Sperm traverse the epididymis and acquire new proteins and glycoproteins on their plasma membranes even though they are incapable of nuclear DNA transcription and mRNA translation. We have been working for the past few years on two rat epididymis-specific secretory glycoproteins (proteins D & E) that are greater than 95% homologous at the amino acid level and are commonly considered to be identical, differing only in their glycosylation. Our preliminary data indicate that there are both carbohydrate and amino acid differences between the two proteins that could be very important to their behavior in the epididymis. We have raised a monoclonal antibody (4E9 MAb) that recognizes purified protein E, but not purified protein D. This MAb also localizes to the tail plasma membrane of caudal epididymal sperm, but not of caput epididymal sperm. All of our data, and published data, are consistent with the hypothesis that the 4E9 MAb antigen, is synthesized by epididymal epithelium, secreted into the epididymal lumen, and subsequently binds to sperm. The association of the antigen with sperm is very tight, since common biochemical assays for integral vs peripheral membrane proteins indicate that 4E9 antigen behaves as though it is an integral membrane molecule. Antibodies to protein D are available in other laboratories, and their immunolocalization studies indicate that protein D is primarily synthesized in regions of the epididymis different from those of protein E and the antibody localizes to the head of the sperm rather than to the tail. Therefore, the major question being asked in this grant is what signals mediate differential targeting to domain-specific regions on the sperm surface of two proteins with greater than 95% homology. In order to investigate this we propose five specific aims: The first is to study quantitatively and qualitatively binding of proteins D & E to the sperm surface and to determine structural motifs that mediate differential targeting. Secondly, it is important to complete studies on the primary amino acid sequences and oligosaccharide structures of proteins D and E and of their membrane counterparts. Thirdly, it is important to ascertain whether there are post-translational modifications of proteins D and E, both from fluid and sperm, other than glycosylation, since these modifications might act in some way to regulate signalling in the system. Fourth, there is evidence that protein E is processed prior to associating with the sperm surface and it is important, therefore, to study this process. Finally, I propose to study regulation of expression of proteins D&E.
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