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research-article |
Department of Biology, University of Bergen, N-5020 Bergen, Norway
ABSTRACT
A structural analysis of the differential proteolysis of vitellogenin (Vtg)-derived yolk proteins in the maturing oocytes of a marine teleost that spawns very large pelagic eggs is presented. Two full-length hepatic cDNAs (hhvtgAa and hhvtgAb) encoding paralogous vitellogenins (HhvtgAa and HhvtgAb) were cloned from nonestrogenized Atlantic halibut, and the N-termini of their subdomain structures were mapped to the oocyte and egg yolk proteins (Yps). The maturational oocyte Yp degradation products were further mapped to the free amino acid (FAA) pool in the ovulated egg. The deduced amino acid sequences conformed to the linear NH2-(LvH-Pv-LvL-beta'-CT)-COO– structure of complete teleost Vtgs. However, the Yps did not match the expected cleavage products of complete Vtgs. Specifically, the phosvitin subdomain of the HhvtgAa paralogue remains covalently attached to the lipovitellin light chain, while the phosvitin subdomain of the HhvtgAb paralogue remains covalently attached to a C-terminal fragment of the lipovitellin heavy chain (LvH). During oocyte hydration, the LvH of the HhvtgAa paralogue is disassembled and extensively degraded to FAA. In the HhvtgAb paralogue, the LvH is nicked in the C-sheet in a manner similar to that seen in lamprey and other teleosts. A small part of the C-teminal end of the LvH-Ab undergoes proteolysis to FAA, together with the phosvitin, beta' component, and much (
65%) of the lipovitellin light chain (LvL-Ab). The independently measured FAA pool in the ovulated egg corroborates that calculated from differential proteolysis of the Yps. Based on the 3:1 (HhvtgAb:HhvtgAa) Yp expression ratio, each paralogue contributes approximately equal amounts of FAA to the organic osmolyte pool of the hydrating oocyte during maturation.
gamete biology, gametogenesis, meiosis, oocyte development, ovulation
Oocyte hydration is a developmentally programmed event that coincides with meiotic resumption in marine teleosts. We have argued that it is a vital reproductive process in marine teleosts in that it preadapts the eggs to the hyperosmotic spawning environment [1–3]. In response to external and maternal signals that involve the brain-pitutary-gonad axis, a suite of co-ordinated events are set in motion, leading to the osmotic flow of water from the maternal plasma to the oocyte via specialized aquaporins that are temporally inserted in the oocyte plasma membrane during this period [4, 5]. This provides the embryo with a vital reservoir of water until alternative osmotic mechanisms arise during embryogenesis.
In order to understand the evolution of these events and those reported herein, we have recently characterized all of the available vertebrate vtg genes (expressed transcripts and genomic variants) using Bayesian and other methods of phylogenetic inference [3]. We have found that all complete (versus phosvitin-less) teleost vtg genes appear to be derived from an ancestral vtgA type but have experienced post-R3 lineage-specific duplication to generate paralogous gene clusters (vtgAa and vtgAb), which are highly correlated to the pelagic or benthic character of the spawned egg. We have argued that the neofunctionalization of the vtgAa paralogue allows its expressed protein (VtgAa) and derivative yolk proteins (Yp) to undergo proteolysis to FAA, thereby causing hydration of the maturing oocytes. The implications of these separate findings, in conjunction with the data presented here, lead us to propose that the success of the marine teleosts (mainly Acanthomorpha) is rooted in the physiological solutions that have evolved in the pelagic egg [3]. The new gene (protein) nomenclature proposed by Finn and Kristoffersen [3], i.e., vtgAa (VtgAa) and vtgAb (VtgAb) instead of vtga (VtgA) and vtgb (VtgB), respectively (see also Materials and Methods), is adopted in the present contribution.
Our previous study [1] demonstrated that oocyte hydration in Atlantic halibut (Hippoglossus hippoglossus), which is an acanthomorph marine teleost that spawns the largest pelagic eggs known, is driven by transient hyperosmolarity of the yolk in relation to the ovarian fluid. The transient hyperosmolarity is caused by the differential uptake of inorganic ions, mainly Cl–, K+, and phosphate (Pi), and the appearance of a large pool of free amino acids (FAA). The appearance of the latter pool of FAA, the largest known for any teleost egg (2300 nmol), has been correlated to the disappearance of several oocyte Yps, in particular the 110-kDa Yp, during oocyte maturation. In the present report, a structural analysis of the differential proteolysis of dual vitellogenins (Vtg) that contribute to oocyte hydration in the maturing oocytes of Atlantic halibut is presented. The data are derived from biopsies [1] of nonestrogenized, naturally maturing females, and therefore this represents the first documentation of the natural in vivo expression ratios of Vtg-derived Yps during the maturational proteolytic events that occur in marine pelagophil teleosts.
As in other vertebrates and most invertebrates, the major Yps arise from precursor Vtgs [6–8], which are among the largest known peptide monomers with predicted molecular masses of up to 290 kDa before posttranslational modification. The precursor molecules are synthesized cotranslationally, predominantly in the liver of vertebrates, as multi-subdomain structures. Beginning at the N-terminus, the precursor of a complete vertebrate Vtg consists of a signal peptide, a lipovitellin heavy chain (LvH), a phosvitin (Pv), a lipovitellin light chain (LvL), and a von Willebrand factor type D domain (Vwfd). In teleosts, the Vwfd is cleaved into a beta component (ß') and a C-terminal coding region (CT). Once assembled, Vtgs are posttranslationally glycosylated and phosphorylated, and then secreted as dimers to the circulating plasma. Upon uptake into the ovary following clathrin-mediated endocytosis [9] and intracellular sorting to early endosomes in the oocyte [10–12], vertebrate Vtgs are consistently cited as being cleaved into lipovitellins (Lv), Pv, and ß' [8, 13].
