Human ribonuclease 9, a member of ribonuclease A superfamily, specifically expressed in epididymis, is a novel sperm-binding protein.
To explore the functions of human ribonuclease 9 (RNase 9), we constructed a mammalian fusion expression vector pcDNA-hRNase9, prepared recombinant human RNase 9-His fusion protein from HEK293T cells and determined its N-terminal amino acid sequences. According to the determined mature protein, recombinant human RNase 9 was prepared in E. coli. Ribonucleolytic activity and antibacterial activity of recombinant human RNase 9 were detected, and the distribution of human RNase 9 on tissues and ejaculated spermatozoa and in vitro capacitated spermatozoa were analyzed via indirect immunofluorescence assay. The results showed that recombinant human RNase 9 did not exhibit detectable ribonucleolytic activity against yeast tRNA, but exhibited antibacterial activity, in a concentration/time dependent manner, against E. coli. Immunofluorescent analyses showed that the predicted human RNase 9 was present throughout the epididymis, but not present in other tissues examined, and human RNase 9 was also present on the entire head and neck regions of human ejaculated spermatozoa and in vitro capacitated spermatozoa. These results suggest that human RNase 9 may play roles in host defense of male reproductive tract.
Cheng
Gui-Zhi
GZ
Department of Immunology, Dalian Medical University, Dalian 116044, China. guizhicheng2002@yahoo.com.cn
Li
Jian-Yuan
JY
Li
Fang
F
Wang
Hai-Yan
HY
Shi
Guang-Xia
GX
D000328:Adult; D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000378:metabolism*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004822:Epididymis / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism*; D029589:Seminal Plasma Proteins / Q000737:chemistry / Q000378:metabolism*; D013094:Spermatozoa / Q000378:metabolism*; D013737:Testis / Q000201:enzymology; D055815:Young Adult
D000890:Anti-Infective Agents; D016601:RNA-Binding Proteins; C543068:RNase9 protein, human; D011993:Recombinant Fusion Proteins; D029589:Seminal Plasma Proteins; C425042:spermadhesin; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic