Association of Eppin With Semenogelin on Human Spermatozoa

Biol Reprod. 2005 May;72(5):1064-70. doi: 10.1095/biolreprod.104.036483. Epub 2004 Dec 8.

Abstract

Eppin (SPINLW1; GeneID, 57119) is a single-copy gene encoding a cysteine-rich protein found only in the testis and epididymis, which contains both Kunitz-type and WAP-type four disulfide core protease inhibitor consensus sequences. This study demonstrates that, in seminal plasma and on human spermatozoa following ejaculation, Eppin is bound to semenogelin I (Sg). Six different experimental approaches: 1) immunoprecipitation from spermatozoa and seminal plasma with anti-Eppin, 2) colocalization in semen and spermatozoa, 3) incubation of recombinant Eppin (rEppin) and rSg and immunoprecipitation with either anti-Eppin or anti-Sg, 4) far-Western blotting of Eppin and Sg, 5) Saturation binding of 125I-Sg to Eppin, which is competed by unlabeled Sg, and 6) direct binding of 125I-Sg to Eppin on a blot, all demonstrate that Eppin and Sg bind to each other. To study the specificity of binding, recombinant fragments of Eppin and Sg were made and demonstrate that the Eppin(75-133) C-terminal fragment binds the Sg(164-283) fragment containing the only cysteine in human Sg I (Cys-239). Reduction and carboxymethylation of Cys239 blocks binding of 125I-rEppin, indicating that a disulfide bond may be necessary for Eppin binding. The physiological significance of the Eppin-semenogelin complex bound on the surface of ejaculate spermatozoa lies in its ability to provide antimicrobial activity for spermatozoa, which has been reported for both Eppin and semenogelin-derived peptides, and in its ability to provide for the survival and preparation of spermatozoa for fertility in the female reproductive tract.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Female
  • Humans
  • Immunohistochemistry
  • In Vitro Techniques
  • Male
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Multiprotein Complexes / chemistry
  • Multiprotein Complexes / metabolism
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Binding
  • Proteinase Inhibitory Proteins, Secretory
  • Proteins / chemistry
  • Proteins / genetics
  • Proteins / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Semen / metabolism
  • Seminal Vesicle Secretory Proteins / chemistry
  • Seminal Vesicle Secretory Proteins / genetics
  • Seminal Vesicle Secretory Proteins / metabolism*
  • Spermatozoa / metabolism*

Substances

  • Eppin protein, human
  • Membrane Proteins
  • Multiprotein Complexes
  • Peptide Fragments
  • Proteinase Inhibitory Proteins, Secretory
  • Proteins
  • Recombinant Proteins
  • Seminal Vesicle Secretory Proteins
  • seminal vesicle-specific antigen