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Abstract:

Rat sperm epididymal glycoprotein DE belongs to the cysteine-rich secretory protein (CRISP) family and participates in sperm-egg fusion through its binding to complementary sites on the egg surface. To investigate the molecular mechanisms underlying the role of DE in gamete fusion, in the present work we expressed DE in a prokaryotic system, and examined the relevance of carbohydrates and disulfide bonds for the biological activity of the protein. Immunofluorescence and sperm-egg fusion assays carried out in the presence of recombinant DE (recDE) revealed that this protein exhibits the ability to bind to the DE-egg binding sites and to inhibit gamete fusion, as does native DE (nDE). Comparison of the proteins indicated, however, that the inhibitory ability of recDE was significantly lower than that of nDE. This difference would not be due to the lack of carbohydrates in the bacterially expressed protein because enzymatically deglycosylated nDE was as able as the untreated protein to inhibit gamete fusion. To examine whether disulfide bridges are involved in DE activity, the presence of sulfhydryls in nDE and recDE was evaluated by the biotin-maleimide technique. Results indicated that, unlike nDE, in which all cysteines are involved in disulfide bonds, recDE contains free thiol groups. Subsequent experiments showed that reduction of nDE with dithiothreitol significantly decreased the ability of the protein to inhibit gamete fusion. Together, these results indicate that whereas carbohydrates do not have a role in DE-mediated gamete fusion, disulfide bridges are required for full biological activity of the protein. To our knowledge, this is the first study reporting the relevance of structural components for the function of a CRISP member.

Registro:

Documento: Artículo
Título:Expression and structure-function analysis of DE, a sperm cysteine-rich secretory protein that mediates gamete fusion
Autor:Ellerman, D.A.; Da Ros, V.G.; Cohen, D.J.; Busso, D.; Morgenfeld, M.M.; Cuasnicú, P.S.
Filiación:Inst. de Biol./Medicina Experimental, Vuelta de Obligado 2490, 1428 Buenos Aires, Argentina
Palabras clave:Epididymis; Fertilization; Fusion; Ovum; Sperm; biotin; carbohydrate; cysteine rich secretory protein; disulfide; dithiothreitol; glycoprotein de; maleimide; recombinant protein; recombinant protein de; secretory protein; thiol derivative; unclassified drug; animal cell; article; binding site; controlled study; deglycosylation; disulfide bond; drug activity; enzyme glycosylation; epididymis; female; fertility; fertilization; gamete; immunofluorescence; inhibition kinetics; male; molecular mechanics; nonhuman; priority journal; prokaryote; protein expression; rat; sperm; structure activity relation; structure analysis; Animalia; Prokaryota
Año:2002
Volumen:67
Número:4
Página de inicio:1225
Página de fin:1231
DOI: http://dx.doi.org/10.1095/biolreprod67.4.1225
Título revista:Biology of Reproduction
Título revista abreviado:Biol. Reprod.
ISSN:00063363
CODEN:BIREB
CAS:biotin, 58-85-5; disulfide, 16734-12-6; dithiothreitol, 3483-12-3; maleimide, 541-59-3; thiol derivative, 13940-21-1
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00063363_v67_n4_p1225_Ellerman

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Citas:

---------- APA ----------
Ellerman, D.A., Da Ros, V.G., Cohen, D.J., Busso, D., Morgenfeld, M.M. & Cuasnicú, P.S. (2002) . Expression and structure-function analysis of DE, a sperm cysteine-rich secretory protein that mediates gamete fusion. Biology of Reproduction, 67(4), 1225-1231.
http://dx.doi.org/10.1095/biolreprod67.4.1225
---------- CHICAGO ----------
Ellerman, D.A., Da Ros, V.G., Cohen, D.J., Busso, D., Morgenfeld, M.M., Cuasnicú, P.S. "Expression and structure-function analysis of DE, a sperm cysteine-rich secretory protein that mediates gamete fusion" . Biology of Reproduction 67, no. 4 (2002) : 1225-1231.
http://dx.doi.org/10.1095/biolreprod67.4.1225
---------- MLA ----------
Ellerman, D.A., Da Ros, V.G., Cohen, D.J., Busso, D., Morgenfeld, M.M., Cuasnicú, P.S. "Expression and structure-function analysis of DE, a sperm cysteine-rich secretory protein that mediates gamete fusion" . Biology of Reproduction, vol. 67, no. 4, 2002, pp. 1225-1231.
http://dx.doi.org/10.1095/biolreprod67.4.1225
---------- VANCOUVER ----------
Ellerman, D.A., Da Ros, V.G., Cohen, D.J., Busso, D., Morgenfeld, M.M., Cuasnicú, P.S. Expression and structure-function analysis of DE, a sperm cysteine-rich secretory protein that mediates gamete fusion. Biol. Reprod. 2002;67(4):1225-1231.
http://dx.doi.org/10.1095/biolreprod67.4.1225