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Purification and partial characterization of vitellogenin from shorthead redhorse (Moxostoma macrolepidotum) and copper redhorse (Moxostoma hubbsi) and detection in plasma and mucus with a heterologous antibody

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Abstract

Vitellogenin (VTG), the egg yolk precursor protein, was purified from plasma of estradiol-3-benzoate (E2B)-treated male shorthead redhorse (Moxostoma macrolepidotum) and immature copper redhorse (Moxostoma hubbsi) by a two-step chromatographic procedure without precipitation. Intact VTGs appeared as dimers with apparent molecular masses, determined by gel filtration, of ∼425 kDa (copper redhorse) and ∼450 kDa (shorthead redhorse). In native polyacrylamide gel electrophoresis (PAGE), dimeric redhorse VTGs appeared as a 520 kDa band. Both VTGs were reduced to a single monomer of ∼150 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions, indicating that monomers are not linked by disulfide bonds in the dimer form. The purified proteins were characterized as phospholipoglycoproteins. Isoelectric focusing of both VTGs revealed components with isoelectric points ranging from 5.3 to 6.0, suggesting charge heterogeneity. The amino acid composition of both VTGs contains a high proportion of nonpolar amino acids and was similar to those of other teleosts. An antibody developed against carp (Cyprinus carpio) VTG showed cross-reactivity with VTG from both redhorse species. Using this antibody, VTG was detected in plasma and surface mucus of E2B-treated redhorse. This is the most extensive report on purification and characterization of vitellogenin from catostomidid species.

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Abbreviations

CR-VTG:

Copper redhorse vitellogenin

E2B:

Estradiol-3-benzoate

IEF:

Isoelectric focusing

SR-VTG:

Shorthead redhorse vitellogenin

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Acknowledgements

Yves Gauthier of the Montréal Biôdome kindly provided the copper redhorse. Bernard Chenard of the Institut Maurice-Lamontagne advised on fish culture. Véronique Desborbes assisted with SDS-PAGE and Western blot. Initial work with copper redhorse was financed by a matching grant from Fisheries and Oceans Canada, under the Species at Risk program. We thank two reviewers whose comments improved the manuscript.

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  1. Pêches et Océans Canada, Institut Maurice-Lamontagne, 850 route de la Mer, P.O. Box 1000, Mont-Joli, QC, Canada, G5H 3Z4

    D. Maltais & R. L. Roy

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  1. D. Maltais
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Maltais, D., Roy, R.L. Purification and partial characterization of vitellogenin from shorthead redhorse (Moxostoma macrolepidotum) and copper redhorse (Moxostoma hubbsi) and detection in plasma and mucus with a heterologous antibody. Fish Physiol Biochem 35, 241–254 (2009). https://doi.org/10.1007/s10695-008-9205-6

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