Human RING finger protein ZNF645 is a novel testis-specific E3 ubiquitin ligase

Abstract

A large number of testis-specific genes are involved in the complex process of mammalian spermatogenesis. Identification of these genes and their roles is important for understanding the mechanisms underlying spermatogenesis. Here we report on a novel human RING finger protein, ZNF645, which contains a C3HC4 RING finger domain, a C2H2 zinc-finger domain, and a proline-rich region, indicating that it has a structure similar to that of the c-Cbl-like protein Hakai. ZNF645 was exclusively expressed in normal human testicular tissue. Immunohistochemical analysis confirmed that ZNF645 protein was present in spermatocytes, round and elongated spermatids, and Leydig cells. Immunofluorescence staining of mature sperms further showed that the ZNF645 protein was localized over the postacrosomal perinuclear theca region and the entire length of sperm tail. An in vitro ubiquitination assay indicated that the RING finger domain of the ZNF645 protein had E3 ubiquitin ligase activity. Therefore, we suggest that ZNF645 might act as an E3 ubiquitin-protein ligase and play a role in human sperm production and quality control.

Figure 1

Expression pattern of ZNF645 gene in various human tissues and cells. (A): Of the multiple human tissue samples that were tested, ZNF645 mRNA expression was detected only in human testis by polymerase chain reaction analysis. The integrity of the examined cDNA was assessed by measuring the mRNA expression of G3PDH. (B): A single 49-kDa band of ZNF645 protein was detected in normal human testis by Western blot analysis using rabbit anti-ZNF645 polyclonal antibody as the primary antibody. (C): ZNF645 protein was present only in normal human testis.

Figure 2

Immunohistochemical localization of ZNF645 in normal human testis. The presence of ZNF645 protein was revealed by brown staining. (A): Negative control with preimmune rabbit serum; (B): ZNF645 protein was present in the cytoplasm of spermatocytes and Leydig cells of stages II–IV; (C): ZNF645 protein was present in spermatocytes, round spermatids and Leydig cells of stages VII–VIII. Bars = 50 μm.

Figure 3

ZNF645 protein localizes over the postacrosomal perinuclear theca region and tail region of human sperm. (A)–(C), (H): Sperm incubated with rabbit anti-ZNF645 polyclonal antibody. (D)–(G): Sperm incubated with preimmune rabbit serum as a negative control. (A, D) Indirect immunofluorescence microscopy image (green). (B), (E): Sperm nuclei stained with DAPI (blue). (C), (F): Merged images of A and B, and D and E, respectively. (G), (H): DAB staining indicating ZNF645 immunoreactivity on human sperm. The arrows indicate the domain-specific location of the ZNF645 protein on human sperm. Bars = 5 μm.

Figure 4

Immunodetection of ZNF645 protein in the different protein fractions of human semen. (A): ZNF645 was detected only in the theca and tail protein fractions of human sperm; (B): Rnf141 was observed only in the acrosome and tail protein fractions of human sperm.

Figure 5

Alignment of the amino-acid sequences of ZNF645 and its homologues. Sequence alignment of human ZNF645 with monkey Znf645 (Q4R361, 85% identity), human c-Cbl-like protein (Human_Cbll, B7ZM03, 52% identity) and mouse c-Cbl-like protein (Q9JIY2-2, 52% identity) was performed with the EBI ClustalW web tool (http://www.ebi.ac.uk/Tools/clustalw2/index.html). The conserved C3HC4-type RING domain, C2H2 zinc-finger domain and proline-rich region are underlined in red.

Figure 6

SDS-PAGE analysis of the expression and purification products of GST, GST-ZNF645, GST-UBC4 and GST-c-Cbl fused proteins (A) and the ubiquitination of GST-ZNF645 and GST-c-Cbl in the in vitro ubiquitination assay (B). M: Protein ladder; Lane 1: Soluble protein extract containing induced GST-ZNF645; Lane 2: purified GST-ZNF645 fused protein; Lane 3: purified GST-UBC4 fused protein; Lane 4: soluble protein extract containing induced GST-UBC4 fused protein; Lane 5: purified GST-c-Cbl fused protein; Lane 6: soluble protein extract containing induced GST-c-Cbl fused protein; Lane 7: purified GST protein; Lane 8: soluble protein extract containing GST protein. Polyubiquitin chains were only observed in the presence of the GST-ZNF645 and GST-c-Cbl (positive control) fusion proteins. E1: E1 ubiquitin-activating enzyme; GST-UBC4: purified GST fused UBC4 protein, an E2 ubiquitin-conjugating enzyme; GST: purified GST protein, a negative control; GST-ZNF645: purified GST fused ZNF645 protein, a putative E3 ubiquitin-protein ligase; GST-c-Cbl: purifed GST fused c-Cbl protein, an E3 ubiquitin-protein ligase.

Supplementary Figure 1

Nucleotide and deduced aa sequences of human ZNF645 gene. The deduced aa sequences of human ZNF645 ORFs are shown beneath the DNA sequences. The numbers in the left and right margins indicate the nt and aa positions, respectively. The predicated ring finger C3HC4 motif is boxed, Zinc finger C2H2 motif is shadowed, and the proline-rich region is indicated in the shadowed boxes. The primers of human ZNF645 gene for the construction of GST-ZNF645 RING fused protein are underlined and the PCR primers for human ZNF645 gene expression analysis are double underlined. The 29 italic letters are nt longer than the sequence of ZNF645 cDNA clone (FLJ25735 fis). The tailing signal AATAAA is underlined and shaded. The nt sequence of this gene has been deposited in Genbank under the Acession No. GQ355336.