The physiological significance of Vtg and the primary yolk derivatives is usually attributed to nutrition of the embryo in the form of yolk [14]. However, recent studies have shown that up to three forms of Vtg are expressed and differentially processed in pelagophil and benthophil teleosts, which spawn pelagic and benthic eggs, respectively [15–21]. A unique secondary proteolytic event, first noted by Wallace and Selman [22], occurs during oocyte maturation in marine teleosts. The degree of proteolysis has been shown to be related to the type of egg spawned, e.g., benthic or pelagic, and also to the differential expression of the vtg genes [15, 21, 23]. In benthic eggs, the yolk proteins are either not processed or are partially cleaved and hydrolyzed, while in pelagic eggs, the yolk proteins are not only cleaved but undergo extensive proteolysis, resulting in the buildup of the FAA pool [1, 2]. The FAA are the major osmolytes that drive the maturational influx of water via specific aquaporins [4, 5], thereby providing the early embryo of marine teleosts with a vital water reservoir before a drinking mechanism is developed [1].
Despite several examinations of the electrophoretic profiles of teleost egg proteins, it is only recently that the structural bases of the secondary cleavage events have been reported [15, 21, 23]. Direct evidence linking the different Vtg forms to the FAA pool is lacking. In the present study, molecular evidence is presented for a functional precursor-product relationship between the dual parent vtg gene transcripts, the expressed oocyte yolk proteins and the egg FAA pool. Based on recent structural studies of related vertebrate apolipoproteins and lamprey Lv [24–29], 3-D models of the paralogous halibut Vtgs and degraded Yps are presented, and these models are used to discuss the mechanism of yolk protein degradation.
Post-vitellogenic, pre-hydrated oocytes (PH ooc) and ovulated eggs (OV eggs) were dissected from biopsies obtained from female Atlantic halibut at the Austevoll Marine Research Station, Norway, as described previously by Finn et al. [1]. Samples were either fresh-frozen at –80°C or subsequently lyophilized for later analysis. Vitellogenic livers were obtained from harvested Atlantic halibut at the Austevoll Marine Research Station and immediately frozen at –80°C for later analysis. These investigations were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals, as promulgated by the Society for the Study of Reproduction.
Hepatic RNA extraction, cloning, and sequencing were performed as described by Reith et al. [17]. PolyA+ RNA was extracted from fresh-frozen (–80°C) livers of female Atlantic halibut using the FastTrack 2.0 RNA isolation kit (Invitrogen). Reverse-transcriptase-PCR was used to recover the vtg sequences. Fragments from the 5'-end of the mRNA were amplified using the primer sets vtg5', 5'-ATGARRGYGSTTKTRCT-3' and vtg2, 5'-TANACRTTYTGNGTYTTYTT-3' or vtg1, 5'-GGNYTNCCNGARGARGG-3' and vtg2, and cDNA from reverse transcriptase reactions primed with vtg2. The PCR products were purified by gel electrophoresis, cloned into pCR2.1 (Invitrogen) by TA cloning, and sequenced. The clones from the vtg1+vtg2 amplification encoded the hhvtgAa sequence, while those from vtg5'+vtg2 amplification contained the hhvtgAb sequence. The following gene-specific primers were designed from these sequences: Hal1 from hhvtgAa, 5'-CTCATCAGCGCCGCAGAGCAAA-3'; Hal2 from hhvtgAb, 5'-TGATCCATGCTG CCGCTGCCG-3'. These primers were used with an oligo(dT) anchor primer (from the Boehringer Mannheim 5'/3' RACE kit) to amplify the region between the 5'-segment and the 3'-end of the mRNA. These products were cloned into the pCR-XL-TOPO vector and sequenced. To obtain the 5'-end of the hhvtgAa sequence, 5'-RACE was carried out (Boehringer Mannheim 5'/3' RACE kit) using nested primers from the hhvtgAa sequence (Hal1-Sp2, 5'-GGTTCTGCTGGCACTTGGTA-3' and Hal1-Sp3, 5'-CTCTGGATGTTTAGCACCAGTGTG-3'). For all regions, at least four independent clones were sequenced to avoid PCR sequence artifacts. All sequencing was carried out on an ABI 373 automated sequencer with Big Dye dideoxy terminators.
Yolk proteins were solubilized from PH ooc and OV eggs as described by Finn et al. [1]. Proteins were denatured in reducing buffer (8% ß-mercaptoethanol, 20% glycerol, 2.5% SDS, 1% bromophenol blue) and separated electrophoretically using the following separating gel systems: 5–20% precast gradient Laemmli gels (ATTO AE-6050A rectangular gel system), 15% homogeneous Laemmli gels [30], and 7.5% homogeneous Tris-Tricine [31] buffer systems. These latter gels were run with 4% stacking gels and mounted in Bio-Rad Protean II vertical slab-gel systems. Visualization of protein bands was accomplished using Coomassie brilliant blue G-250, silver stains (Wako), and the Gelcode phosphoprotein staining kit (Pierce), with serine trypsin inhibitor (STI) as the negative control. This latter kit specifically stains highly phosphorylated proteins, such as Pv, by in-gel hydrolysis of phosphoprotein phosphodiester linkages using 0.5 M NaOH in the presence of calcium ions. The gel that contains the newly formed insoluble calcium phosphate is treated with ammonium molybdate in dilute nitric acid and the resultant insoluble nitrophospho-molybdate complex is stained with the basic dye methyl green. The kit states a Pv detection limit of 80 ng, although in our experiments, we needed to load the gels more heavily (25 µg per lane) followed by extended destaining to visualize the putative Pv bands. The extended destaining sometimes obliterated the Pv standard, which was applied at lower concentrations than the yolk proteins. We further noted that different gel systems yielded slightly different Pv band mobilities. In order to clarify the molecular mass of each putative Pv band, freshly extracted PH ooc and OV eggs were simultaneously electrophoresed in duplicate in the three gel systems and after slicing the gels through the central molecular markers, the left half of each gel was stained with Coomassie brilliant blue G-250 and the right half of each gel was placed in the Gelcode phosphoprotein stain. For the estimation of band molecular masses, Bio-Rad Precision Plus prestained markers (250, 150, 100, 75, 50, 37, 25, 15, and 10 kDa) were applied to each gel.
