Association of eppin with semenogelin on human spermatozoa.
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.
Wang
Zengjun
Z
Laboratories for Reproductive Biology and Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Widgren
E E
EE
Sivashanmugam
P
P
O'Rand
M G
MG
Richardson
R T
RT
D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012661:Semen / Q000378:metabolism; D029607:Seminal Vesicle Secretory Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013094:Spermatozoa / Q000378:metabolism*
C431553:Eppin protein, human; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D010446:Peptide Fragments; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D011994:Recombinant Proteins; D029607:Seminal Vesicle Secretory Proteins; C424482:seminal vesicle-specific antigen