The coccolithophorids have calcified scales called coccoliths on their surface that include abundant acid polysaccharides. To determine the localization and associative strength of acid polysaccharides Ph-PS-1, -2, and -3 incoccoliths of Pleurochrysis haptonemofera, we analyzed the acid polysaccharides extracted with urea from partially decalcified coccoliths. On treatment of coccoliths with8.0 Murea at pH8.0 inboiling water, Ph-PS-2 was not extracted, but parts of Ph-PS-1 and -3 were from the surface without the crystal morphology being affected. When coccoliths were partially decalcified at various pHs (pH 8.0 - 5.0), Ph-PS-1, -3, and -2 were extracted with urea in that order as the calcite crystals dissolved. Detection of the acid polysaccharides using fluorescein-isothiocyanate-labeled lectin (ConA) and anti-Ph-PS-2 antibodies demonstrated that Ph-PS-2 exists on the surfaces of both untreated and urea-treated coccoliths. Moreover, NaClO-treatment under moderate conditions extracted only Ph-PS-1, -3, and part of Ph-PS-2 from coccoliths, without separation of the crystal units, suggesting that at least some part of Ph-PS-2 acts as a glue connecting crystal units and/or a crystal unit and a base plate inside coccoliths. These results suggest that Ph-PS-2 exists not only on the surface but also inside of the coccoliths, most of it being strongly bound to coccoliths, while Ph-PS-1 and -3 are weakly associated close to the coccolith surface, in that order from the surface. This strongly supports a scheme in which PS-2 type acid polysaccharides play an important role in the crystal nucleation and PS-3 type functions during crystal growth.
The coccolithophorids are unicellular microalgae belonging to the Haptophyta that have calcified scales, called coccoliths, on their surface. A coccolith in many coccolithophorides, including most extensively examined genera Pleurochrysis and Emiliania, consists of elaborate calcite crystals, a crystal coat comprising organic matter, and an organic base plate. The calcite crystals are composed of two distinct interlocking units, V and R units. Coccoliths are produced intracellularly in special organelles called coccolith vesicles. TEM observation of coccolith-forming cells has been extensively performed, and thereby the process of coccolith formation has been demonstrated morphologically. However, it remains unclear how calcite crystals are formed and how the coccolith morphology is determined [1-3].
Biomineralization is a phenomenon that is observed in a wide variety of organisms, and in most cases, acidic organic matter such as acid proteins is involved in it [
Coccolith-forming cells of P. haptonemofera were obtained and cultured axenically as described previously [
The coccoliths liberated from the cell surface into the medium were isolated by the following procedure. A culture at the early-stationary phase was centrifuged, and then the white pellet of released coccoliths on the whitish brown pellet of cells was collected with the tip of a micropipet or a spatula and resuspended in 50 mM HEPES buffer (pH 8.0). Contaminating cells in the suspension were disrupted by sonication (at range 2 for 8 sec, using a Sonifier 250 D (Branson Ultrasonics Co., Danbury, CT, USA)), and then the suspension was passed through a Percoll (Amersham Pharmacia Biotech, Chalfont, UK) gradient (1.2 ml of 50% Percoll per 0.3 ml of crude coccolith suspension). The purified coccolith pellet was washed with the HEPES buffer (pH 8.0). Ca2+ included in the coccolith suspension was determined by the direct EDTA titration method, as described previously [13,14].
For urea-treatment of coccoliths, isolated coccoliths containing 10 μmol Ca2+ were suspended in 0.1 ml of a urea solution (8.0 M urea, 100 mM HEPES (pH 8.0)), and then boiled for 10 min in a water bath. For guanidine hydrochloride (GdnHCl)-treatment, 6.0 M GdnHCl was used in place of 8.0 M urea. When various pH-treatments were performed before the urea-treatment, the coccoliths (containing 10 μmol Ca2+) were suspended in 0.5 ml of 100 mM HEPES or MES buffer (MES buffer, pH 5.0 - 6.5, or HEPES buffer, pH 7.0 - 8.0), and then mixed well by vigorous shaking for 30 min. Sodium hypochlorite (NaClO)-treatment was performed basically following the method of [
The extracted acid polysaccharides were quantified by the carbazole method [
Acid polysaccharides on the surface of the treated coccoliths were detected and quantified using fluoresceinisothiocyanate (FITC)-labeled lectin, ConA [7,14], and anti-CMAP (Ph-PS-2) antibodies. For detection with FITC-labeled ConA, 10 μg of ConA was added to 200 μl of a treated coccolith suspension, which had been prepared with coccoliths containing 10 μmol Ca2+, and then the mixture was incubated for 1 h in the dark. After centrifugation (18,500 g, 5 min, 4˚C), the pellet was washed twice with 200 μl of 50 mM Tris-HCl (pH 8.0) to remove free lectin, and then dissolved in 300 μl of 500 mM EDTA (pH 8.0). After samples had been transferred to the wells of a 96-well microtiterplate, the relative fluorescence of each sample was measured with Fluoroskan Ⅱ (Dainippon Pharmaceutical Co., Osaka, Japan).
