Purification and Characterization of a Putative Vitellogenin from the Ovary
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____________________________________________________________________________www.paper.edu.cn Comparative Biochemistry and Physiology Part B 129Ž. 2001 121᎐127 Purification and characterization of a putative vitellogenin from the ovary of amphioxus ž/Branchiostoma belcheri tsingtaunese Xutong Sun, Shicui ZhangU Department of Marine Biology, Ocean Uni¨ersity of Qingdao, Qingdao 266003, PR China Received 18 August 2000; received in revised form 21 January 2001; accepted 29 January 2001 Abstract An oocyte-yolk protein was purified by double-step chromatography from amphioxus ovaries. The purified protein appeared to exist as a homodimer of approximately 320 kDa in native polyacrylamide gel electrophoresisŽ. PAGE , and was reduced to a single monomer of approximately 160 kDa in sodium dodecyl sulfate-PAGEŽ. SDS-PAGE . The protein was characterized as a phospholipoglycoprotein by native PAGE and staining of gels for phosphorus with methyl green, for lipids with oil red O and Sudan black B, and for carbohydrates using periodic acidrSchiff reagent. In addition, the amino acid composition of the oocyte-yolk protein was generally similar to that of vitellogeninsŽ. Vgs isolated from different phyla of animals including both vertebrates and invertebrates. The purified phospholipoglycoprotein is thus considered as putative amphioxus Vg. ᮊ 2001 Elsevier Science Inc. All rights reserved. Keywords: Amphioxus; Branchiostoma; Ovary; Vitellogenin; Phospholiglycoprotein; Purification; Characterization; Evolution 1. Introduction Ž.Wallace, 1985; Byrne et al., 1989 . In the oocytes, Vg is usually cleaved proteolytically to form yolk Vitellogenesis, the production of vitellogenin proteins, which are later used as the nutritive Ž.Vg , is a significant event in the reproductive material by the developing embryos and larvae cycle of all egg-laying animals. Vg, the egg yolk Ž.Wallace, 1985 . protein precursor, is usually synthesized by ex- Vg is a high molecular mass protein, containing traovarian tissues, secreted into the circulatory covalently linked carbohydrates and phosphates system and subsequently internalized by growing and non-covalently bound lipids, it is thus a phos- oocytes through receptor-mediated endocytosis pholipoglycoprotein. The protein has been isolated from different phyla of animals including insectsŽ.Ž Pan et al., 1969 , arthropods Lui and U O’Connor, 1977.Ž , nematodes Klass et al., 1979 . , Corresponding author. Tel.: q86-532-203-2787; fax: q86- 532-203-2276. sea urchinsŽ.Ž Amant et al., 1986 , fish Roubal et E-mail address: [email protected]Ž. S. Zhang . al., 1997.Ž , amphibians Rudack and Wallace, 1096-4959r01r$ - see front matter ᮊ 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 6 - 4 9 5 9Ž. 0 1 00310-4 ____________________________________________________________________________中国科技论文在线 www.paper.edu.cn 122 X. Sun, S. Zhang rComparati¨e Biochemistry and Physiology Part B 129() 2001 121᎐127 1968.Ž , snakes Janeiro-Cinquini et al., 1999 . and mM Tris᎐HClŽ. pH 8.0 and dialyzed against 20 birdsŽ. Greengard et al., 1964 . The amino acid mM Tris᎐HClŽ. pH 8.0 for 24 h at 4ЊC. After composition analysis reveals that Vgs purified centrifugation at 8000=g for 20 min at 4ЊC, the from different animal phyla are generally rich in supernatant was applied to a 2.0=6.0 cm column Ala, Glu, Asp, Ser, Leu, Val and Lys residues. of Sephadex G-200Ž. Pharmacia equilibrated with Vg has been investigated most extensively in 20 mM Tris᎐HClŽ. pH 8.0 and the proteins were vertebrates including fish, amphibians and birds eluted at a flow rate of 15 mlrh with 20 mM and in invertebrates including nematodes and Tris᎐HClŽ. pH 8.0 . Protein in the eluted fractions arthropods. In these animals, Vg generally circu- was measured by the method of BradfordŽ. 1976 lates as a dimer consisting of two identical po- using ovalbumin as the standard protein. Eluted lypeptide chains of ;170᎐200 kDaŽ Klass et al., fractions containing putative Vg protein were 1979; Wallace, 1985; Montorzi et al., 1994; Rup- identified on 7.5% native polyacrylamide gel elec- pert, 1997. Amphioxus or lancelet, a cephalo- trophoresisŽ. PAGE , pooled, concentrated with chordate, has long been regarded as the living polyethyleneglycol 20 000 and chromatographed invertebrate most closely related to the archetype on a 10-ml bed-volume column of DEAE-23 cel- of vertebrates. It has been widely known as the luloseŽ. Whatman equilibrated with 50 mM most important animal to study the origin of Tris᎐HClŽ. pH 8.0 . Proteins bound to the column vertebrates and extensively studied embryologi- were eluted at a constant flow rate of 40 mlrh cally, morphologically and physiologicallyŽ Willey, with 50 mM Tris᎐HClŽ. pH 8.0 containing 0, 0.05, 1894; Ruppert, 1997. However, there has been 0.1, 0.15, 0.2, 0.25 and 0.3 M NaClŽ. 