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.
Herr
J C
JC
Department of Cell Biology, The Center for Recombinant Gamete Contraceptive Vaccinogens, University of Virginia, Charlottesville, Virginia 22908, USA. jch7k@viginia.edu
Thomas
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D
Bush
L A
LA
Coonrod
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S
Khole
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V
Howards
S S
SS
Flickinger
C J
CJ
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