Journal of Comparative Physiology B

, Volume 184, Issue 5, pp 589–599 | Cite as

Development and partial characterisation of an antiserum against apolipoprotein B of the short-finned eel, Anguilla australis

  • Erin L. Damsteegt
  • Hiroko Mizuta
  • Yuichi Ozaki
  • Naoshi Hiramatsu
  • Takashi Todo
  • Akihiko Hara
  • Shigeho Ijiri
  • Shinji Adachi
  • P. Mark Lokman
Original Paper

Abstract

Despite its key role in transportation of triacylglycerides in blood, the distribution, localisation and molecular weight variants of apolipoprotein B (Apob) in teleost fish have essentially escaped study. To address this, a specific short-finned eel (Anguilla australis) Apob antiserum was produced by an immunised rabbit, purified and partially characterised. Localisation of Apob at both the mRNA (in situ hybridisation) and protein (immunohistochemistry) levels mirrored that of mammals; thus immunostaining was confined to the interstitial spaces of the liver and the vascular core of the intestinal villi. Immunostaining of proteins by Western blotting, followed by high-resolution LC–MS, indicated that peptide sequence coverage of Apob in low-density lipoproteins spanned the full-length protein. We conclude that only full-length Apob is produced by eels and that both liver and intestine are key sites for its synthesis.

Keywords

Apolipoprotein B Antiserum Anguilla australis In situ hybridisation Immunohistochemistry 

