The organic matrix of coleoid cephalopod shells - Springer Link

Marine Biology (1996) 125:525-529

9 Springer-Verlag 1996

Y. Dauphin

The organic matrix of coleoid cephalopod shells: molecular weights and isoelectric properties of the soluble matrix in relation to biomineralization processes

Received: 5 July 1995 / Accepted: 1 December 1995

Abstract Soluble matrices of coleoid cephalopod shells, namely the organic pen of Loligo sp. and mineralized calcified shells of Spirula spirula and Sepia sp., were extracted and studied by HPLC (high-performance liquid chromatography) gel filtration and isoelectrofocusing (IEF). Molecular weights of Spirula spirula and Sepia sp. extracts are not well separated, whereas Loligo sp. shows several well-defined peaks. In Loligo sp. and Sepia sp., molecular weights of > 100 kdaltons were found. Spirula spirula yielded a profile devoid of high molecular material. The main part of the extracted matrix of the organic pen of Loligo sp. is basic. In mineralized shells of both Spirula spirula and Sepia sp., the soluble organic matrices are acidic. These results support the view that the acidity of the organic matrix is related to mineralization processes. Introduction

In cephalopods, the general evolutionary trend has been towards progressively smaller shells, and in many cases they have disappeared completely. Recent cephalopods are separable into two unequal groups. One group (Tetrabranchiata) is comprised only of Nautilus sp. which is characterized by a chambered external shell while the other group consists of all other Recent cephalopods (Dibranchiata or Coleoidea), all of which have shells that are internal and usually vestigial or absent. The reduced shell of coleoids has been shown to contain chitin (Peters 1972). Among the Coleoidea, only species of the genera Spirula and Sepia possess a rigid mineralized shell. Analyses of recent cephalopod shell carbohydrates (soluble and insoluble matrices) Communicated by A. Rodriguez, Puerto Real Y, Dauphin Laboratoire de Pal6ontologie, URA 723, Bfitiment 504, Universit6 Paris XI-Orsay, F-91405 Orsay cedex, France

suggest that the loss of mineralized shell is correlated with an increase in the "chitinous" nature of the insoluble matrix (Dauphin and Matin 1995). Insight into the mineralization processes depends strongly on knowledge of the characteristics of the organic matrices of the shells. Proteins and polysaccharides are the two major constituents of the organic matrix of mineralized mollusc shells. It has been postulated that mineralization is nucleated by specific chemical groups of the organic matrix (Wilbur 1964; Wilbur and Simkiss 1968; Gr6goire 1972). Crenshaw (1972) isolated a water-soluble glycoprotein from Mercenaria mercenaria and suggested that the soluble matrix plays a role in initiation of calcification. The proteins are generally rich in acidic amino acids and glycine (Gr6goire et al. 1955; Hare 1963; Degens et al. 1967). Polysaccharides are also acidic. The major portion of the soluble fraction consists of sulfated glycoprotein (Simkiss 1965; Crenshaw 1972). The soluble matrices have been shown to be rich in aspartic and glutamic acids (Weiner and Hood 1975; Samata et al. 1980). Histochemical localization of the central region of nacreous tablets in Nautilus pompilius has shown that these sites are acidic sulfated polysaccharides (Crenshaw and Ristedt 1976). It has been postulated that only a small part of the matrix, probably acidic proteins, plays a role in the nucleation of minerals (Weiner and Traub 1984). Support for this hypothesis is the prevalence of acidic matrix components in molluscs. The amino acid composition of the soluble organic matrix indicates its acidity: theoretical isoelectric points (pI) can be calculated from amino acid compositions (Sillero and Ribeiro 1989). However, these only indicate the average pI of the soluble matrix. Direct pI measurements of the organic matrices of invertebrate shells are very rare: in some cases (Crassostrea gigas) "... neither peak could be applied successfully to PAGE or isoelectrofocusing because of the extreme high molecular weight and the acidic pH of their solution. This material did not penetrate into the gels." (Krampitz

