Characterization of Secreted and Membrane-Bound Forms of 2D6 antigen
The demonstration that 2D6 McAb recognizes an O-linked carbohydrate epitope has important implications for identification of its cognate antigens in luminal fluid and on spermatozoa. One interpretation of the multiple bands on Western blots shown in Figure 3 is that 2D6 antigen is secreted in the distal caput epididymidis as a high-molecular-mass complex (—250 kDa) that is “processed” in a variety of ways (e.g., by endoproteolytic cleavage or de-glycosylation) to produce several lower-molecular-weight components, some of which are found in the luminal fluid and some on the sperm surface.
One of these processed proteins (the —24-kDa component, which is a subunit of a cross-linked homodimer of —48 kDa) is the major 2D6-reactive antigen on cauda sperm. The remaining proteins that were present initially on distal caput sperm are either released or processed further so that the carbohydrate epitope is no longer recognized by 2D6 McAb. Support for this interpretation is provided by the finding that the purified —24-kDa component from spermatozoa self-associates to form a high-molecular-mass aggregate (—250 kDa) when incubated for 48 h at 4°C. This tendency for aggregation could conceivably produce a profile of related glycoproteins of different molecular weights.