Abbreviations

2ME

2-Mercaptoethanol

Apob

Apolipoprotein B

BCIP

5-Bromo-4-chloro-3-indolyl phosphate

CBB

Coomassie brilliant blue

DEPC

Diethylpyrocarbonate

DIG

Digoxigenin

Eef1α

Eel elongation factor one alpha

IPTG

Isopropyl β-d-1-thiogalactopyranoside

LC–MS

Liquid chromatography–mass spectrometry

Ldl

Low-density lipoprotein

NBT

4-Nitro blue tetrazolium chloride

PBS

Phosphate-buffered saline

PFA

Paraformaldehyde

PVDF

Polyvinylidene fluoride

qPCR

Quantitative polymerase chain reaction

SDS-PAGE

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

tag

Triacylglyceride

Vldl

Very low-density lipoprotein

References

  1. Alexander CA, Hamilton RL, Havel RJ (1976) Subcellular localization of B apo-protein of plasma lipoproteins in rat liver. J Cell Biol 69:241–263PubMedCrossRefGoogle Scholar
  2. Ando S, Matsuzaki M (1996) A unique lipoprotein profile found in the plasma of cultured Japanese eel Anguilla japonica: very low density lipoprotein, but not high density lipoprotein, is the main component of plasma. Fish Physiol Biochem 15:469–479PubMedCrossRefGoogle Scholar
  3. Babin PJ (1987) Plasma lipoprotein and apolipoprotein distribution as a function of density in the rainbow trout (Salmo gairdneri). Biochem J 246:425–429PubMedCentralPubMedGoogle Scholar
  4. Blue ML, Protter AA, Williams DL (1980) Biosynthesis of apolipoprotein B in rooster kidney, intestine, and liver. J Biol Chem 255:10048–10051PubMedGoogle Scholar
  5. Boren J, Wettesten M, Rustaeus S, Anderson M, Oloesson S-O (1993) The assembly and secretion of Apob-100 containing lipoproteins. Biochem Soc Trans 21:487–493PubMedGoogle Scholar
  6. Bostrom K, Borbn J, Wettesten M, Sjoberg A, Bondjers G, Wiklund O, Carlsson P, Olofsson S-O (1988) Studies on the assembly of apo B-100-containing lipoproteins in HepG2 cells. J Biol Chem 263:4434–4442PubMedGoogle Scholar
  7. Braissant O, Wahli W (1998) A simplified in situ hybridization protocol using non-radioactively labeled probes to detect abundant and rare mRNAs on tissue sections. Biochemica 1:10–16Google Scholar
  8. Callan CK, Laidley CW, Kling LJ, Breen NE, Rhyne AL (2012) The effects of dietary HUFA level on flame angelfish (Centropyge loriculus) spawning, egg quality and early larval characteristics. Aquac Res 44:1–11CrossRefGoogle Scholar
  9. Cardin AD, Witt KR, Chao J, Margolius HS, Donaldson VH, Jackson RL (1984) Degradation of apolipoprotein B-100 of human plasma low density lipoproteins by tissue and plasma kallikreins. J Biol Chem 259:8522–8528PubMedGoogle Scholar
  10. Chapman MJ, Goldstein S, Mills GL, Leger C (1978) Distribution and characterization of the serum lipoproteins and their apoproteins in the rainbow trout (Salmo gairdneri). Biochem 17:4455–4464CrossRefGoogle Scholar
  11. Chen SH, Habib G, Yang CY, Gu ZW, Lee BR, Wang S, Silberman SR, Cai S, Deslypere JP, Rosseneu M, Gorro AM, Li W, Chan L (1987) Apolipoprotein B-48 is the product of a messenger RNA with an organ-specific in-frame stop codon. Science 238:363–366PubMedCrossRefGoogle Scholar
  12. Davis RA, Prewett AB, Chan DCF, Thompson JJ, Borchardt RA, Gallaher WR (1989) Intrahepatic assembly of very low density lipoproteins: immunologic characterization of apolipoprotein B in lipoproteins and hepatic membrane fractions and its intracellular distribution. J Lipid Res 30:1185–1196PubMedGoogle Scholar
  13. Dullaart RPF, Speelberg B, Schuurman H-J, Milne RW, Havekes LM, Marcel YL, Geuze HJ, Hulshof MM, Erkelens DW (1986) Epitopes of apolipoprotein B-100 and B-48 in both liver and intestine expression and evidence for local synthesis in recessive abetalipoproteinemia. J Clin Invest 78:1397–1404PubMedCentralPubMedCrossRefGoogle Scholar
  14. Gibbons GF (1990) Assembly and secretion of hepatic very-low density lipoprotein. Biochem J 268:1–13PubMedCentralPubMedGoogle Scholar
  15. Havel RJ, Eder HA, Bragdon JH (1955) The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 34:1345–1353PubMedCentralPubMedCrossRefGoogle Scholar
  16. Hayashi S, Yu F-G (1993) Lipoprotein synthesis and secretion by cultured eel (Anguilla japonica) hepatocytes. Comp Biochem Physiol B Comp Biochem 105:443–448CrossRefGoogle Scholar
  17. Hoeg JM, Sviridov DD, Tennyson GE, Demosky SJ, Meng MS, Bojanovski D, Safonova IG, Repin VS, Kuberger MB, Smirnov VN, Higuchi K, Gregg RE, Brewer HB (1990) Both apolipoproteins B-48 and B-100 are synthesized and secreted by the human intestine. J Lipid Res 31:1761–1769PubMedGoogle Scholar
  18. Hopkins B, Brice AL, Schofield PN, Baralle FE, Graham CF (1987) Identity of cells containing apolipoprotein B messenger RNA, in 6- to 12-week postfertilization human embryos. Development 100:83–93PubMedGoogle Scholar
