficher and Aronson, 1989; White et al. 1985; Muller-Taubenbergrt et al. 1989). The sequence PSKLQTHKWENC at residues 283–294, corresponded to the consensus sequence of the α-l-fucosidase, PXXLXXXKWEXC.

Fig. 1

Alignment of amino acid sequences of fucosidase homologue of human, rat and Dictiosterium. Amino acid identities for representative genes are compared with HrFuc'ase. The signal sequences are indicated with italics and the fucosidase motif is shown boxed.

To confirm the existence of the HrFuc'ase in the ascidian genome, southern blot analysis of the ascidian genomic DNA was performed. Hybridization with HrFuc'ase cDNA gave single bands for DNA digested with EcoRI, BamHI and SalI (data not shown), suggesting that HrFuc'ase is encoded by a single gene in the ascidian genome.

Identification of HrFuc'ase on western-blots with polyclonal antibody

To identify the gene product encoded by the HrFuc'ase cDNA, the antiserum was directed against a fusion protein. The resulting antiserum was used for the Western blot analyses of proteins extracted from mature sperm (Fig. 2). The antibody crossed with the 54 kDa band strongly and the 37 kDa band weakly. The molecular size of the band which crossed strongly with the antiserum was most similar to the molecular weight of 52.4 kDa calculated from this cDNA clone. The HrFuc'ase protein was contained more in the preparation of 100,000 × g supernatant than in 15,000 × g supernatant (Fig. 2, lane 1 and lane 2).

Fig. 2

Detection of HrFuc'ase protein in sperm using HrFuc'ase polyclonal antibody. The CBB staining (lanes 1 and 2) and the Western blot with HrFuc'ase polyclonal antibody (lanes 3 and 4) of SDS-PAGE of sperm membrane are shown. Loaded samples: 15,000 g sup (lanes 1 and 3), 100,000 g sup (lanes 2 and 4).

Inhibition of fertilization with HrFuc'ase antiserum

To elucidate the function of HrFuc'ase further, the effect of HrFuc'ase antserum upon fertilization was examined (Fig. 3). Fertilization was inhibited by HrFuc'ase antibody in a dose-dependent manner.

Fig. 3

Inhibition of HrFuc'ase polyclonal antibody on fertilization. Panel shows the effect of the antibody concentration. Control experiments are shown with ○ and the experiments with HrFuc'ase polyclonal antibody are shown with □.