Indian Journal of Experimental Biology Vol. 51, June 2013, pp. 411-420

Isolation, purification and characterization of the egg-yolk proteins from the oocytes of the Indian freshwater murrel, Channa punctatus (Bloch) Om Prakasha, N Sehgalb, K V Ranib & N Aggarwalb* a

Department of Zoology, SriVenkateswara College, University of Delhi, New Delhi 110 021, India b Department of Zoology, University of Delhi, Delhi 110007, India Received 19 September 2012; revised 8 March 2013

In oviparous organisms, yolk accumulation in the oocytes is critical and indispensable for the development of the newly hatched young ones. In fish and many other oviparous vertebrates, the major constituents of the egg-yolk are synthesized as a precursor in the liver. The precursor is transported to the oocyte for uptake and cleaved into major yolk proteins lipovitellin, phosvitin and β’-components. The eggs of Channa punctatus are pelagic, have large oil globule and exceptionally high lipid content. Lipovitellin was isolated by single step gel filtration chromatography on Sepharose 6B. Purified native lipovitellin showed immunological reactivity with vitellogenin antiserum. Phosvitin isolated by phenol extraction method could not be visualized with routine protein staining methods, whereas incorporation of trivalent ions in the coomassie brilliant blue stained phosvitin. It was characterized by in vivo labeling of egg-yolk proteins with 32P. The molecular mass of murrel phosvitin was less than 14,000 kDa. Keywords: Channa punctatus, Egg-yolk extract, Lipovitellin, Murrel, Phosvitin

In oviparous and ovo-viviparous teleosts, egg-yolk proteins are the main source of nutrition during embryonic development. The yolk proteins are derived from vitellogenin, which is synthesized in liver, under the influence of estrogens1. Vitellogenin is processed to produce lipovitellin, a lipoprotein, and phosvitins and phosvettes which are phosphoproteins1-6. Even though yolk formation is a highly conserved physiological strategy of reproduction, the composition of egg-yolk differs widely from species to species because vitellogenin is a multimember family gene7. Though lipovitellin, phosvitin and lipids are the major constituents of fish eggs, vitamins, metal binding proteins and induction factors are also present in the egg-yolk8,9. Lipovitellin, the largest vitellogenin-derived yolk product, consists of two polypeptides, the N-terminal lipovitellin heavy chain (LvI or LvH) and the C-terminal lipovitellin light chain (LvII or LvL). It serves mainly as a source of amino acids and lipids required for embryonic development. Phosvitin is widely found in the vertebrate egg-yolk and consists of long chains of serine residues interrupted by short ___________ *Correspondent author Telephone: 91 9958880622 Fax: +91 011 27666254. E-mail: [email protected]

stretches of basic amino acids10. Characteristically, phosvitin acts as a phosphate and metal ion carrier associated with bone formation in vertebrates. Fish phosvitin is reported to be smaller in size in comparison to Xenopus and chicken phosvitin11. Voluminous information is available on the isolation, purification and characterization of vitellogenin in fishes7,12-16 but information regarding yolk proteins is scanty17,18. Not much work with respect to isolation and characterization of egg-yolk proteins has been carried out in fishes native to Indian subcontinent. In fishes whose yolk proteins have been characterized, information mainly pertains to lipovitellin19,20. Considering the existence of VgA and VgB21 in the form of charge isomers19,22, the present study has been undertakan to isolate, purify and partially characterize lipovitellin and phosvitin from eggs of murrel. Also, a relatively simple method has been devised to visualize phosvitin protein. The investigation was conducted on the Indian freshwater murrel, Channa punctatus, an economically important fish known for low fat, few intramuscular spines and survival in oxygen-depleted waters23. They are considered to be a delicacy and demand high prices24. Channa punctatus is an annual breeder and spawns during monsoon season. It releases pelagic eggs with a large oil globule containing exceptionally high lipid content.

