Cloning and sequencing of cDNAs coding for the human intra-acrosomal antigen SP-10.
cDNAs coding for the intra-acrosomal protein SP-10 were cloned and characterized as a first step in understanding the expression of this antigen during spermatogenesis. Three overlapping SP-10-specific cDNAs were isolated from a human testes cDNA expression library. These cDNAs hybridized to a 1.35-kb mRNA that was present in human testes but was not found in liver or placenta. Complete sequencing of these cDNAs, designated SP-10-5, SP-10-8, and SP-10-10, produced an 1117-bp sequence containing a 265-amino acid-coding region for the SP-10 protein. Hydrophobicity plots generated from the deduced amino acid sequence showed a very hydrophobic amino terminus characteristic of a signal peptide. Sequence data showed that three different amino acid repeats occurred a total of 16 times in the central third of the SP-10 protein. Interestingly, cDNA SP-10-10 has an internal 57-base pair (19 amino acids) in-frame deletion that is not present in SP-10-5, suggesting that alternative splicing generates more than one SP-10 mRNA. The SP-10 protein appears to be a unique acrosomal protein, based on previous immunohistological data and the observation that SP-10 cDNA sequences did not show any significant homology to other sequences found in the Genbank, National Biomedical Research Foundation, or Swiss sequence banks. A recombinant SP-10 fusion protein was produced in an Escherichia coli expression vector and used to generate a polyclonal antiserum. This antiserum stained the acrosomal cap in situ and reacted with a similar set of peptides on Western blots as did a monoclonal antibody to SP-10.
Wright
R M
RM
Department of Anatomy and Cell Biology, University of Virginia, Charlottesville 22908.
John
E
E
Klotz
K
K
Flickinger
C J
CJ
Herr
J C
JC
D000177:Acrosome / Q000276:immunology*; D000595:Amino Acid Sequence; D000941:Antigens / Q000032:analysis*; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D012739:Gonadal Steroid Hormones / Q000032:analysis*; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008297:Male; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D012313:RNA / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D013091:Spermatogenesis / Q000276:immunology; D013094:Spermatozoa / Q000276:immunology*
C512729:ACRV1 protein, human; D000941:Antigens; D012739:Gonadal Steroid Hormones; D008565:Membrane Proteins; D012313:RNA; D004247:DNA