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et al. 1983, p. 234). The organic soluble matrix of the gastropod Nassa reticulata has been extracted and purified by gel permeation (Krampitz et al. 1976). The only peak obtained was subjected to ion-exchange chromatography, each; "ion-exchange" peak was then analyzed by isoelectrofocusing. The first "ion-exchange" peak displayed three isolectric points between 2 and 5 after isoelectrofocusing, the second "ion-exchange" peak showed two isoelectric points, the first near 2, the second at 10. The third ion-exchange peak displayed six isoelectric points between 2 and 10 (Krampitz et al. 1976). These data indicate that the acidity of the soluble matrix is heteregeneous. However, chromatography of the Ca-ligands from N. reticulata and their isoelectrofocusing revealed a pI in the range of pH 5.5. All the above data were obtained from calcified shells. Comparisons between the amino acid composition in calcified and non-calcified molluscan shells have been made only on the insoluble organic matrix (Wada 1966). No comparable information exists for the soluble part of the organic matrix of non-mineralized shells. Thus, to establish whether or not a compositional change occurs in the organic matrix during loss of the hard shell is vital to our understanding of mineralization processes. Some living cephalopods possess a mineralized shell, the majority possesses an organic shell. In order to test the hypothesis of a specific role of acid-soluble matrix in mineralization, molecular weights and the pI of the soluble matrix of the organic pen of LoIigo sp. were compared to those of the mineralized shells of Spirula spirula and Sepia sp., the morphological characteristics of which have been reviewed by Hopkins and von Boletzky (1994). Nautilus sp. shells were excluded because this genus is not a coleoid, and because the structure of the nacreous layer differs between the shells of the Tetrabranchiata and Coleoidea (Dauphin and Keller 1982). Materials and methods

allows the separation of protein from 5 to 100 kdaltons. The spectromonitor 5000 (Thermo Instrument Systems) photodiode-array detector provides measurements over its full 190 to 360 nm range, following separation by HPLC. The isoelectric properties of the soluble matrix were estimated by non-denaturing isoelectric-focusing electrophoresis. One-dimensional microslab isoelectric focusing (IEF) was performed in homogeneous polyacrylamide small gel (25% total acrylamide concentration, 3% degree of crosslinking) with 100 x 65 mm gels (0.4 mm thick), 5% carrier ampholytes Bio-Lyte pH 3 to 10, containing 25% w/v glycerol. A three-phase catalyst system of ammonium persulfate, riboflavin-Y-phosphate and temed was used. This system is catalyzed by light. Gels are cast on a sheet of gel-support film for polyacrylamide. With the Bio-Rad's Model 111 Mini IEF cell, the gel is run upside down, in direct contact with the electrodes, to eliminate the need for electrode buffers. The lyophilised samples were redissolved in 1% gtycine. Running conditions were 15 rain at 100 V, 15 rain at 200 V, and ~ 60 min at 450 V. Following IEF, the gels were placed in 27% ethanol, 10% acetic acid, 0.04% Coomassie Blue R-250, 0.5% CuSO~ and 0.05% Crocein scarlet for ~ lh 30 rain (fixing and staining solution). The first destaining solution contained 12% ethanol, 7% acetic acid and 0.5% CuSO~, the second destaining solution contained 25% ethanol and 7% acetic acid. Dried gels were scanned and digitalized with a CCD (chargecoupled device) camera (256 grey levels) in order to locate the stained bands. The numerical data acquired were transfered to a computer program to obtain profiles. No densitometric scanning was performed, thus the surfaces under the peaks cannot been used for quantification.

Results

Fig. 1 shows the results of the gel filtration of the soluble matrix of Loligo sp., Spirula spirula and Sepia sp. shells. When the same shell extract was read at different wavelengths (224, 240, 256 and 280 nm), the positions of the peaks sometimes differed, indicating that several substances were included in those peaks: Moreover, the peaks were not always bell-shaped and symmetrical. In Loligo sp. and Sepia sp., molecular weights > 100 kdaltons were recorded. The profile for Spirula spirula differs from those of the other genera:

E

c

The following shells were used: Loli9o sp. (probably Atlantic), Spirula spirula (New Caledonia) and Sepia sp. (New Caledonia). The different layers of the mineralized shells were not isolated for SpiruIa spiruIa and Sepia sp., since some layers were too thin. Although the LoIigo sp. pen is not calcified, all samples were prepared by the same procedure. BSA (bovine serum albumin, Eurobio) and ribonuclease A (Sigma) were used for calibration. BSA has several pIs: 4.9, 5.2, 5.4, 5.5 and 5.6; ribonuclease A has two main pIs: 8.9 and 9.3. The samples were washed first with sodium hypochlorite to remove contaminating superficial amino acids and peptides, then with distilled, deionized water. They were crushed, decalcified with acetic acid (pH 4), and centrifuged at 3000 • for 45 rain. The supernatant containing the soluble matrix (SM) was filtered against distilled water using a Filtron membrane with a molecular weight cut-off at 3 kdaltons, and was then lyophilized. Gel filtration chromatography [TSK (Tosohaas) G2000 SW (silice water) XL] using citrate buffer (pH 4.7) and eluted at 15 mlh i

OJ

>100 kdaltons

0

=o c~ o

'

Spirula

! 20 rain

, ,2o,..4 32.50 rain

elution time

Fig. 1 Loligo sp., Spirula spirula, Sepia sp. High-performance liquid chromatography of soluble extracts of cephalopod shells. Spirula spirula and Sepia sp. molecular weights are not well separated. (Not to scale)

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there was no soluble high-molecular material, the profile was initially horizontal, changing to a low regular slope. Peaks were faint and only low-molecular weights were present (10 kdaltons). Previous chromatographs of the soluble matrix of Sepia sp. (Superosel2 and 6) revealed two main peaks: a small peak at ~ 220 kdaltons, and a main peak at --~22 kdaltons (Dauphin and Kervadec 1994). The Sepia sp. HPLC profile shows a stronger, smooth slope, peaking at ~ 19 and ~ 13.5 kdaltons. These faint peaks were more distinct when first- and second-order derivatives of the profile were used. The peaks of the Loligo sp. profile were more distinct, and indicated molecular weights of ~ 29.5, 16 and 12 kdaltons, respectively. The results of the IEF of the soluble matrix of LoIigo sp., Spirula spiruIa and Sepia sp. shells are shown in Fig. 2. In Lotigo sp., the most prominent heavilystained band was seen at basic pIs between 7 and 9. The highest basic band was broad and composed of four minor bands. The band at pI 7.8 is unique to Loligo sp. The acidic band (5.7 to 6.3) was composed of two faint bands. In Spirula spirula and Sepia sp., the main bands were located on the acidic side only. Several bands were scattered between pI 4 and 6 in the Spirula spiruta

BSA

Ribonuclease A

soluble organic matrix. In occurred at pI 100 kdaltons) recorded for this species, but not for Spiruta spirula Fig. 1). This idea is supported by the fact that the acidic amino acids of the insoluble part of non-mineralized horny plate of Sepia sp. are less abundant than in the spongy, aragonitic, ventral part (Wada 1966; Drozdova and al. 1971). Even if it is difficult to correlate the molecular weights and pI of the soluble matrices of the studied species, comparison of chromatograms and electrophoretic profiles of the two mineralized shells (Spirula spirula and Sepia sp.) are more similar than those of the Loligo sp. pen: molecular weights and pIs are better separated in the soluble matrix of Loligo sp. than in the matrices of Spirula spirula and Sepia sp. Only the amino-acid compositions of the insoluble matrices of some coleoid shells are available in the

529

Literature. Thus a comparison between theoretical pI (calculated from the amino-acid contents) and the observed pl is not possible for the soluble matrices. It is well known that in molluscan shells the acidic amino acid content of soluble matrix is higher than that of insoluble matrix (Gr6goire et al. 1955; Hare 1963; Degens et al. 1967; Weiner 1979; Watabe et al. 1993). However, the acidity of the insoluble matrices of various molluscan shells is heterogeneous (Wada 1966). Total acidic amino acid contents are higher in the mineralized part of a shell. For example, the aspartic acid content in the insoluble part of the Loligo sp. pen is 7 % (Ghiselin et al. 1967; Hunt and Nixon 1981), and in the insoluble part of the horny plate of Sepia sp. it is 6 % (Drozdova and al. 1971). Glutamic acid contents are ~ 4 and 8%, respectively, in the same shells. The aspartic acid of the ventral part of Sepia sp. is ~ 17%, and glutamic acid content is 7% (Wada 1966; Drozdora and al. 1971). The basic pI of the soluble matrix of the Loligo sp. pen and the acidic pI of the soluble matrices of the mineralized shells of Spirula spirula and Sepia sp. support the view that the acidity of the organic matrix is related to mineralization processes.

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