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Materials and Methods AnimalsGravid murrel, Channa punctatus (body wt. 100–150 g) collected from the backwaters of river Yamuna around Delhi (Lat. 28o35' N, Long. 77o12' E) were maintained at 25 ± 1 ºC and a photoperiod of 12 L:12 D for at least 7 days prior to use in experiments25. Fish were fed ad libitum every evening with minced beef. Polyclonal antibodies against vitellogeninPurified vitellogenin from murrel was emulsified with complete Freund’s adjuvant and administered subcutaneously four times at weekly intervals into sensitized New Zealand white rabbits25. Blood was collected after booster dose, serum was separated, decomplimented and stored at -20 °C. This antiserum was referred to as VgAs. The specificity of VgAs was ascertained by immunoprecipitation technique25. Preparation of egg-yolk extractGravid females were sacrificed by decapitation; ovarian covering was removed and ovaries transferred to 0.9% saline containing 2 mM phenyl methyl sulphonyl fluoride (PMSF). Oocytes were weighed and homogenized with an equal volume of 1 M NaCl containing 3 mM PMSF (1:1 w/v) using a Teflon coated homogenizer. The homogenate was passed through 100 µm sieve and centrifuged for 1 h at 10,000 g at 4 °C. Subnatant was recentrifuged till a clear subnatant was obtained. The clear subnatant was centrifuged at 106,000 g for 3 h at 4 °C to remove low-density lipoproteins. The resultant subnatant was egg-yolk extract (EYE) which was processed to isolate lipovitellin and phosvitin by different methods. Gravid fish were injected with estradiol (10 µg/100 g body weight) along with an intra-peritoneal injection of 0.25 mCi of 32P in 0.6% saline, to obtain 32Plabeled egg-yolk extract (32P*EYE). Fish were sacrificed 24 h after treatment. Fish were intraperitoneally injected with aprotinin, a protease inhibitor, fifteen minutes prior to decapitation. Isolation of lipovitellinEYE was subjected to gel filtration chromatography on Sepharose 6B equilibrated with Tris–Cl buffer (0.02 M Tris-Cl, pH 8.0 containing 0.35 M NaCl and 0.2% sodium azide) at 4 °C. Proteins were eluted at a flow rate of 20 mL/h, 5 mL fractions were collected and absorbance was recorded at 280 nm. Yolk proteins were precipitated from the EYE by adding ammonium sulphate to 67% saturation followed by ultracentrifugation at 134,000 g for 1 h at 4 °C26-28. Subnatant was discarded and the pellet was

dissolved in Tris-Cl buffer, dialyzed against the same buffer and fractionated on to Sepharose 6B column. Proteins were eluted at a flow rate of 20 mL/hour, five-ml fractions were collected and absorbance was recorded at 280 nm. Another method used to isolate egg-yolk proteins was distilled water precipitation29. The yolk extract was diluted by adding 30 volumes of chilled distilled water and kept overnight at 4° C. The extract was centrifuged at 2000 g at 4° C; the pellet was dissolved in Tris-Cl buffer and subjected to gel filtration chromatography on Sepharose 6B as described above. Partial characterization of lipovitellinFractions from the three absorbance peaks after gel filtration chromatography of EYE were subjected to native PAGE. Gels were stained for proteins separately with coomassie brilliant blue R-250 (CBB) and CBB containing Al3+ ions; for glycoproteins with Basic Fuchsin; and for lipids with Sudan Black B. Stained gels were scanned and photographed. Proteins from other corresponding unstained gel were transferred to nitrocellulose membrane. After blocking the membrane with 1% casein in phosphate buffer saline (PBS, pH 7.4), membrane was incubated with primary vitellogenin antiserum (VgAs). This was followed by incubation with secondary antibody labeled with horseradish peroxidase, and colour development using specific substrate25. Protein samples and standard molecular weight markers (range 14 to 97 kDa) were electrophoresed under denaturing conditions. The stained gels were analysed by Quantity One D Analytical software (BioRad) to compute molecular mass of lipovitellin with reference to standards. Isolation of phosvitin32P-labeled egg-yolk extract was chromatographed on Sepahrose 6B column as described above. 100 µL from each fraction was applied to individual filter paper disk (Whatman 3 mm). Disks were air-dried and fixed for 30 min at 4 °C in 10% trichloroacetic acid-10% phosphotungstic acid mixture and then washed with ethanol:ether (3:1) followed with ether to remove the lipids30. The disks were placed in scintillation vials and counted for 32P activity. Phosvitin was separated from EYE as well as from 32 P*EYE by a modification of the phenol extraction method31. The EYE was passed through 0.22 µ millipore filter paper and equal volume of 90% phenol was added gradually with constant stirring at room temperature. The solution was centrifuged at 10,000 g for 1 h at 4 °C and aqueous phase was collected. The