Western immunoblotting was performed as described by Matsubara et al. [15], using polyvalent antisera raised in rabbits against the following purified yolk proteins (with molecular masses) of barfin flounder (Verasper moseri): aVm-LvH-Aa, 107 kDa; aVm-LvH-Ab, 94 kDa; aVm-LvL-Aa, 30 kDa; aVm-LvL-Ab, 28 kDa; and aVm-ß', 17 kDa. Following semi-dry electrical transfer of electrophoresed proteins (Trans-Blot SD; Bio-Rad) to polyvinylidene fluoride (PVDF) membranes (Millipore Immobilon-P), nonspecific binding of antibodies was blocked with 5% skimmed milk for 30–60 min at ambient temperature. The membranes were incubated overnight at ambient temperature with 30 ml of 1:2000 to 1:5000 dilutions of primary antibody. After washing in Tris-buffered saline that contained Tween-20 (TBST; 20 mM Tris, 0.5 M NaCl, 0.05% Tween-20), the membranes were exposed to 1:20 000 dilutions of secondary antibody (alkaline phosphatase-conjugated goat-anti-rabbit IgG; Sigma). Immunoreactive bands were visualized using the BCIP/NBT color development substrate (Promega).
N-terminal microsequencing was conducted following semi-dry electrical transfer of electrophoresed proteins to PVDF membranes (Millipore Immobilon-P >20 kDa; ImmobilonP ISEQ10100 <20 kDa). Several instruments were used, including the Shimadzu PPSQ-21, Applied Biosystems HP477A, and Applied Biosystems Procise cLC 492 N-terminal peptide sequencer. Only the PPSQ-21 and Procise cLC 492 systems yielded unambiguous results in the 0.1–10 pmol range. Each instrument utilizes sequential Edman degradation followed by HPLC analysis of phenylthiohydantoin (PTH)-stabilized amino acids. Computer analysis of the peptide sequences was performed using the Applied Biosystems model 610a v2.1a software. Samples with molecular masses less than 20 kDa were analyzed in the presence of Biobrene (Applied Biosystems). Under the described conditions, cysteine (Cys) residues were not detected, and a Cys was assumed to be present in the N-terminal signal only when no peak change was observed in the chromatogram, although a Cys was present in the cDNA. For this reason, the Cys residues are boxed in Figure 1. All peptides were analyzed more than once.
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Finn and Kristoffersen [3] have proposed a reclassification of the vertebrate vtg gene family based on whole genome duplication (WGD) and lineage-specific gene duplication. For WGD, a capital letter is used to denote paralogues, while a small letter is used to denote lineage-specific paralogues in accordance with the Hox gene nomenclature. This new gene (protein) nomenclature is used in the present report. Among Paracanthopterygian teleosts (e.g., haddock), vtga (VtgA = Had2) is now classified as vtgAa (VtgAa), while vtgb (VtgB = Had1) is now classified as vtgAb (VtgAb). Among the Acanthopterygian teleosts, vtg1(I) or vga (Vtg1(I) or VgA) is now also classified as vtgAa (VtgAa), and vtg2(II) or vgb (Vtg2(II) or VgB) is classified as vtgAb (VtgAb). Among all teleosts, the phosvitin-less type of vtg, which is concluded to be a product of R2 WGD [3], continues to be classified as vtgC (VtgC).
Two full-length vtg sequences were obtained from Atlantic halibut liver cDNA using a PCR-based approach with redundant primers. One sequence contained an open reading frame (ORF) of 4899 nucleotides (nt) that encoded a predicted protein of 1633 amino acids (aa). The other contained an ORF of 4941 nt that encoded a predicted protein of 1647 aa. Homology searches using the NCBI BLAST interfaces confirmed that both sequences belonged to the Vtg family of proteins. Closer analysis of the predicted proteins using multiple alignment programs and Bayesian analysis revealed paralogous clustering among the paracanthopterygian and acanthopterygian classes of teleosts. The 1633-aa protein with a predicted molecular mass of 178 kDa is named HhvtgAa, and the 1647 aa protein with a predicted molecular mass of 179.9 kDa is named HhvtgAb, in accordance with the scheme proposed by Finn and Kristoffersen [3].
The deduced primary structure of the HhvtgAa paralogue of Atlantic halibut was 96.6% identical and 98.3% similar to the VtgAa of barfin flounder (accession no. AB181833), its Japanese cousin, while that of HhvtgAb was 95.6% identical and 98.0% similar to the VtgAb orthologue of barfin flounder (AB181834). This high sequence identity provides a sound basis for utilizing the antibodies raised against the barfin flounder yolk proteins [15]. Furthermore, the immunodetection of the specific forms of each Vtg-derived yolk protein was validated by the N-terminal data (see next section).
In order to understand the expression and domain structure of each Vtg, and to determine how each is differentially disassembled and degraded during oocyte maturation, oocyte and egg yolk proteins (Yps) were electrophoretically separated and subjected to N-terminal microsequencing and Western immunoblotting. Six major bands (Yp1–6) were observed in the electrophoretic profile of PH ooc, while a further four major bands (Yp7, Yp10, Yp11, and Yp12) and three minor bands (Yp8, Yp9, and Yp13) were observed in the electrophoretic profile of OV eggs (Fig. 2). Yp13 was present in most gels (see Fig. 3) but ran off the gel presented in Figure 2. Each Yp yielded sequence information and could be mapped precisely to its location in the parent Vtg paralogue (Figs. 1 and 2). As previously noted [1], Yp1 had an estimated molecular mass of 110 kDa and mapped to aa position 16 of HhvtgAa. This protein showed specific immunoreactivity to aVm-LvH-Aa but was not observed in the OV egg (Fig. 2). Thus, Yp1 represents the lipovitellin heavy chain (LvH-Aa) after removal of the putative signal peptide (MRVVALALTLALVAG). Several variants were noted for this protein, with up to three further aa missing from the N-terminus. However, although these variants may exist in nature, the most-N-terminal position 16 (counted from the putative signal Met) was interpreted to be the N-terminus of the mature HhvtgAa. This conclusion was independently supported by multiple alignments of the known vertebrate Vtgs for this region [3] and by the submission of each paralogue to the available signal peptide prediction services [32].
