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Disulfide linked αoLH-gelonin conjugate failed to recombine with βoLH subunit to generate bioeffective hormonotoxin

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Abstract

Since, linking of ovine luteinizing hormone (oLH) to ribosome inactivating protein gelonin (in oLH-gelonin conjugate) occur via the alpha-subunit, αoLH, an attempt has been made to develop a ‘universal’ hormonotoxin for selective targeting to specific cells in the gonads. Four different molar ratios of oLH and N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) were used to activate the epsilon amino (ψ-NH2) groups of αoLH. The αoLH-SPDP derivatives recombine to native beta subunit of oLH (βoLH) and the purified recombinants retained substantial receptor binding, steroidogenic activity and immunoreactivity to native oLH. The disulfide linked αoLH-S-S-gelonin conjugates prepared by SPDP method were purified by gel filtration chromatography and analysed by reverse-phase high performance liquid chromatography (RP-HPLC). In order to obtain specificity and bioeffectivity, the αoLH-S-S-gelonin conjugates were allowed to recombine to native βoLH and the recombination mixture was further purified by gel-filtration chromatography. The RP-HPLC analysis of these recombinants indicated that αoLH-S-S-gelonin did not recombine to βoLH. The failure of recombination may be due to the reasons. (i) The site of ε-NH2 activation by SPDP may be different in the αoLH than the native oLH. (ii) The activation site may be in close proximity to the annealing site which facilitates the recombination of β-subunit but failured to reassociate to αoLH-S-S-gelonin conjugate. (iii) The introduction of gelonin (30 kDa basic protein) might have induced some steric hinderence for βoLH to recombine to the αoLH site which might have been masked in αoLH-S-S-gelonin conjugates. (Mol Cell Biochem120: 95–102, 1993)

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Abbreviations

oLH:

ovine Luteinizing Hormone

αoLH:

alpha subunit of oLH

βoLH:

beta subunit of oLH

BSA:

Bovine Serum Albumin

DTT:

Dithiothreitol

RP-HPLC:

Reverse Phase High Performance Liquid Chromatography

TSH:

Thyroid Stimulating Hormone

FSH:

Follicle Stimulating Hormone

LH:

Luteinizing Hormone

eCG:

equine Chorionic Gonadotropin

DMEM:

Dulbecco's Modified Eagles Medium

HEPES:

4-(2-Hydroxyethyl)-1 Piperazine Ethane Sulfonic acid

PAP:

Pokeweed Antiviral Protein

RIA:

Radioimmunoassay

hCG:

human Chorionic Gonadotropin

TRH:

Thyrotropin Releasing Hormone

CRF:

Corticotropin Releasing Factor

hPL:

human Placental Lactogen

TFA:

Trifluroacetic Acid

αoLH-SPDP:

SPDP activated derivative of αoLH

References

  1. 1.

    Frankel AE (ed): Immunotoxins. Kluwer Academic Publishers, Norwell MA, 1988

  2. 2.

    Vogel CW (ed): Immunoconjugates: Antibody conjugates in radioimaging and therapy of cancer. Oxford University Press, New York, 1987

  3. 3.

    Moller G (ed): Immunol Rev 62: 1–255, 1982

  4. 4.

    Vitetta ES, Krolick KA, Miyama-Inaba M, Cushely W, Uhr JW: Immunotoxins: A new approach to cancer therapy. Science 219: 644–650, 1984

  5. 5.

    Vitetta ES, Foulton RJ, May RD, Till M, Uhr JW: Redesigning nature's poisons to create anti tumor reagents. Science 238: 1098–1104, 1987

  6. 6.

    Chang T, Dazord A, Neville DM Jr: Artificial hybrid protein containing a toxin protein fragment and a cell membrane receptor binding moiety in a disulfide complex. J Biol Chem 252: 1515–1522, 1977

  7. 7.

    Miskimins WK, Shimizu N: Synthesis of a cytotoxic insulin cross-linked to diphtheria toxin fragment A capable recognising insulin receptors. Biochem Biophys Res Commun 91: 143–151, 1979

  8. 8.

    Oeltmann TN, Heath EC: A hybrid protein containing a toxic subunit of ricin and the cell specific subunit of human chorionic gonadotropin. I. Synthesis and characterization. J Biol Chem 254: 1022–1027, 1979

  9. 9.

    Oeltmann TN: Synthesis andin vitro activity of a hormone-diphtheria toxin fragment A hybrid. Biochem Biophys Res Commun 133: 430–435, 1985

  10. 10.

    Bacha P, Murphy JR, Reichlein S: Thyrotropin-releasing hormone-diphtheria toxin related polypeptide conjugates: Potential role of the hydrophobic domain in toxin entry. J Biol Chem 258: 1565–1570, 1983

  11. 11.