OM PRAKASH et al.: EGG-YOLK PROTEINS FROM THE OOCYTES OF CHANNA PUNCTATUS

molarity of aqueous phase was adjusted with 0.1 M CaCl2. The aqueous phase was kept overnight at 4 °C and centrifuged at 12,000 g to collect the precipitate. The precipitate was dissolved in 1 M NaCl and the solution was characterized by electrophoresis. The obtained solution and crude EYE were separately dialyzed against Tris-Cl buffer. Partial characterization of phosvitinDialysed as well as undialysed samples of crude EYE and phenolextracted fraction were electrophoresed under native conditions on 15% gels. Gels were stained with CBB dissolved in methanol and acetic acid for proteins. For staining acidic phosphoproteins, CBB was dissolved in 0.1 M aluminium nitrate containing 25% isopropyl alcohol, 10% acetic acid and 1% triton X-100. Gels were also silver stained32. All the stained gels were scanned and photographed using GS-800 calibrated densitometer, combined with Image analyzer software (Bio Rad). Phenol-extracted phosvitin fraction and standard molecular weight markers (range 14 to 97 kDa) were electrophoresed under denaturing conditions on 20% gels. The stained gels were analysed by Quantity One D Analytical software (BioRad) to compute molecular weights of the different subunits of phosvitin with reference to standards. 32 P labeled phenol-extracted phosvitin was electrophoresed and unstained gel was processed for radioactivity analysis by cutting gel transversally into 2 mm thick slices. Individual gel slices were dried in scintillation vials overnight at room temperature. Hydrogen peroxide (0.5 mL; 30% w/v) was added and the vials were incubated at 60 oC to solubilize the gel slices. Radioactivity was measured in scintillation counter. AutoradiographyCrude EYE as well as phenolextracted phosvitin (radiolabeled and unlabeled) was subjected to native gel electrophoresis in two sets. One set was stained with CBB containing Al3+ ions for acidic phosphoproteins. The proteins from the second set of gels were transferred to nitrocellulose membrane. The nitrocellulose membrane was washed thoroughly with 0.1 M phosphate buffer, pH 7.4 containing 0.9% NaCl. Washed membrane was placed over X-ray film in a cassette and kept at –20 ºC for 7 days. The film was developed in dark, washed and fixed. Results Isolation and characterization of lipovitellinThe crude egg-yolk extract (radiolabeled and unlabeled),

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on subjecting to gel filtration chromatography, resolved into three absorbance peaks at Ve 310 mL, 370 mL and 425 mL (Fig. 1a). Protein-bound 32Pradioactivity was absent in all the three peaks, although free (unbound) radioactivity was associated with the last peak (Fig. 1b). Addition of saturated ammonium sulphate or chilled distilled water to the egg-yolk extract resulted in the precipitation of lipovitellin. The gel filtration chromatography of ammonium sulphate precipitated proteins resulted in an elution pattern similar to that of crude egg yolk extract (Fig. 1c). Gel filtration chromatography of chilled distilled water precipitated proteins resulted in a single homogenous absorbance peak (Ve 320 mL) followed by two minor contaminants with very little absorbance (Fig. 1d). Fractions from the three peaks were electrophoresed under native conditions. The gels were stained separately with CBB and CBB containing Al3+ ions. A single protein band was detected in peak I fraction, whereas no protein band could be detected in remaining two peaks (Fig. 2A, B). The same band showed positive staining with Basic Fuchsin (for carbohydrates) and Sudan Black B (for lipids). The protein in peak I showed immunoreactivity with the vitellogenin antiserum (Fig. 2C). On SDS PAGE, the protein from peak I resolved into two peptides (110 and 100 kDa) (Fig. 3). Isolation and characterization of phosvitinPhosvitin isolated by phenol extraction of egg yolk extract (radiolabeled and unlabeled) was electrophoresed on polyacrylamide gel (15%) under native conditions (Fig. 4). Phosvitin could not be stained with CBB stain or with silver stain. When Al3+ ions were incorporated in the CBB stain, five protein bands were visualized. Even in the crude egg yolk extract, protein bands corresponding to purified phosvitin could be visualized only after Al3+ incorporation. Autoradiography of crude and purified phosvitin suggested that radioactivity was associated with these bands (Fig. 5). This observation was further substantiated with radioactivity count in gel slices (Fig. 6). Purified phosvitin resolved into four peptides under denaturing conditions (Fig. 7). Dialysis membrane with molecular weight cut-off of 10 kDa was unable to retain purified phosvitin suggesting it to be of low molecular mass (Fig. 8). Protein bands were not visualized in dialyzed purified phosvitin while they were visible in the dialyzed and undialyzed egg yolk extract, and non-dialyzed purified phosvitin.