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Analysis of Yp2 revealed that it mapped to position 16 of the HhvtgAb protein, which also represented the lipovitellin heavy chain of this paralogue (LvH-Ab). The specific immunoreactivity for aVm-LvH-Ab (Fig. 3) corroborated this conclusion. However, the two cDNAs encoded putative LvHs of similar molecular mass, which suggests that part of the C-terminal region of LvH-Ab, of the order of 10–15 kDa, had been cleaved off. Two peptides (Yp5 and Yp6) with identical N-terminal sequences (LKIPKGFEKKMCVAI) were found to map to position 967 in HhvtgAb. With the identification of the N-terminus of the Pv domain (see below), Yp5 with an in-gel molecular mass of 11 kDa contained 98 aa with a calculated molecular mass of 10.8 kDa. It is thus concluded that LvH-Ab is cleaved at position 967 to yield a large N-terminal subunit (LvHn-Ab) and a small C-terminal subunit (LvHc-Ab). This terminology (n and c suffixes) for the two ends of the LvH-Ab is used throughout the present report. Yp6 was concluded to be a degradation product of Yp5.
Initial examination of Yp3, which had an estimated in-gel molecular mass of 27–30 kDa, revealed that it contained multiple peptides with a dominant signal that mapped to position 1141 in HhvtgAb (Fig. 1). This represents the N-terminus of LvL-Ab. In order to enhance the separate signals, membrane chips cut from separate regions (a, b, and c) were subjected to sequencing. A weak secondary signal for Yp3a (KKILAP) was mapped to position 1067 of HhvtgAa. This represents the N-terminus of Pv-Aa. However, this signal became undetectable after six residues due to the strong valine signal at the fifth residue from LvL-Ab, and it was thus not possible to determine whether Pv-Ab, which has an N-terminal sequence of KKILVP, was also located in this region. The N-terminal sequence of Yp3b mapped to position 1130 in HhvtgAa, which represents the N-terminus of lipovitellin light chain (LvL-Aa), while the N-terminal sequence of Yp3c mapped to position 1141 in HhvtgAb, representing the N-terminus of LvL-Ab. The Western immunoblot experiments further corroborated these findings. The upper region of a 27–30-kDa band showed specific antigenicity for aVm-LvL-Aa in the PH ooc, while the lower region of this band showed specific antigenicity for aVm-LvL-Ab in the PH ooc (Fig. 3).
Analysis of Yp4, which had an estimated in-gel molecular mass of 17 kDa (Fig. 2), also revealed two strong sequences (Fig. 1), one of which mapped to position 1363 in HhvtgAa and the other to position 1372 in HhvtgAb. These peptides represent the N-termini of the ß'-Aa and ß'-Ab, respectively. The majority of the sequences for ß'-Ab had the N-terminus of AKAG, while some variants were noted that had N-termini of TAKAG or LTAKAG, indicating that minor cleavage variants with one or two extra aa may exist in the yolk protein pool. These findings were further corroborated by the specific immunoreactivity of this band with aVm-ß' (Fig. 3). However, the major ß' peptides were both too small to explain the remaining aa in the C-terminal region of each parent Vtg paralogue. The most-C-terminal segment of each Vtg appeared to be missing from the electrophoretic profiles. Using multiple alignment of the vertebrate Vtgs [3], a conserved QEY cleavage site was indentified in all but two of the aligned Acanthopterygian species. The QEY site occurs at position 1515 in HhvtgAa and at position 1525 in HhvtgAb. Knowing the N-termini of each ß' component and counting the aa residues to the QEY site, it could be estimated that the ß'-Aa (position 1363–1514) contains 152 aa and has a calculated molecular mass of 17.1 kDa, while the ß'-Ab (position 1372 – 1524) contains 153 aa and has a calculated molecular mass of 17.2 kDa. These calculated molecular masses are very close to the observed in-gel molecular mass of Yp4 of 17 kDa. The remaining amino acids in each paralogue should represent two C-terminal coding region (CT) peptides of approximately 13–14 kDa. Such peptides were not observed by electrophoresis or microsequencing.
Several other PH ooc proteins were occasionally noted in some of the gels. Yp2b with an in-gel molecular mass of 62 kDa (Figs. 1 and 3) had an identical N-terminal sequence to Yp2, i.e., LvH-Ab, and may represent an artifact of extraction. A separate protein at 80 kDa (Fig. 3) immunoreacted with aVm-LvH-Ab (see below) and was also deemed to be an artifact of extraction. A 46-kDa band (Yp2c; Fig. 3) appeared in some extractions but did not yield sequence data (Fig. 1). A minor band (Yp3d; Fig. 2) that was also sometimes observed was subjected to N-terminal microsequencing. The peptides contained in this band were a mixture of Yp3 and Yp4 and assumed to be artifacts of the extraction process or the Tris-Tricine gel system.