  19. Ikeuchi T, Mochida K, Ura K, Adachi S, Yamauchi K (1995) Specific antibodies against synthetic peptides of salmonid GTH I and II β subunits. Zool Sci 12:317–323CrossRefGoogle Scholar
  20. Kane JP, Hardman DA, Paulus HE (1980) Heterogeneity of apolipoprotein B: isolation of a new species from human chylomicrons. Proc Natl Acad Sci USA 77:2465–2469PubMedCentralPubMedCrossRefGoogle Scholar
  21. Kjolby M, Andersen OM, Breiderhoff T, Fjorback AW, Pedersen KM, Madsen P, Jansen P, Heeran J, Willnow TE, Nykjaer A (2010) Sort1, encoded by the cardiovascular risk locus 1p13.3, is a regulator of hepatic lipoprotein export. Cell Metab 12:213–223PubMedCrossRefGoogle Scholar
  22. Knott TJ, Rail SC, Innerarity TL, Jacobson SF, Urdea MS, Levy-Wilson B, Powell LM, Pease RJ, Eddy R, Nakai H, Byers M, Priestley LM, Robertson E, Rall LB, Betholtz C, Shows TB, Mahley RW, Scott J (1985) Human apolipoprotein B: structure of carboxylterminal domains, sites of gene expression and chromosomal localization. Science 230:37–43PubMedCrossRefGoogle Scholar
  23. Knott TJ, Pease RJ, Powell LM, Wallis SC, Rall SC, Innerarity TL, Blackhart B, Taylor WH, Marcel Y, Milne R, Johnson D, Fuller M, Lusis AJ, McCarthy BJ, Mahley RW, Levy-Wilson B, Scott J (1986) Complete protein sequence and identification of structural domains of human apolipoprotein B. Nature 323:734–738PubMedCrossRefGoogle Scholar
  24. Kovar J, Havel RJ (2002) Sources and properties of triglyceride-rich lipoproteins containing Apob-48 and Apob-100 in postprandial blood plasma of patients with primary combined hyperlipidemia. J Lipid Res 43:1026–1034PubMedCrossRefGoogle Scholar
  25. Kusakabe M, Todo T, McQuillan HJ, Goetz FW, Young G (2002) Characterization and expression of steroidogenic acute regulatory protein and MLN64 cDNAs in trout. Endocrinology 143:2062–2070PubMedCrossRefGoogle Scholar
  26. Lee DM, Valente AJ, Kuo WH, Maeda H (1981) Properties of apolipoprotein B in urea and in aqueous buffers, the use of glutathione and nitrogen in its solubilization. Biochim Biophys Acta 666:133–146PubMedCrossRefGoogle Scholar
  27. Levy E, Rochette C, Londono I, Roy CC, Mihe RW, Marcel YL, Bendayant M (1990) Apolipoprotein B-100: immunolocalization and synthesis in human intestinal mucosa. J Lipid Res 31:1937–1946PubMedGoogle Scholar
  28. Linton MF, Gish R, Hubl ST, Butler E, Esquivel C, Bry WI, Boyles JK, Wardell MR, Young SG (1991) Phenotypes of apolipoprotein B and apolipoprotein E after liver transplantation. J Clin Invest 88:270–281PubMedCentralPubMedCrossRefGoogle Scholar
  29. Lokman PM, Vermeulen GJ, Lambert JGD, Young G (1998) Gonad histology and plasma steroid profiles in wild New Zealand freshwater eels (Anguilla dieffenbachii and A. australis) before and at the onset of the natural spawning migration. I. Females. Fish Physiol Biochem 19:325–338CrossRefGoogle Scholar
  30. Lokman PM, Kazeto Y, Ozaki Y, Ijiri S, Tosaka R, Kohara M, Divers SL, Matsubara H, Moore LG, Adachi S (2010) Effects of reproductive stage, GH, and 11-ketotestosterone on expression of growth differentiation factor-9 in the ovary of the eel, Anguilla australis. Reproduction 139:71–83PubMedCrossRefGoogle Scholar
  31. Milne RW, Weech PK, Blanchette L, Davigon J, Alaupovic P, Marcel YL (1984) Isolation and characterization of apolipoprotein B-48 and B-100 very low density lipoproteins from type III hyperlipoproteinemic subjects. J Clin Invest 73:816–823PubMedCentralPubMedCrossRefGoogle Scholar
  32. Morrow JA, Arnold KS, Dong J, Balestra ME, Innerarity TL, Weisgraber KH (2000) Effect of arginine 172 on the binding of apolipoprotein E to the low density lipoprotein receptor. J Biol Chem 275:2576–2580PubMedCrossRefGoogle Scholar
  33. Ndiaye D, Hayashi S (1997) A lipoprotein secreted by cultured hepatocytes of silver or yellow eel: comparison with their plasma lipoproteins Comp. Biochem Physiol B Biochem Mol Biol 116:209–216CrossRefGoogle Scholar
  34. Nielsen LB, McCormick SPA, Pierotti V, Tam C, Gunn MD, Shizuya H, Young SG (1997) Human apolipoprotein B transgenic mice generated with 207- and 145-kilobase pair bacterial artificial chromosomes. Evidence that a distant 59-element confers appropriate transgene expression in the intestine. J Biol Chem 272:29752–29758PubMedCrossRefGoogle Scholar
  35. Paolucci M, Callard IP (1995) Distribution and characterization of apolipoproteins in Chrysemys picta plasma. Comp Biochem Physiol B Biochem Mol Biol 110:583–588PubMedCrossRefGoogle Scholar
  36. Paolucci M, Guerriero G, Botte V, Ciarcia G (1998) Apolipoproteins and their electrophoretic pattern throughout the reproductive cycle in the green frog Rana esculenta. Comp Biochem Physiol B Biochem Mol Biol 119:647–654PubMedCrossRefGoogle Scholar