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Fig. 1Elution profiles of egg-yolk proteins from murrel, Channa punctatus chromatographed on Sepharose 6B. Egg-yolk extract (a) or 32 _ P labelled egg-yolk extract (b) was directly subjected to chromatography. Egg-yolk proteins were precipitated with either ammonium sulphate (c) or distilled water (d) prior to chromatography. Proteins were resolved into three peaks (I, II and III).

Fig. 2Electrophoretograms (A and B) of fractions (lane 1, 2, 3) from respective peaks (I, II, III) separated by gel filtration chromatography of egg-yolk extract of the murrel, Channa punctatus (refer Fig. 1a) and corresponding Western blots (C) using vitellogenin antiserum.

Discussion In several teleost fishes, the egg-yolk proteins, lipovitellin and phosvitin, are reported to exist as a complex in the form of non-granular fluid yolk. Various fractionation schemes have been proposed to isolate purified egg-yolk proteins. A commonly used method is the selective precipitation of one or both of these proteins with ammonium sulphate33,34. In addition, the characteristic insolubility of fish yolkproteins at low ionic strength has been used for selectively precipitating these proteins by extreme dilution of egg-yolk extract with distilled water26,29. Lipovitellin and phosvitin precipitate as a complex and can be separated by chromatography in Heteropneustes fossilis35, Sebastes mystinus36 and Mugil cephalus34. In the present study, both the methods effectively precipitate lipovitellin from egg-yolk extract leaving phosvitin in solution. However the gel filtration profile reveals contamination in precipitated proteins,

OM PRAKASH et al.: EGG-YOLK PROTEINS FROM THE OOCYTES OF CHANNA PUNCTATUS

with distilled water precipitate being less contaminated than ammonium sulphate precipitate. Further purification was required to obtain pure lipovitellin. This is in contrast to the observation of de Vlaming37 on the goldfish, Carassius auratus, Sundararaj P35 on Heteropneustes fossilis and Carnevali38 on the gilthead seabream, Sparus aurata where, uncontaminated lipovitellin could be isolated

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by repeated precipitation method only. In herring, Clupea harengus39 and in grunion, Leuresthes tenuis36 dilution of egg-yolk extract did not result in precipitation of yolk proteins. The egg-yolk extract of the murrel resolved into three discrete absorbance peaks on subjecting to gel filtration on Sepharose 6B. The gel filtration profiles of precipitated lipovitellin on Sepharose 6B reveal an identical pattern. 32P radiolabeled egg-yolk extract of murrel was subjected to gel filtration chromatography to monitor the phosvitin fraction. Of the three proteins isolated by this procedure, two exhibited high absorbance at 280 nm whereas the last fraction absorbed poorly at 280 nm but had very high 32P radioactivity and possibly represents phosvitin. Phosvitin fraction has low molecular weight as it elutes at the bed volume. Gel filtration chromatography

Fig. 3Determination of molecular mass of murrel lipovitellin. Electrophoretic banding pattern of purified lipovitellin (lane I) and marker proteins (lane M: 14 kDa to 97 kDa) on 10% resolving gel under denaturing conditions.