    Schwartz J, Penke B, Rivier J, Vale W: A new cytotoxin specific for the target of corticotropin releasing factor. Endocrinology 121: 1454–1460, 1987

  12. 12.

    Singh V, Sairam MR, Bhargavi GN, Akhras RG: Hormonotoxins: Preparation and characterization of ovine luteinizing hormone-gelonin conjugate. J Biol Chem 264: 3089–3095, 1989

  13. 13.

    Singh V, Sairam MR: Hormonotoxins: Conjugation of human chorionic gonadotropin with the ribosome inactivating protein, gelonin and comparison with a lutropin conjugate. Mol Cell Endocrinol 67: 217–229, 1989

  14. 14.

    Singh V: Hormonotoxin: Synthesis, characterization and bioefficacy of some defined disulfide linked conjugates of ovine luteinizing hormone with a ribosome inactivating protein gelonin. Indian J Exp Biol 29: 916–925, 1991

  15. 15.

    Singh V, Das C:In vitro selective killing of gonadal cells by hormonotoxin composed of ovine luteinizing hormone linked by a disulfide bond to the ribosome inactivating protein, gelonin. Biochemistry International 24: 689–699, 1991

  16. 16.

    Singh V: Design and synthesis of bioeffective hormonotoxins for selective elimination of gonadal cells. In Horizons in Endocrinology, Vol II, Edited by M Maggi and V Geenen, Serono Symposia publications, Raven Press, Vol. 76, pp 197–202, 1991

  17. 17.

    Singh V: Hormonotoxins In Recent Frontiers in Reproduction Research. Edited by C Das and J Sengupta, Wiley-Eastern Publishers, New Delhi, 1992. In press

  18. 18.

    Singh V, Sairam MR: Hormonotoxins. I. Strategy for synthesis of ovine luteinizing hormone-toxin conjugate bearing toxin in the beta subunit. Int J Peptide & Protein Res 33: 22–28, 1989

  19. 19.

    Stripe F, Olsnes S, Phil A: Gelonin, a new inhibitor of protein synthesis, non-toxic to intract cell. Isolation, characterization and preparation of cytotoxic complexes with Concanavalin A. J Biol Chem 255: 6947–6953, 1980

  20. 20.

    Singh V, Sairam MR: Effect of thiolation on immunoreactivity of the ribosome inactivating protein, gelonin. Biochem J 263: 417–423, 1989

  21. 21.

    Singh V, Kar SK: Properties of a ribosome inactivating protein, gelonin purified from three different methods. Indian J Biochem & Biophys 29: 31–41, 1992

  22. 22.

    Singh V, Curtiss R, III: Hormonotoxins: Abrogation of ribosome inactivating property of gelonin in the disulfide linked ovine luteinizing hormone-gelonin conjugate. Biochemistry International 25: 531–536, 1991

  23. 23.

    Singh V, Sairam MR: Effect of thiolation of amino groups of ovine lutropin on immunoreactivity, receptor binding and bioactivity. Mol Cell Endocrinol 63: 255–262, 1989

  24. 24.

    Carlsson J, Drevin H, Axen R: Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl-3-(2-pyridyl dithio) propionate, a new hetero bifunctional reagent. Biochem J 173: 723–737, 1978

  25. 25.

    Pierce JG, Parsons TF: Glycoprotein hormones: Structure and function. Ann Rev Biochem 50: 465–495, 1981

  26. 26.

    Sairam MR: Gonadotropic hormones: relationship between structure and function with emphasis on antagonists. In: Hormonal Proteins and Peptides, Li CH, Editor. Academic Press, New York, pp 1–79, 1983

  27. 27.

    Bewely TA, Sairam MR, Li CH: Circular dichorism of ovine interstital stimulating hormone and its subunits. Biochemistry 11: 932–936, 1972

  28. 28.

    Sairam MR, Papkoff H, Li CH: Reaction of ovine interstitial cell stimulating hormone with tetranitromethane. Biochem Biophys Acta 278: 421–432, 1972

  29. 29.

    Mendelson C, Dufau M, Catt K: Gonadotropin binding and stimulation of Cyclic Adenosine 3′∶5′-monophosphate and testosterone production in isolated Leydig cells. J Biol Chem 250: 8818–8823, 1975

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Correspondence to Vinod Singh.

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Singh, V., Curtiss, R. Disulfide linked αoLH-gelonin conjugate failed to recombine with βoLH subunit to generate bioeffective hormonotoxin. Mol Cell Biochem 120, 95–102 (1993). https://doi.org/10.1007/BF00926081

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Key Words

  • gonadotropin
  • immunoreactivity
  • receptor binding
  • steroidogenesis