  37. Perez LE, Williams P, Callard IP (1992) Putative apolipoprotein B-100 in the freshwater turtle Chrysemys picta: effects of estrogen and progesterone. Comp Biochem Physiol B Comp Biochem 103:707–713CrossRefGoogle Scholar
  38. Phillips ML, Pullinger C, Kroes I, Kroes J, Hardman DA, Chen G, Curtiss LK, Intestineierrez MM, Kane JP, Schumaker VN (1997) A single copy of apolipoprotein B-48 is present on the human chylomicron remnant. J Lipid Res 38:1170–1177PubMedGoogle Scholar
  39. Powell LM, Wallis SC, Pease RJ, Edwards YH, Knott TJ, Scott J (1987) A novel form of tissue-specific RNA processing produces apolipoprotein-B48 in intestine. Cell 50:831–840PubMedCrossRefGoogle Scholar
  40. Schuh J, Fairclough GF, Haschemeyer RH (1978) Oxygen-mediated heterogeneity of apo-low-density lipoprotein. Proc Natl Acad Sci USA 75:3173–3177PubMedCentralPubMedCrossRefGoogle Scholar
  41. Segrest JP, Garber DW, Brouillette CG, Harvey SC, Anantharamaiah GM (1994) The amphipathic alpha helix: a multifunctional structural motif in plasma apolipoproteins. Adv Protein Chem 45:303–369PubMedCrossRefGoogle Scholar
  42. Setiawan AN, Lokman PM (2010) The use of reference gene selection programs to study the silvering transformation in a freshwater eel Anguilla australis: a cautionary tale. BMC Mol Biol 11:75–86PubMedCentralPubMedCrossRefGoogle Scholar
  43. Skinner ER, Rogie A (1978) The isolation and partial characterization of the serum lipoproteins and apolipoproteins of the rainbow trout. Biochem J 173:507–520PubMedCentralPubMedGoogle Scholar
  44. Sorci-Thomas M, Wilson MD, Johnson FL, Williams DL, Rudel LL (1989) Studies on the expression of genes encoding apolipoproteins B100 and B48 and the low density lipoprotein receptor in nonhuman primates: comparison of dietary fat and cholesterol. J Biol Chem 264:9039–9045PubMedGoogle Scholar
  45. Spector AA (1984) Plasma lipid transport. Clin Physiol Biochem 2:123–134PubMedGoogle Scholar
  46. Srivastava RA, Tang J, Baumann D, Schonfeld G (1992) Hormonal and nutritional stimuli modulate apolipoprotein B mRNA editing in mouse liver. Biochem Biophys Res Commun 188:135–141PubMedCrossRefGoogle Scholar
  47. Steele JCH, Reynolds JA (1979a) Characterization of the apolipoprotein B polypeptide of human plasma low density lipoprotein in detergent and denaturant solutions. J Biol Chem 245:1633–1638Google Scholar
  48. Steele JCH, Reynolds JA (1979b) Molecular weight and hydrodynamic properties of apolipoprotein B in guanidine hydrochloride and sodium dodecyl sulfate solutions. J Biol Chem 245:1639–1643Google Scholar
  49. Tarugi P, Albertazzi L, Nicolini S, Calandra S (1990) Absence of apolipoprotein B-48 in the chick, Gallus domesticus. J Lipid Res 31:417–427PubMedGoogle Scholar
  50. Weisgraber KH (1994) Apolipoprotein E: structure-function relationships. Adv Protein Chem 45:249–302PubMedCrossRefGoogle Scholar
  51. Wiegand MD (1996) Composition, accumulation and utilization of yolk lipids in teleost fish. Rev Fish Biol Fisher 6:259–286CrossRefGoogle Scholar
  52. Williams DL (1979) Apoproteins of avian very low density lipoprotein: demonstration of a single high molecular weight apoprotein. Biochemistry 18:1056–1063PubMedCrossRefGoogle Scholar
  53. Yamamoto M, Ranganathan S, Kottke BA (1985) Structure and function of human low density lipoproteins: studies using proteolytic cleavage by plasma kallikrein. J Biol Chem 260:8509–8513PubMedGoogle Scholar
  54. Yanes-Roca C, Rhody N, Nystrom M, Main KL (2009) Effects of fatty acid composition and spawning season patterns on egg quality and larval survival in common snook (Centropomus undecimalis). Aquaculture 287:335–340CrossRefGoogle Scholar
  55. Yu F-G, Ando S, Hayashi S (1991) Characterization of lipoprotein secreted by cultured eel hepatocytes and its comparison with serum lipoproteins. Cell Struct Funct 16:347–355PubMedCrossRefGoogle Scholar
  56. Yu F-G, Ando S, Hayashi S (1992) Effects of insulin, eel serum very low density lipoprotein, and high density lipoprotein on lipoprotein synthesis and secretion by cultured hepatocytes of eel (Anguilla japonica). Comp Biochem Physiol B Comp Biochem 101:333–339CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Erin L. Damsteegt
    • 1
  • Hiroko Mizuta
    • 2
  • Yuichi Ozaki
    • 1
  • Naoshi Hiramatsu
    • 2
  • Takashi Todo
    • 2
  • Akihiko Hara
    • 2
  • Shigeho Ijiri
    • 2
  • Shinji Adachi
    • 2
  • P. Mark Lokman
    • 1
  1. 1.Department of ZoologyUniversity of OtagoDunedinNew Zealand
  2. 2.Faculty of Fisheries SciencesHokkaido UniversityHakodateJapan

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