Organization and chromosomal localization of the murine Testisin gene encoding a serine protease temporally expressed during spermatogenesis

Abstract

The recently characterized human serine protease, Testisin, is expressed on premeiotic testicular germ cells and is a candidate type II tumor suppressor for testicular cancer. Here we report the cloning, characterization and expression of the gene encoding mouse Testisin, Prss21. The murine Testisin gene comprises six exons and five introns and spans < 5 kb of genomic DNA with an almost identical structure to the human Testisin gene, PRSS21. The gene was localized to murine chromosome 17 A3.3-B; a region syntenic with the location of PRSS21 on human chromosome 16p13.3. Northern blot analyses of RNA from a range of adult murine tissues demonstrated a 1.3 kb mRNA transcript present only in testis. The murine Testisin cDNA shares 65% identity with human Testisin cDNA and encodes a putative pre-pro-protein of 324 amino acids with 80% similarity to human Testisin. The predicted amino-acid sequence includes an N-terminal signal sequence of 27 amino acids, a 27 amino-acid pro-region, a 251 amino-acid catalytic domain typical of a serine protease with trypsin-like specificity, and a C-terminal hydrophobic extension which is predicted to function as a membrane anchor. Immunostaining for murine Testisin in mouse testis demonstrated specific staining in the cytoplasm and on the plasma membrane of round and elongating spermatids. Examination of murine Testisin mRNA expression in developing sperm confirmed that the onset of murine Testisin mRNA expression occurred at < day 18 after birth, corresponding to the appearance of spermatids in the testis, in contrast to the expression of human Testisin in spermatocytes. These data identify the murine ortholog to human Testisin and demonstrate that the murine Testisin gene is temporally regulated during murine spermatogenesis.Keywords: Prss21; serine protease; spermatogenesis; testis.Human Testisin is a recently identified protein that is expressed by spermatocytes and may function as a nonclassical type II tumor suppressor gene [1]. Testisin is a member of the large multigene family of serine proteases, enzymes characterized by a triad of histidine, aspartate and serine residues necessary for catalytic activity [2]. Testisin mRNA was also identified from human eosinophils using reverse transcription polymerase chain reaction (RT-PCR) and called esp-1 [3]. The human Testisin gene (PRSS21) has been localized to human chromosome 16p13.3 [1,4], a region associated with high genomic instability. Testisin belongs to an emerging group of serine proteases that possess a hydrophobic C-terminal domain which acts as a direct anchor for membrane attachment, likely via a glycosyl-phosphatidylinositol anchor. This structural feature is also present in the serine proteases CAP1, a putative mediator of sodium channel activity [5], prostasin, which is thought to function in the activation of cell surface proteins [6], and the murine serine proteases TESP1 and TESP2 [7].Testicular germ cell maturation, or spermatogenesis, is a complex process by which diploid spermatogonial stem cells differentiate ...

A Mouse Serine Protease TESP5 Is Selectively Included into Lipid Rafts of Sperm Membrane Presumably as a Glycosylphosphatidylinositol-anchored Protein

“…The nucleotide sequence of the TESP5 gene demonstrates the identity of this gene with the testisin (18) and esp-1 (19) genes previously reported. TESP5/testisin/ esp-1 is localized on the sperm membrane probably as a glycosylphosphatidylinositol (GPI)-anchored protein, and corresponds to the 42-and 41-kDa gelatin-hydrolyzing enzymes.…”

Trypsin is a multifunctional factor in spermatogenesis

“…Hence, eel sperm heads have trypsin activity although the protein in question differs from the testicular trypsin that we have cloned, since the molecular mass of sperm head trypsin (80 and 95 kDa) differs from that of testicular type trypsin (27 kDa). Since it has been reported also that membrane-type serine protease exists in elongated spermatids in mammals (27,28), there is a possibility that sperm head trypsin in eel is similar to this serine protease in mammals.…”

Proteolytic Activation of the Protease-activated Receptor (PAR)-2 by the Glycosylphosphatidylinositol-anchored Serine Protease Testisin

“…Cell surface testisin is known to be localized within lipid raft microdomains (22,52), and unlike other membrane-anchored serine proteases, testisin has not been found to be naturally shed from the cell surface, although we and others (23) find that testisin is released from cells in culture using exogenous bacterial phosphatidylinositol-specific phospholipase C. Lipid rafts provide signaling modules that can enhance interactions of proteins even at low concentrations within the rafts by bringing them into close proximity, as has been suggested for activation of PAR-2 by FXa-TF in lipid rafts (53,54). Of note, activation of PAR-2 by soluble proteases is unlikely to be specific for receptors present in lipid rafts versus other microenvironments on the membrane, whereas a PAR-2 agonist co-localized with the receptor within a lipid raft provides the opportunity for biased signaling dependent on the presence of specific lipid raft-associated adaptor proteins.…”