Analysis of furin ectodomain shedding in epididymal fluid of mammals: demonstration that shedding of furin occurs in vivo.
Sperm cell surface proteins and proteins of their surrounding fluids are reported to be proteolytically processed in relation to acquisition of sperm fertility during epididymal transit. Several of these proteins might be potential targets for subtilisin-like pro-protein convertase. Using immunochemistry and mass spectrometry analysis, we found that an 80 kDa form of furin (EC 3.4.21.75) is present in the fluid from the mid-caput to the distal corpus regions of the epididymis of various domestic mammals. This protein is absent from the fluid of the caudal region, suggesting that it is reabsorbed or degraded. The cDNA sequence of ovine furin was obtained and the mRNA was found throughout this organ, although in greater amounts in the mid and distal caput regions. Metabolic labeling with (35)S-amino acids indicated that the protein was synthesized and released from the epithelium only in a restricted area of the mid-caput, suggesting a specific regionalized mechanism of secretion. The fluid protein is not pelleted at 100 000 g and did not react with a C-terminal antibody indicating that it is not bound to membranous materials. These findings demonstrate that a furin ectodomain shedding occurs naturally in vivo in the epididymis where this enzyme could be involved in fluid and/or sperm membrane protein processing.
Thimon
Véronique
V
Equipe Gamètes Males et Fertilité, UMR 6175 INRA, CNRS-Université de Tours-Haras Nationaux, Station de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, 37380 Nouzilly, France.
Belghazi
Maya
M
Dacheux
Jean-Louis
JL
Gatti
Jean-Luc
JL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D004822:Epididymis / Q000201:enzymology*; D004848:Epithelium / Q000201:enzymology / Q000378:metabolism; D045683:Furin / Q000032:analysis* / Q000235:genetics; D006736:Horses; D008297:Male; D008322:Mammals / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012661:Semen / Q000201:enzymology*; D012756:Sheep; D013094:Spermatozoa / Q000502:physiology; D013552:Swine
D045683:Furin