ABSTRACT
Fertilin alpha and beta (previously known as PH-30 alpha and beta) are sperm surface proteins implicated in sperm-egg binding and membrane fusion. In this work we report the purification, characterization, and cloning of the bovine homologues of fertilin alpha and beta. Immunofluorescence and Western blotting, using polyclonal antibodies made against guinea pig fertilin, identified likely bovine fertilin alpha and beta homologues. These candidates were purified under nondenaturing conditions using lectin and ion-exchange chromatography and were subjected to amino acid sequence analysis. Degenerate oligonucleotides made from bovine N-terminal protein sequences and from guinea pig fertilin sequences were used to isolate clones encoding bovine fertilin alpha and beta from a bovine testis cDNA library. The predicted proteins have the same domain organization as guinea pig fertilin alpha and beta: pro, metalloprotease-like, disintegrin-like, and cysteine-rich domains, followed by an EGF-like repeat, a transmembrane domain, and a cytoplasmic tail. In contrast to the reported sequence of guinea pig fertilin alpha, the mature, proteolytically processed bovine fertilin alpha subunit contains a complete disintegrin-like domain, and a potential amphipathic beta strand, which may be involved in sperm-egg fusion. Gradient sedimentation experiments suggest that the fertilin alpha/beta heterodimer may be present on the sperm surface as a higher-order oligomer.
Subject(s)
Membrane Glycoproteins/chemistry , Membrane Glycoproteins/physiology , Metalloendopeptidases/chemistry , Metalloendopeptidases/physiology , Sperm-Ovum Interactions/physiology , ADAM Proteins , Amino Acid Sequence , Animals , Antibodies , Base Sequence , Cattle , Cell Fusion/physiology , Cloning, Molecular , DNA Primers/genetics , Female , Fertilins , Guinea Pigs , Male , Membrane Glycoproteins/genetics , Metalloendopeptidases/genetics , Molecular Sequence Data , Molecular Structure , Polymerase Chain Reaction , Protein Conformation , Sequence Homology, Amino AcidABSTRACT
Intermembrane protein transfer between erythrocytes and phospholipid vesicles was examined under a variety of conditions to investigate physical factors governing this process. Human erythrocytes were incubated with sonicated dimyristoylphosphatidylcholine vesicles containing trace [14C]dipalmitoylphosphatidylcholine. Protein-vesicle complexes were separated from cells and from membrane fragments by density gradient centrifugation. The yield of isolated protein vesicles was determined from the 14C-vesicle marker; protein compositions were analyzed by SDS-polyacrylamide gel electrophoresis. Enzymatic removal of portions of the cytoplasmic or exoplasmic domains of cell membrane proteins had little effect on the extent of protein transfer. Membrane additives such as cholate produced a 2-fold increase in protein-vesicle yield. The selectivity of protein transfer from erythrocytes was influenced by the lipid composition of recipient vesicles: inclusion of cholesterol increased band 3 content while the presence of anionic phospholipids reduced transfer. Proteins transferred from 32P-labeled cells differed in specific radioactivity from bulk cell proteins: glycophorin, highly phosphorylated in the cell membrane, showed no detectable labeling in the corresponding protein-vesicle band. These observations suggest that cell-to-vesicle protein transfer is insensitive to bulk steric and electrostatic properties of cell membranes, but enhanced by membrane defects. Recipient membrane composition influences the selectivity of transferred proteins and may reveal subtle differences in the membrane association of protein subpopulations.