For detection with anti-Ph-PS-2 antibodies, the ELISA method involving horseradish peroxidase (HRP)-linked secondary antibodies was performed. As the primary antibody, rabbit antiserum against Ph-PS-2, which was purified by application to a TSG-gel DEAE-5PW HPLC column (7.5 × 75 mm; Tosoh, Tokyo, Japan) several times and whose purity was confirmed by NMR, was used [
The coccolith morphology was observed by SEM (JCM- 5700; JEOL, Tokyo, Japan), as described previously [
On urea-treatment of isolated coccoliths, a part of the acid polysaccharides (parts of Ph-PS-1 and -3) was extracted (
on SEM (Figures 2(a)-(d), suggesting that the extracted acid polysaccharides were localized close to the coccolith surface. Thus, these treatments were expected to be novel methods applicable to analysis of the localization of acid polysaccharides in coccoliths.
For further investigation of localization of acid polysaccharides in coccoliths, the partially decalcified coccoliths were applied to urea-extraction. When exposed to low pH were subsequently treated with urea, the amount of extracted acid polysaccharides increased as the amount of dissolved CaCO3 increased (
and -2 were extracted with urea, in that order, as the calcite crystals dissolved (
crystals are intact.
When almost 40% of the CaCO3 in the coccoliths had been dissolved at pH 6.5, Ph-PS-1 and -3 were completely extracted with urea (Figures 3 and 4). The sharp increase in uronic acids in extracts from pH 8.0 to 7.0 coincided with the extraction of Ph-PS-1 and -3. On the other hand, more than half of the Ph-PS-2 remained in the coccoliths, even after they had been completely decalcified at pH 5.0 and subsequently treated with urea (Figures 3 and 4). These findings suggest that Ph-PS-1 and -3 are weakly associated close to the coccolith surface, in that order from the surface, while some part of Ph-PS-2 (relatively small-size molecules) is weakly bound but exist deeply inside the coccoliths as if being protected from the detergent by CaCO3, the remainder of it (relatively large-size molecules) being strongly bound directly onto the base plate and/or in the crystal coat. The binding stability of Ph-PS-2 on coccoliths might be affected by the amount of associated divalent cations per molecule.
To obtain a clue about the localization sites of Ph-PS-2, coccoliths were treated with the oxidizing reagent NaClO, which separates crystals from each other [
To determine whether or not Ph-PS-2 exists in the crystal coat and remains there even on urea-treatment, we tried to detect acid polysaccharides using FITC-labeled ConA [
Then, to more determine whether or not Ph-PS-2 exists on the coccolith surface, anti-Ph-PS-2 antibodies were used. Its binding to the coccolith surface could be detected by the modified ELISA method sensitively and the amount was not decreased by urea-treatment (122% ± 9%; mean ± SD, n = 6) (
Marsh et al. [10,15] have found that PS-1 and PS-2 are localized in the crystal coats of mature coccoliths and in electron-dense Golgi particles (often called coccolithosomes), while PS-3 is localized at the interface between growing crystals and the membranes of coccolith vesicles, using anti-polysaccharide antibodies. Based on these findings, and the putative functions of PS-2 and PS-3 predicted on the observation of the immature coccolith morphology in variants devoid of acid polysaccharides [9,10], they proposed the following interesting scheme for the coccolith formation process in P. carterae [
aPh-PS-2 on the surface of urea-treated and untreated coccoliths was detected with anti-Ph-PS-2 antibodies. A one-hundredth to one-tenth volume of each antigen-antibody reaction mixture containing 4 μmol Ca2+ of coccoliths was used for the detection with the HRP-linked secondary antibodies. Data are means ±SD (n = 3 - 6).
the crystals acquire an amorphous polysaccharide coat derived through the dissociation of PS-1/PS-2-containing coccolithosomes.
On the other hand, in this study, the localization of acid polysaccharides in coccoliths was examined by a new extraction method involving denaturants that do not appear to decalcify them. Our results suggested that PhPS-1 and -3 are localized close to the coccolith surface, while at least some Ph-PS-2 is buried deep in the coccoliths. This finding supports her idea that Ph-PS-2 plays an important role in the crystal nucleation and Ph-PS-3 functions during the growth phase. Also, our results suggest that most Ph-PS-2 is strongly associated with the coccoliths, probably with both the coccolith surface and the base plate, and that a little of it is incorporated between crystal units and/or between crystal units and base plates (Figures 4 and 5). Furthermore, Ph-PS-3 is suggested to be incorporated at a relatively late stage of crystal growth, i.e., a little before the attachment of PS-1 (
However, whether or not Ph-PS-1 and Ph-PS-2, both being included in coccolithosomes [
In this paper, the localization and associative strength of acid polysaccharides in coccoliths were investigated, using novel extraction method, urea-extraction, which could extract part of acid polysaccharides without coccolith decalcification. The results obtained by urea-extraction and other denaturant treatment indicated that the three acid polysaccharides are not localized uniformly:
Ph-PS-1 and -3 are weakly associated close to the coccolith surface, while Ph-PS-2 exists not only on the surface but also inside of the coccoliths, most of it being strongly bound to coccoliths. The results also suggested that PhPS-2 plays an important role in maintenance of coccolith oval shape. These strongly support a scheme in which PS-2 type acid polysaccharides play an important role in the crystal nucleation and PS-3 type functions during crystal growth.
We are grateful to Drs. I. Inouye and M. Kawachi of Tsukuba University for kindly providing the P. haptonemofera cells, to Mr. H. Akiyama of Tokyo University of Pharmacy and Life Sciences for the kind support in the SEM observations and to Mr. N. J. Halewood for correcting the English version of this paper. This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan (grant nos. 20570059 and 24570114).