As observed earlier [1], the electrophoretic profiles of the OV eggs were very different from those of the PH ooc (Figs. 2 and 3). Each of the major and minor Yps yielded sequence data and it was possible to map their positions to the precursor Vtg paralogues. Yp7, the dominant protein, was mapped to position 17 in HhvtgAb. This is the N-terminus of LvHn-Ab but it lacks the Asn at position 16 in its prehydrated form. Yp7 also showed greater gel mobility (88 kDa) compared to Yp2 (92 kDa), which suggests that part of its C-terminal region (4 kDa) has been cleaved. A peptide of this magnitude that mapped to the C-terminal region of LvHn-Ab was not observed. The two minor proteins, Yp8 and Yp9, had identical N-terminal sequences (Fig. 1) and mapped to position 20 of LvHn-Ab. In our earlier analysis, these proteins (66 kDa and 62 kDa [1]) appeared only during the maturational phase. Yp9 appeared temporarily, while Yp8, as seen in the present study, persisted in the OV egg. Both Yps show immunoreactivity to aVm-LvH-Ab (Fig. 3) and are degradation products of LvHn-Ab, with slight processing at their N-termini (Fig. 1) and more substantial processing at their C-termini. In the present study, the difference in mass between Yp7 and Yp8 was 22 kDa. Yp11 with a molecular mass of 16 kDa was found to map to position 643 in HhvtgAb, while Yp13 with a molecular mass of 6 kDa and an identical N-terminal sequence to those of Yp5 and Yp6 in the oocyte mapped to position 967 in HhvtgAb. These data demonstrate that LvHn-Ab and LvHc-Ab are cleaved but predominantly not proteolyzed during oocyte maturation. Since Yp13 in the OV egg was significantly smaller than either Yp5 or Yp6 in the PH ooc, it is concluded that part of the C-terminal region of LvHc-Ab is proteolyzed. Two other proteins, Yp10 (18 kDa) and Yp12 (8 kDa) were mapped to positions 1171 in HhvtgAa and 1176 in HhvtgAb, respectively, and demonstrated antigenicity for aVm-LvL-Aa and aVm-LvL-Ab, respectively (Fig. 3). Based on mass, antigenicity, and N-terminal sequence, it is concluded that Yp10 is a truncated form of LvL-Aa, while Yp12 is a more heavily processed and truncated form of LvL-Ab (Figs. 2 and 4).
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Both paralogues contained nt sequences between the LvH and LvL domains (190 bp in hhvtgAa; 224 bp in hhvtgAb) that coded for polyserine subdomains. These subdomains represent putative phosvitins that are heavily phosphorylated [33]. The difference in the nt length of these tracts explains most of the molecular mass difference between the paralogues.
The mapping of Yp3a to position 1067 in HhvtgAa and the mapping of Yp3b to position 1130 in HhvtgAa, mean that the putative Pv-Aa should have a molecular mass of 6–7.5 kDa. However, no such protein was observed in any of the Coomassie- or silver-stained gels (silver gel data not shown). Simultaneous analyses of PH ooc and OV egg yolk proteins by Coomassie staining and phosphoprotein staining in the three gel systems also did not reveal Yps with a molecular mass of approximately 7 kDa. Instead, three or four separate bands appeared in each of the gel systems visualized with the phosphoprotein staining kit (Fig. 5). The molecular mass of each band was slightly different in each gel system but the bands approximated 30 kDa, 24–26 kDa, 14–18 kDa, and 12–16 kDa. Although each gel system showed slightly different band mobilities for the phosphoprotein-stained gels, each gel duplicated the result, and only slight yield differences were noted between fresh-frozen and lyophilized samples. The different band mobilities and lack of Pv standard in some of the gels prompted new experiments to localize the Pv domains. A 7.5% Tris-Tricine gel and 15% Laemmli gel were run and chicken egg yolk, which is known to contain Pv [34–40], was included. The gels revealed further anomalies, whereby bands of 30 kDa and 23 kDa were visualized in the Tris-Tricine gel and two different bands appeared in the 15% Laemmli gel for PH ooc (Fig. 6). The Pv standard was observed in both gel systems; it ran as a 25 kDa band in the Tris-Tricine system but as a 33-kDa band in the Laemmli system. The chicken Pv was observed as a major 50-kDa band and a minor 27-kDa band. The smaller halibut PH ooc bands observed in the 15% Laemmli gel system were correlated to bands of similar molecular masses in the Coomassie stain of the 15% Laemmli gel (Fig. 6, right panel). Since Pv bands were not detected in the 7–8 kDa range and the Pv domain of each paralogue falls between the LvH and LvL domains, it is concluded that nonspecific processing of Vtg paralogues occurs during incorporation into the oocyte, such that different amounts of the Pv domain remain covalently attached to different Yps. Thus, in some oocytes, the Pv domain from the HhvtgAa paralogue seems to form a Pv-LvL conjugate of 25–30 kDa (Yp3a). It is possible that this also occurs for Pv from the HhvtgAb paralogue (Yp3c). However, in the latter case, the Pv domain also remains covalently attached to the C-terminal subunits of LvHc, thereby forming LvHc-Pv complexes (Yp5 and Yp6). Despite the anomalous behavior of Pv in the different gel systems, only PH ooc contained Pv and in no gel was Pv ever observed in the OV eggs (Figs. 5 and 6).
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Disassembly and Differential Proteolysis
Each paralogue was aligned with the known vertebrate Vtgs and the structure mask of lamprey Vtg [24, 27], in order to determine the structural homologies of the halibut proteins [3]. This permitted identification of the key features of the Vtgs present in the PH ooc but absent in the OV eggs. The identity and similarity scores of the halibut paralogues compared to the crystallographically resolved lamprey Vtg structures (Lv1 and Lv2) were highest for secondary structures compared to loops (Fig. 7). The good homology seen between the halibut Vtg paralogues and the lamprey structure mask shows that the secondary, tertiary, and quaternary structures of the Lv molecules are conserved. To examine this in detail, 3-D structures of each halibut paralogue were modeled using the modeler6 webservice (http://salilab.org/modeller). The LvH domains of the halibut paralogues conformed to the (N-sheet)-(
-helix)-(C-sheet)-(A-sheet) structure of the LvH (Lv1) present in lamprey Vtg. It was not possible to match the Pv, ß', and CT domains for the halibut Vtgs, as these were missing from the lamprey 3-D model. The assembled PH ooc proteins are therefore illustrated on lamprey Lv monomers (Fig. 4). Using the N-terminal data and the aligned vertebrate Vtgs [3], the cleavage sites of the halibut paralogues were precisely mapped to the lamprey model (Fig. 4). The N-terminus of the halibut egg Yp11 (Gly643) is located at Gly639 in lamprey Lv1n, at the start of the second ß-sheet of the C-sheet in the model. This position is 49 aa upstream of the LV1n-LV1c cleavage site in lamprey Vtg [24]. The N-termini of halibut PH ooc Yp5, Yp6, and egg Yp13 (Leu967) were located at Met1002 in lamprey Lv1c, just after a disordered loop, but 12 aa upstream of the first of two fully conserved Cys residues that juxtapose between ß-sheet 17 and ß-sheet 18 in the A-sheet of the lamprey model. The maturational proteolytic processing of the N-termini of the LvLs proved virtually identical in both halibut paralogues. In the PH ooc, the LvL-Aa N-terminus (Lys1130) was one aa upstream of a highly conserved Phe, while the LvL-Ab N-terminus (Thr1141) was one aa downstream of the same highly conserved Phe. During oocyte maturation and hydration, both LvLs had their N-termini truncated by 40 aa in LvL-Aa and by 35 aa in LvL-Ab to a position two aa downstream of a highly conserved Phe/Tyr (Tyr1168, HhvtgAa; Tyr1173, HhvtgAb). An examination of these sites in all vertebrate Vtgs revealed that they are fully conserved cleavage sites and they were separately termed the lipovitellin light chain cleavage site (LvL-CS), and the oocyte maturational LvL N-terminal cleavage site (LvL-MCS) [41]. Unlike the N-terminal processing, the C-termini of the LvLs were differentially processed. The C-terminus of LvL-Aa was much less degraded than that of LvL-Ab (Fig. 4).
