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Identification and characterization of membrane cofactor protein of human spermatozoa
F Cervoni1, T J Oglesby, E M Adams
1Institut National de la Santé et de la Recherche Médicale U210, Faculté de Médecine, Nice, France.
Abstract:
Membrane cofactor protein (MCP) regulates C activation by serving as a cofactor for the cleavage of C3b and C4b by the serine protease factor I. An MCP-like molecule on the inner acrosomal membrane of human spermatozoa has been characterized. Three mAb and a rabbit polyclonal antibody against MCP recognized the sperm protein. On SDS-PAGE, it migrated as a single band with a molecular mass of 38,000 and 44,000 Da under nonreducing or reducing conditions, respectively. The molecular mass was 10,000 to 20,000 Da less than the two forms of MCP expressed on others cells. The electrophoretic pattern, by one- and two-dimensional gel analysis, and the isoelectric point profile (4.5 to 5.0) of the sperm protein were similar among multiple individuals. In contrast to MCP of other cells, digestion with endoglycosidases did not alter either the m.w. or the pI of the protein, suggesting that it is a poorly or nonglycosylated form of MCP. The solubilized sperm protein bound C3 with broken thioester bond to Sepharose and possessed cofactor activity for factor I-mediated cleavage of C3 with the broken bond. A mAb that blocks the regulatory function of MCP inhibited the cofactor activity of the sperm lysate. Thus, the sperm protein is an antigenic and functional homologue of MCP but has the distinct structural features of a lower m.w. and an apparent lack of glycosylation. MCP may play an essential role in the survival of the acrosome-reacted spermatozoa by modulating C activation in the female genital tract.
Insights
A novel Membrane Cofactor Protein (MCP) homologue found on human sperm exhibits functional complement regulatory activity. This sperm MCP, distinct in structure, may aid acrosome-reacted sperm survival in the female reproductive tract.
Area of Science:
- Immunology
- Reproductive Biology
- Complement System
Background:
- Membrane Cofactor Protein (MCP) is crucial for regulating complement (C) activation.
- MCP acts as a cofactor for Factor I in cleaving C3b and C4b.
- The role of MCP in sperm function is not well understood.
Purpose of the Study:
- To characterize an MCP-like molecule on human spermatozoa.
- To investigate the functional and structural properties of this sperm-associated MCP.
- To determine the potential role of sperm MCP in reproductive processes.
Main Methods:
- Utilized monoclonal antibodies (mAbs) and polyclonal antibodies for protein recognition.
- Employed SDS-PAGE and 2D gel electrophoresis to analyze molecular mass and isoelectric point.
- Performed endoglycosidase digestion to assess glycosylation.
- Assessed cofactor activity using C3 binding assays and Factor I-mediated cleavage.
Main Results:
- Identified an MCP-like protein on the inner acrosomal membrane of human sperm.
- The sperm protein has a lower molecular weight and appears poorly or non-glycosylated compared to MCP on other cells.
- The sperm protein demonstrated functional cofactor activity for C3 cleavage.
- Antibodies blocking MCP function inhibited the sperm protein's cofactor activity.
Conclusions:
- Human spermatozoa possess an antigenic and functional homologue of MCP with unique structural characteristics.
- This sperm MCP may be vital for the survival of acrosome-reacted spermatozoa.
- Modulation of complement activation by sperm MCP could be important in the female genital tract.