Sperm mitochondria-associated cysteine-rich protein (SMCP) is an autoantigen in Lewis rats.
A common repertoire of rat sperm antigens have previously been identified by Western blotting of sperm proteins with sera obtained after vasectomy or isoimmunization with sperm. Aside from a determination of their apparent masses, however, the biochemical characteristics of these antigens have remained unknown. In this study, a rat testis cDNA expression library was screened with polyclonal antibodies obtained from rats immunized with isologous spermatozoa to identify and sequence a full-length clone encoding rat sperm mitochondria-associated cysteine-rich protein (SMCP). The open reading frame of SMCP was expressed in the pET22b vector, and recombinant SMCP (rec-SMCP) was purified. Sera from rats that had been vasectomized or hyperimmunized with isologous sperm specifically recognized rec-SMCP whereas preimmune sera from these experimental groups did not react. Rabbit antiserum produced to rec-SMCP recognized rec-SMCP on Western blots and precisely immunolocalized SMCP to the mid-piece of rat sperm. On Western blots against sperm extracts, the rabbit antibody recognized a major protein band of approximately 22-25 kDa that co-migrated with bands of identical mass that were recognized by sera from hyperimmune or vasectomized rats. These findings demonstrate that SMCP is a sperm autoantigen, recognized following vasectomy, and an isoantigen, recognized by antibodies generated through isologous immunization with sperm.
Department of Cell Biology, The Center for Recombinant Gamete Contraceptive Vaccinogens, University of Virginia, Charlottesville, Virginia 22908, USA. jch7k@viginia.edu
J C
JC
Herr
D
D
Thomas
L A
LA
Bush
S
S
Coonrod
V
V
Khole
S S
SS
Howards
C J
CJ
Flickinger
D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000276:immunology*; D001483:Base Sequence; D015153:Blotting, Western; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D008297:Male; D008928:Mitochondria / Q000276:immunology; D008969:Molecular Sequence Data; D011506:Proteins / Q000276:immunology*; D011817:Rabbits; D051381:Rats; D011917:Rats, Inbred Lew / Q000276:immunology*; D051140:Selenoproteins; D013094:Spermatozoa / Q000276:immunology; D013737:Testis / Q000378:metabolism
D001324:Autoantigens; D018076:DNA, Complementary; D011506:Proteins; C099545:SMCP protein, rat; D051140:Selenoproteins