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Vitellogenin and Yolk Protein Structures
In the present study, two full-length hepatic cDNAs that encode paralogous Vtgs in the Atlantic halibut were isolated and cloned for the first time, and their subdomain structures were mapped to the PH ooc and OV egg Yps expressed in this species. The maturational Yp degradation products were further mapped to the FAA pool in the OV eggs. Using N-terminal microsequencing coupled with phosphoprotein-specific stains, immunoblotting of the isolated Yps, multiple alignment tools, and 3-D modeling, it was possible to identify many salient features not only of the halibut Vtg paralogues, but also of Vtgs in general. The deduced aa sequences of each cDNA conformed to the linear NH2-(LvH-Pv-LvL-ß'-CT)-COO– structure that is typical of complete Vtgs [8]. However, the Yps did not strictly match the expected cleavage products of complete Vtgs. The consensus view is that the primary CatD-mediated cleavage event should result in the production of Lv, Pv, and ß'. However, the present data do not adhere to the notion that Lv and Pv are independent products of the primary cleavage event.
The N-terminus of the HhvtgAa PH ooc LvH was identified as His16 and was localized to the 110-kDa Yp1 by Western immunoblotting. This is in close agreement with the predicted molecular mass of 114.5 kDa. However, a Pv with deduced content of 63 aa (49% Ser) and predicted molecular mass of 6.3 kDa was not observed in any gel. Instead, phosphoprotein staining revealed bands of 25 kDa and 30 kDa. A separate analysis of the 30-kDa Yp3a (Fig. 2) revealed the N-terminus of Pv-Aa (Lys1067), while analysis of the 28-kDa Yp3b revealed the N-terminus of LvL-Aa (Lys1130). The band with molecular mass of 30 kDa was not always observed, which suggests that the presence at this location of Pv, with its highly anionic phosphates, inhibits the binding of Coomassie Brilliant blue, a well-known phenomenon [38]. The most likely explanation is that this band is a Pv-LvL-Aa conjugate with a calculated molecular mass of 31.9 kDa, which is in very good agreement with the observed upper band (Figs. 5 and 6). A similar finding was made for the VtgAa form of the Yps in barfin flounder [15] and red sea bream [21]. The lower 25-kDa phosphoprotein-stained band is probably a C-terminally processed variant of this conjugate. In addition, the high degree of phosphorylation of the Pv domain may inhibit binding of SDS, thereby altering the charge densities and in-gel conformations of these proteins. This would explain the anomalous migration of peptides conjugated to Pv fragments in the phosphoprotein-stained gels.
The deduced aa regions of the Pv-Ab fractions contained 75 aa (56% Ser), with a predicted molecular mass of 7.6 kDa. Again, no such proteins were observed by electrophoresis and it was not possible to verify that the N-terminus of Pv-Ab also occurred at 30 kDa due to the presence of Val1145 in LvL-Ab. Similarly, although the N-terminus of LvH-Ab (Asn16) was identified and verified as the 92-kDa Yp2 by Western immunoblotting, this Yp does not match the molecular mass of 114.4 kDa predicted from the cDNAs for LvH-Ab. However, the mapping of Yp5 and Yp6 to Leu967 in HhvtgAb revealed the C-terminal fraction (LvHc) of Yp2, i.e., LvHc-Ab. The in-gel molecular masses of Yp5 and Yp6 were 12 kDa and 9 kDa, respectively, while the predicted molecular mass of the peptide fragment between Leu967 and Lys1066 at the Pv KKIL site was 10.8 kDa. Therefore, the sum of the predicted LvHc-Ab and the observed Yp2, which represents LvHn-Ab, is about 103 kDa, which is somewhat different from the 114.4 kDa predicted for uncleaved LvH-Ab. From this analysis, it is reasonable to surmise that the gels underestimate the molecular masses of the LvH Yps. More interesting is the presence of two phosphostained bands between 12 kDa and 18 kDa, since these bands coincide with the migration patterns of Yp5 and Yp6 and indicate that uncleaved segments of Pv-Ab are attached to them. This contrasts with the processing of Pv in the HhvtgAa paralogue, whereby Pv-Aa segments remain attached to LvL-Aa. These findings neither violate the linear structure of Vtg nor invalidate the principle of the primary cleavage event, but rather suggest that the primary cleavage event may be temporal, regional, incomplete or a combination thereof.
Once internalized via clathrin-mediated endocytosis, Vtg is exposed to CatD in the early endosomes [11, 42, 43]. The pH of early endosomes is reported to be about 6 [44], a value that has also recently been verified for the oocytes of barfin flounder [16] and localized to cortical yolk globules in the vitellogenic oocytes of the common mummichog [43]. This pH is well above the optimal catalytic pH for CatD [45]. The rapid centripetal sorting of Yps into either the fluid-phase yolk, which seems to have a neutral pH [43], or a dense orthorhombic lattice in the yolk platelets [11, 46, 47] may prevent CatD from fully processing the internalized Vtgs.