Fig. 5Stained electrophoretogram of 32P-labeled egg-yolk extract (lane a), purified unlabeled phosvitin (lane b) and purified 32 P labeled phosvitin (lane b′) of the murrel, Channa punctatus (A) followed by autoradiography (B).

Fig. 4Native polyacrylamide gel electrophoresis (15% resolving gel and 3.5% stacking gel) of egg-yolk extract (lane a), phenol extracted proteins (lane b) and bovine serum albumin as marker protein (lane c). Gels were stained with coomassie brilliant blue containing trivalent aluminum ions.

Fig. 6Radioactive measurement following native PAGE of 32Plabeled purified phosvitin of the murrel, Channa punctatus. Gel was sliced (2mm) and 32P activity was counted.

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Fig. 7Determination of molecular mass of murrel phosvitin. Electrophoretogram of purified phosvitin (lane a) and marker proteins (lane M; 14 kDa to 66 kDa) on 20% resolving gel under denaturing conditions.

Fig. 8Native polyacrylamide gel electrophoresis (15% resolving gel and 3.5% stacking gel) of purified phosvitin (lane a), egg-yolk extract (lane b), dialyzed purified phosvitin (lane a′) and dialyzed egg-yolk extract (lane b′).

separates phosvitin from major yolk proteins in murrel, but is unable to fractionate it in a pure form, whereas, with selective precipitation, phosvitin remains in the supernatant. Therefore, these methods are not recommended for purification of murrel phosvitin. The fractions from first absorbance peak in the gel filtration chromatography of crude yolk extract and

precipitated EYE resolve into a single protein band in native state. This protein showed immunological relationship with vitellogenin antibody, thus confirming its identity as lipovitellin and indicating a precursor product relationship of vitellogenin with lipovitellin40. On SDS-PAGE murrel lipovitellin resolved into peptides of 100 and 110 kDa, possibly representing products of VgA and VgB. This assumption has to be further investigated. The present study indicates that murrel lipovitellin can be purified by subjecting the egg-yolk extract directly to gel filtration and precipitation step is not essential. A review of literature reveals that lipovitellinphosvitin complex obtained by extreme dilution of egg-yolk with chilled distilled water or by salting out using ammonium sulphate, can be used to purify phosvitin26,36,41,42. In few fishes, including the murrel (present study), phosvitin is not precipitated as a complex but remains in solution after selective precipitation and has to be either dialysed43,44 or subjected to chromatography in order to obtain purified phosvitin34. Dialysis of soluble fraction leads to the loss of phosvitin in many cases due to its low molecular weight36,37. In addition, a phenol extraction procedure has also been employed frequently27,31. In the present study, phenol extracted precipitate resolved into four bands on native PAGE. Phosvitin, being a phosphorylated protein is negatively charged and could not be stained with CBB or with silver stain. Most proteins have absorption maxima in UV-region and this characteristic is used for their detection after routine purification procedure. Since fish phosvitins lack aromatic amino acids and have high content of serine residues, their detection becomes difficult33,36,37. Moreover, on native PAGE phosvitin does not stain with CBB stain33,34,38,45,46, though positive staining with this stain has been reported by Riazi and Fremont47. The inability of phosvitin to bind anionic dyes has been attributed to high negative charge density of clustered phosphorylserine residues in phosvitin molecules48. A cationic dye ‘Stains-All’ has been employed successfully by many investigators to stain phosvitin27,38,45,46. Poor staining with silver has been observed in few species45. Phosvitin showed a negative staining reaction with methyl green49 whereas it has been used for staining phosvitin in some fishes34,50. Incorporation of aluminium ions in the CBB, successfully stained phosvitin. The trivalent ions act like a bridge between the dye and the

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phosphorylserine residues of phosvitin to form a coloured complex. Exceptional affinity of phosphorylserine residues for trivalent metal ions has been reported36,51,52. This property has been utilized to devise a protocol for staining phosvitin36,52,53. The electrophoretic pattern of dialysed egg-yolk extract on native gel shows the presence of lipovitellin as well as phosvitin. Presence of radioactivity only in the bands corresponding to phosvitin reveals that it is the only phosphorylated protein present in the egg-yolk of murrel. The SDS gel electrophoretic pattern reflects

