SAMP32, a testis-specific, isoantigenic sperm acrosomal membrane-associated protein.
To identify novel human sperm membrane antigens, we analyzed two-dimensional gels of sperm extracts containing hydrophobic proteins that partitioned into Triton X-114. Four protein spots with isoelectric points (pIs) ranging from 4.5 to 5.5 and apparent molecular weights from 32 to 34 kDa were sequenced by mass spectrometry and found to contain common peptide sequences. Cloning the corresponding cDNA revealed that these protein spots were products of a single gene (SAMP32), encoding a protein of 32 kDa with a predicted pI of 4.57. SAMP32 has a potential transmembrane domain in the carboxyl terminus and is phosphorylated in vivo on serine 256. Northern blotting of eight human tissues and RNA dot blotting of 76 human tissues showed that SAMP32 expression was testis specific. SAMP32 contained an amino terminal domain homologous to the major malarial circumsporozoite surface protein and a domain similar to that of Krp1 from Schizosaccharomyces pombe in its carboxyl terminus. The SAMP32 locus consists of seven exons on chromosome 6q15-16.2. Antiserum against recombinant SAMP32 recognized protein spots originally cored from a two-dimensional gel. This antiserum strongly stained the equatorial segment and faintly stained the acrosome cap of ejaculated human spermatozoa by immunofluorescence. Immunoelectron microscopy showed that SAMP32 was associated with the inner acrosomal membrane in the principal and the equatorial segments of the sperm acrosome. By immunostaining enzyme-dissociated testicular cells, SAMP32 was localized to Golgi phase round spermatids and subsequent stages of acrosome biogenesis. Recombinant SAMP32 reacted with serum from an infertile man, suggesting that it is isoantigenic. Antibodies against recombinant SAMP32 inhibited both the binding and the fusion of human sperm to zona-free hamster eggs.
Hao
Zhonglin
Z
Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, Virginia 22908, USA.
Wolkowicz
Michael J
MJ
Shetty
Jagathpala
J
Klotz
Kenneth
K
Bolling
Laura
L
Sen
Buer
B
Westbrook
V Anne
VA
Coonrod
Scott
S
Flickinger
Charles J
CJ
Herr
John C
JC
D000177:Acrosome / Q000276:immunology* / Q000502:physiology / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D000918:Antibody Specificity; D000941:Antigens; D001483:Base Sequence; D002462:Cell Membrane / Q000737:chemistry; D002874:Chromosome Mapping; D002896:Chromosomes, Human, Pair 6; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D012739:Gonadal Steroid Hormones; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007248:Infertility, Male / Q000276:immunology; D007519:Isoantigens / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007526:Isoelectric Point; D008297:Male; D013058:Mass Spectrometry; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology; D029589:Seminal Plasma Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D013084:Sperm-Ovum Interactions / Q000187:drug effects; D013094:Spermatozoa / Q000648:ultrastructure; D013737:Testis / Q000276:immunology*
D000906:Antibodies; D000941:Antigens; D018076:DNA, Complementary; D012739:Gonadal Steroid Hormones; D007106:Immune Sera; D007519:Isoantigens; D011994:Recombinant Proteins; C455113:SPACA1 protein, human; D029589:Seminal Plasma Proteins