Another possibility is the temporal expression of CatD during vitellogenesis. Data for seabream show that CatD expression is highest during the earliest stages of vitellogenesis [45, 48], which suggests that Vtg internalized during the later stages of oogenesis may be processed very little or not at all. CatD mRNA expression data have also been reported for rainbow trout [49], which shows continuous expression during oogenesis and a large increase following fertilization. Unfortunately, the functional protein was not investigated. Perhaps the simplest explanation is that recently endocytosed Vtg at the oocyte cortex is incompletely processed, while the processing of the more centrally located Yps is complete. As a consequence, a population of variously processed Yps may exist in the oocytes until further acidification occurs during oocyte maturation or development.
Both halibut vtg paralogues contained relatively short polyserine tracts that coded for Pvs. The 5'-TCx and 3'-AGx synonymous codon structure noted earlier [6, 50, 51] is not as clearly conserved in the shorter halibut Pv domains. As in other vertebrate and invertebrate Pvs, approximately half of the amino acids in each halibut Pv consisted of Ser residues. The Ser residues are heavily phosphorylated (>90% [33]) and thus are highly polar in aqueous solvents. In addition, the core regions of the Pv domains contained many Lys and Arg residues, which also are highly polar, but unlike phosphorylated Ser are basic rather than acidic. Other highly represented aa in the Pv domains were Leu and Ala (9.4% Ala, 10.9% Leu in HhvtgAa; 4% Ala, 4% Leu in HhvtgAb), which are nonpolar residues. Together, these five aa represented 84% and 80% of the aa contents of the HhvtgAa and HhvtgAb Pvs, respectively. Furthermore, the balance between polar and nonpolar aa was highly skewed towards polar aa in both paralogues, a condition that is also reflected in the Pv domains of other animals. The prevalence of Arg can be explained by single base mutations of Ser codons, and Arg together with Lys interacts strongly with phosphorylated Ser residues, to stabilize the tertiary structure.
The conserved receptor domains reported by Li et al. [52], and highlighted in yellow in Figure 4, were located on an exposed ß-sheet of the LvH N-sheet, between a tetrad of fully conserved Cys residues in the newly classified VtgA-type Vtgs [3]. These Cys residues form disulfide bridges [24] that stabilize the conformation of the N-sheet by linking two small ß-sheets and an otherwise disordered loop that interacts with the C-sheet. Interestingly, VtgC-type Vtgs lack these latter Cys doublets, which may have relevance for the expression of a second form of the Vtgr reported in teleosts [52–54].
The electrophoretic band shift of the Yps in the OV eggs replicated our earlier observations [1], and the mapping of each Yp to the parent Vtg paralogue facilitated the determination of which Yp remained for embryonic development and which contributed to the FAA pool (Fig. 2). These data clearly illustrate the differential proteolysis of the HhvtgAa- and HhvtgAb-type Yps in this marine pelagophil. The calculated FAA released from each Vtg paralogue match the previously measured profile [1]. Only three aa (Gln, Pro, and Cys) were found to deviate slightly from the measured profile. However, the very good match noted between the calculated and measured pools of all other FAA indicates that the origin of the FAA pool is as described in Figure 2. These data corroborate recent studies on the maturational proteolysis of Yps in other marine pelagophils [15, 21], in which the majority of the aa in the FAA pool are argued to stem from the LvH domain of VtgAa-type Vtgs. However, due to the lower expression ratio of HhvtgAa to HhvtgAb (1:3) in the present context, approximately half of the FAA pool derives from the differential proteolysis of each respective paralogue. This conclusion is supported by the total protein data, which show that 210 µg/ind protein was lost during oocyte maturation, leaving 600 µg/ind in the OV egg, which represents a drop of 25% of the protein present in the PH ooc [1]. This finding supports earlier suggestions that the expression ratio of VtgAa- to VtgAb-type Vtgs serves to optimize the organic osmolyte pool of FAA required to hydrate the egg during oocyte maturation and the acquisition of buoyancy [16, 21].
The data in the literature suggest that the degree of proteolysis differs between eggs that contain oil globules and those that do not. Such eggs have been classified previously as type I and type II eggs, respectively [55]. Yolk proteolysis was maximal in type I eggs, such as in halibut in the present study, while maturational proteolysis may be lower in type II eggs that contain oil globules [56–58]. Furthermore, in order to achieve buoyancy in a marine environment, the egg must attain a lower specific gravity than the seawater into which it is spawned (e.g., 1.0278 kg/L at 35 ppt, 6°C). Therefore, in order to float, it is necessary for an Atlantic halibut egg to acquire 90% hydration to reduce its specific gravity below this value. The great size of the halibut egg (>3 mm [1]) means that the volume of water needed for hydration is proportionately less than that for smaller eggs, due to a surface:volume effect. The surface of an egg is composed of extracellular proteins in the chorion, which together with the intracellular Yps are heavier than the specific gravity of seawater. Increasing the intracellular volume through the influx of water will thus achieve buoyancy in proportion to 3/r, where r is the radius of the egg. These proposals are supported by the observation that a large halibut OV egg swells only 4-fold, while a small Labrid pelagic egg (0.86 mm) swells 6.5–8.4-fold compared to the PH ooc [1, 2]. In contrast to these type I eggs, the presence of oil globules in type II eggs contributes to buoyancy, and therefore the degree of proteolysis may be lower in such eggs [57, 58]. One can thus predict a dynamic expression ratio of VtgAa- and VtgAb-type Vtgs in relation to temperature, ionic strength, egg size, and the presence of oil globules. This notion requires further investigation.
In the current model, the CT domains were not located in the gels. However, in a separate analysis, we identified a conserved QEY site in all but two of the acanthopterygian species examined [3]. The Gln of the QEY site occurs one aa downstream of a conserved Arg/Lys in the complete Vtgs and two aa upstream of the hydrophic Tyr. It is suggested that this is a putative CatD recognition motif, and that the Gln of the QEY site represents the putative N-terminus of the CT domain that cyclizes to form pyroglutamate during the primary cleavage event. This may explain the absence of N-terminal data in other studies, and the immunological detection of the CT protein in barfin flounder, which is a close relative of the halibut [16]. As a consequence, the CT domain is included in the proteolysis model (Fig. 2), although further studies will be needed to clarify this.