50 ml each , no report on amphioxus Vg so far. In the present respectively, and the A280 of the column frac- study, we report for the first time the purification tionsŽ. 2 ml each was monitored. Fractions were and characterization of a phospholipoglycopro- analyzed by PAGE and those containing putative tein, a putative Vg, from amphioxus ovaries. The Vg protein were pooled and stored at y70ЊC. results will be relevant in future determinations of evolution and comparative trends of am- 2.2. Polyacrylamide gel electrophoresis phioxus Vg with that of both invertebrates and vertebrates. Native PAGE was conducted on a 7.5% separa- tion gel with a 4% spacer gel using the buffer system of DavisŽ. 1964 . After electrophoresis, the 2. Materials and methods total proteins were stained with Coomassie bril- liant blue R-250. To test the presence of carbohy- 2.1. Purification of the putati¨e Vg protein from drate, lipid and phosphorus components, the gels amphioxus o¨aries were stained using periodic acidrSchiff reagent Ž.Ž.Fairbanks et al., 1971 , oil red O Noble, 1969 Ovaries obtained from female amphioxus and Sudan black BŽ. De Vlaming et al., 1977 and Branchiostoma belcheri tsingtauense collected from methyl greenŽ. Cutting and Roth, 1973 , respec- the sandy bottom of the seawater near Shazikou, tively. The molecular mass standards used were Qingdao, China at the breeding season were bovine milk ␣-lactalbuminŽ. 14.2 kDa , carbonic stored at y70ЊC until use. The putative Vg pro- anhydraseŽ. 29 kDa , chicken egg albumin Ž 45 tein was isolated and purified following the proce- kDa.Ž. , bovine serum albumin monomer 66 kDa dures of Hiramatsu and HaraŽ. 1996 and Roubal and dimerŽ. 132 kDa , Jack bean urease trimer et al.Ž. 1997 with slight modifications. Briefly, 2-g Ž.272 kDa and hexamer Ž.Ž. 545 kDa Sigma . ovaries were homogenized in 10 volumes of 20 SDS-PAGEŽ. 0.1% SDS was performed accord- mM Tris᎐HCl bufferŽ. pH 8.0 containing 2 mM ing to LaemmliŽ. 1970 . The gel consisted of a 4% EDTA, 150 mM NaCl, 0.25 mM PMSF and 1 mM spacer gel and a 7.5% separation gel. The protein -mercaptoethanol and centrifuged at 8000=g components were stained with Coomassie bril- for 20 min at 4ЊC. The supernatant was pooled liant blue R-250. The molecular mass standards and mixed with ammonium sulfate to a final con- used were rabbit actinŽ. 43 kDa , bovine serum centration of 67% saturation. The mixture was albuminŽ. 66.2 kDa , rabbit phosphorylase b Ž 97.4 placed and stirred on ice for 2 h, and centrifuged kDa.Ž. , calmodulin binding protein 130 kDa and as above. The pellet was collected, dissolved in 20 myosinŽ. 200 kDa . ____________________________________________________________________________中国科技论文在线 www.paper.edu.cn X. Sun, S. Zhang rComparati¨e Biochemistry and Physiology Part B 129() 2001 121᎐127 123 Fig. 1. Gel filtration of the extracted egg-yolk proteins on a Sephadex G-200 column. Approximately 50 mg of proteins were applied to a 2.0=6.0 cm Sephadex G-200 column and then eluted with bufferŽ. 20 mM Tris᎐HCl, pH 8.0 . Four Fig. 2. DEAE anion exchange column of amphioxus Vg. The peaks A, B, C and D appeared in the elution curve, and the Vg-containing fraction A from the Sephadex G-200 column r Vg was contained in peak A. were pooled and concentrated up to 0.5mg ml, and then applied to DEAE-23 column. Proteins bound to the column were eluted with 50 mM Tris᎐HClŽ. pH 8.0 containing 0.1, 0.15, 0.2 and 0.25 M NaCl, respectively, and the Vg was eluted 2.3. Amino acid analysis with buffer containing 0.2 M NaCl. Samples of the purified Vg-like protein were 3. Results hydrolyzed in 6 N HCl at 110ЊC for 24 hŽ Spack- man et al., 1958. , and amino acid analysis was performed on Beckman 6300E analyzer. The N- Gel filtration of the extracted oocyte-yolk pro- terminal amino acid sequence was also carried teins on Sephadex G-200 yielded A, B, C and D out according to the method of MatsudariaŽ. 1987 . four distinct protein peaksŽ. Fig. 1 and the Vg-like Fig. 3.Ž. a Native PAGE Ž 7.5% . Lane 1, amphioxus Vg purified by Sephadex G-200; lane 2, amphioxus Vg purified by Sephadex G-200 and DEAE-cellulose. The gels were stained with Coomassie brilliant blue R-250.Ž. b Molecular mass determination of putative amphioxus Vg using native PAGE. ____________________________________________________________________________中国科技论文在线 www.paper.edu.cn 124 X. Sun, S. Zhang rComparati¨e Biochemistry and Physiology Part B 129() 2001 121᎐127 Fig. 4.Ž. a SDS-PAGE Ž 7.5% . Lane 1, marker; lane 2, Vg. Ž. b Molecular mass determination of amphioxus Vg using SDS-PAGE. protein was eluted in peak AŽ Fig. 1 and Fig. 3a, under SDS-PAGEŽ. Fig. 4b . It appears that the lane 1. Fig. 2 shows the elution profile of DEAE- putative amphioxus Vg is a homodimer.