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presence of four peptides in murrel phosvitin having a molecular weight less than 14 kDa. Table 1 summarizes the molecular mass of lipovitellin and phosvitin from several teleosts. The molecular weight estimates of native lipovitellin from fish eggs are reported to range from 240-500 kDa14. Three yolk proteins (YP 1-3) have been reported in Morone americana43 whereas four proteins (YP 1-4) were purified from Mugil cephalus34. In general, phosvitins of fish have lower molecular weights as compared to other vertebrates37,43,44,50. The goldfish has two

Table1Molecular mass of lipovitellin and phosvitin from teleosts Species

Lipovitellin Native

Acanthogobius flavimanus Anguilla japonica Carassius auratus Channa punctatus Clupea pallasi Dicentrarhcus labrax L Fundulus heteroclitus Gadus morhua Heteropneustes fossilis Hucho perryi Ictalurus punctatus Labeo rohita Melanogrammus aeglifenus Morone americana

Mugil cephalus

Oreochromis mossambicus Oreochromis mossambicus Oncorhynchus kisutch Oryzias latipes Oryzias latipes Pleuronectes vetulus Pleuronectes americanus Salmo trutta fario Salmo gairdneri Sparus aurata Verasper moseri

Phosvitin

Molecular Mass (kDa) Subunits

480 320 350

Native

References

Molecular Mass (kDa) Subunits

122, 157 127 85, 31, 25

[59]

14.5, 7.6 110, 100 85, 55, 35, 15 122, 103 400 330 330 120 620, 225 110, 30, 20 YP1 310 YP2 27 YP3 YP1 330 YP2 325 YP3 335 YP4 570 116, 105, 18, 17, 14 107,25, 24, 23 390 190, 220 190 YP1: 270 YP2: 380 -

< 14 4.2 30, 26, 20

31, 53, 71, 105,124 150 23 100, 90, 29 29, 17 20, 15, 14 110, 87, 34, 30 99, 87, 29 97, 87, 21.5 110, 87, 70, 54 113, 94 84, 72 115, 95, 67 110, 105, 94 94

-

21, 17, 14 84, 40, 47

-

[22, 62] [63] [35] [33] [64] [65]

-

[66] [43]

25

[34]

38 22

[67] [68] [69] [50]

-

[17] -

-

9.3 40

24

111, 94 350 150, 100, 75, 16 85, 72, 44, 39, 12 107,102, 94, 34, 42,28

[60] [37] [Present study] [54] [61]

[70] [71] [72] [31] [47] [38] [73]

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phosvitins of 7.6 and 14.5 kDa37, herring has a phosvitin of 4.2 kDa54, while in eight salmonids species, phosvitin ranges from 3 to 5 kDa31. In addition to lipovitellin and phosvitin, lipids are also the major constituents of fish egg. Presence of large oil globule in murrel eggs may contribute to buoyancy. The positive buoyancy of pelagic eggs confers clear advantages, since buoyancy allows more efficient oxygen exchange between the developing embryos and the atmosphere and increases their survival55. The egg specific gravity also affects the dispersal and vertical distribution of pelagic eggs within the water column by currents, and thus it may influence the reproductive success at different hydrographic conditions55-57. True egg buoyancy in freshwater is seen in few species, as Amur snakehead (Ophiocephalus argus) and gourami (Colisa lalia), in which an enormous oil droplet occupies two-thirds of the egg volume allowing the eggs to develop at the surface of stagnant, oxygen-depleted waters58. Even in marine teleosts that spawn in freshwater, such as striped bass (Morone saxatilis), the eggs show large oil globuli and have an exceptional high lipid content (55% dry weight). In the present study on the Indian murrel, lipovitellin is seen to consist of two peptides (100 and 110 kDa), whereas phosvitin consists of four peptides with a molecular mass of less than 14 kDa.

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Acknowledgement This work was supported by research grant WOSA/LS-49/2009 from Department of Science and Technology (DST), New Delhi.

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References

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