The differential proteolysis of the Yps in halibut as in other pelagophils is intriguing, since both paralogues are exposed to lysosomal enzymes during the secondary cleavage event. Unlike the primary cleavage event, the secondary and tertiary cleavage events are caused by the activity of cathepsin L (CatL) [42, 45, 59, 60] or cathepsin B (CatB) [16, 43, 61] as a consequence of further acidification of the yolk platelets. The low pH in conjunction with high ionic strength, particularly K+, disassembles the crystalline nature of the yolk [62, 63], while simultaneously activating CatL or CatB [64, 65]. However, our previous data for Atlantic halibut [1] do not support a role for K+ in the disassembly of yolk crystals. Previously, we and others [66] have observed that K+ lags behind the influx of Cl– during oocyte maturation and hydration. Since reacidification is caused by the same or related V-class proton pumps, which are activated during the CatD-mediated primary cleavage event [43, 62, 67], coporting of Cl– may be possible. A different mechanism must be responsible for the movement of K+. Although this mechanism remains to be clarified [68], a plasma membrane Na+/K+-ATPase, [69], as well as heterologous intercellular gap junctions between the oocyte and the granulosa cells [70] seem to be implicated. Under physiological (in vivo) conditions, the increase in H+ may disassemble the yolk crystals and simultaneously activate acid phosphatases and cathepsins. These events could dephosphorylate Pv, resulting in a coincident release of Ca2+, which destabilizes the Lv structures, causing the release of lipids and exposure of the apoprotein to the activated cathepsins. The released Ca2+ could then become available for SNARE protein-mediated fusion of the vesicular membranes. The basis for these suggestions lies in the elevated Cl– and depressed K+ contents [1] while the oocyte remains acidic [16]. Further studies will be necessary to clarify these proposals.
As noted by Fagotto [71], cathepsins are voracious degradative enzymes. This adds to the mystery of how LvH-Ab and the remaining segments of the LvLs withstand degradation. No motif has been identified within the primary structure of the halibut Vtg paralogues that might explain this mystery. However, a clue may come from the related plasma protein ApoB. The stability of ApoB is determined by its cotranslation of the first 1000 aa [28, 29, 72]. The inability to assemble and lipidate the first 1000 aa of the ApoB lipoprotein particle, which is homologous to the LvH of the vertebrate Vtgs, results in degradation, which combined with aberrant forms of microsomal triglyceride transfer protein is the basis of abetalipoproteinemia in humans [73–75]. Thus, the innate binding of lipid within the (C-sheet)-(A-sheet) cavity seems to be vital for the survival of the protein.
Two regions appear to be critical for the survival of the protein. The first region concerns a DRE buried salt bridge in the -helical domain [25, 29]. However, these residues are not conserved in the vertebrate Vtgs. In both halibut paralogues, the motif homologous to DRE is HLE (Fig. 4). The second region represents a hairpin salt bridge (ApoB: R717K718H719E720 - R997E998D999R1000) at the end of the homologous segments of ApoB and vertebrate Vtgs [26, 29]. This bridge would exist at the base of the Pv domain in Vtg, where the Pv domain is proposed to loop up to lock the lipids in the (C-sheet)-(A-sheet)-(LvL) cavity [41]. It is not unreasonable to assume that the dephosphorylation of Pv will unfold the loop, and thus open this salt bridge, causing the loss of lipid from the pocket. It is concluded, in agreement with Hartling and Kunkel [76], that the loss of lipid from this cavity is instrumental in the demise of the LvH-Aa domains. In further support of this notion, it is only during oocyte maturation that the lipid droplets coalesce to form the oil globules in type II eggs. However, the degradation of almost all of the HhvtgAa Yps and survival of most of the LvL-Aa domains indicate that other mechanisms are also involved. This latter domain in the OV egg of pelagophils forms multimeric complexes (400 kDa) with the egg lipids [21] and as such, functionally resembles the exchangeable vertebrate apolipoprotein C and invertebrate apolipophorin III.
Of note is the process of oocyte hydration itself. It is entirely analogous to the lysosomal generation of vacuoles in plants. The major purpose of both is to cause an influx of water that increases cell volume. In plants, this establishes turgor and confers rigidity to the cell wall, as well as providing a vital pool of water in arid conditions. In the pelagophil egg, oocyte hydration also increases cell size and provides vital water for the early embryo in the functionally arid (hyperosmotic) conditions of seawater. The influx of water is also sufficient to enable the egg to float and thus acquire its pelagic nature. This keeps the egg in the euphotic zone amongst the nekton upon which the larva will feed, and the increased cell size is an important adaptation against predation. Of further interest is that upon reactivation of the vacuolar ATPases during oocyte maturation of the halibut egg, the entire endosomal yolk platelet structure fuses to become what may be the largest lysosome in the animal kingdom.
ACKNOWLEDGMENTS
The author thanks Takahiro Matsubara (Hokkaido National Fisheries Research Institute, Japan) for assistance and for providing the barfin flounder antibodies. Michael Reith and Janet Munholland (NRC Institute for Marine Biosciences, Halifax, Canada) are thanked for help with the cDNA sequencing. Birgitta Norberg (Institute of Marine Research, Austevoll, Norway) is thanked for providing the halibut eggs and samples of vitellogenic livers. The Research Council of Norway is thanked for their financial support, and the University of Bergen is thanked for funding the Procise peptide sequencer. Hans Jørgen Fyhn and Ivar Rønnestad (Dept Biol, Univ Bergen, Norway) are thanked for their help in acquiring the sequencer, and Hans Jørgen Fyhn is further thanked for stimulating discussions pertaining to these studies.
FOOTNOTES
1Supported by the Research Council of Norway and in part by the University of Bergen. 
Correspondence: 2FAX: 47 55 589667; e-mail: nigel.finn{at}bio.uib.no
Received: 18 July 2006.
First decision: 24 August 2006.
Accepted: 20 February 2007.
REFERENCES
