RESUMEN
We recently identified a novel testis-enriched receptor guanylyl cyclase (GC) in the mouse, designated mGC-G. To further investigate its protein expression and function, we generated a neutralizing antibody specifically against the extracellular domain of this receptor. RT-PCR and immunohistochemical analyses show that mGC-G is predominantly expressed from round spermatids to spermatozoa in mouse testis at both the mRNA and protein levels. Flow cytometry and confocal immunofluorescence reveal that mGC-G is a cell surface protein restricted to the plasma membrane overlying the acrosome and midpiece of the flagellum in mature sperm. Interestingly, Western blot analysis demonstrates that testicular mGC-G is approximately 180 kDa but is subject to limited proteolysis during epididymal sperm transport, resulting in a smaller fragment tethered on the mature sperm surface. On Fluo-3 cytometrical analysis and computer-assisted sperm assay, we found that serum albumin-induced elevation of sperm intracellular Ca(2+) concentration, protein tyrosine phosphorylation, and progressive motility associated with capacitation are markedly reduced by preincubation of the anti-mGC-G neutralizing antibody. Together, these results indicate that mGC-G is proteolytically modified in mature sperm membrane and suggest that mGC-G-mediated signaling may play a critical role in gamete/reproductive biology.
Asunto(s)
Guanilato Ciclasa/fisiología , Proteínas de la Membrana/fisiología , Espermatozoides/metabolismo , Testículo/metabolismo , Animales , Anticuerpos Bloqueadores/farmacología , Western Blotting , Citometría de Flujo , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/genética , Inmunoglobulinas/metabolismo , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Fosforilación , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Motilidad Espermática/fisiología , Testículo/citología , Tirosina/metabolismoRESUMEN
OBJECTIVE: The aim of the study was to investigate the protein expression and function of a novel secreted protein in the vascular system, named SCUBE1 for signal peptide, CUB (Complement proteins C1r/C1s, Uegf, and Bmp1) and epidermal growth factor-like (EGF)-like domain containing protein 1. METHODS AND RESULTS: Immunohistochemical analysis demonstrated that the SCUBE1 staining is mainly confined to the intravascular platelet-rich thrombus in vascular tissue samples. While quantitative real-time RT-PCR verified that the SCUBE1 mRNA is expressed in human platelets, numerous immunolocalization techniques revealed that the preformed SCUBE1 protein is stored in the alpha-granules and translocated to the surface upon platelet stimulation. A smaller SCUBE1 fragment, possibly formed by limited proteolysis after being released from the storage granules, was detected in thrombus lysate by Western blot analysis. Interestingly, deposition of SCUBE1 into the subendothelial matrix of the atherosclerotic plaques was evidenced by immunohistochemistry. In addition, studies of platelet adhesion and ristocetin-induced platelet agglutination showed that fragments containing the amino-terminal EGF-like repeats were able to support platelet adhesion and enhance the ristocetin-induced platelet agglutination, respectively. CONCLUSION: These data suggest that platelet-derived SCUBE1 could function as a novel adhesive molecule and its matrix-bound and soluble fragments may play critical (patho)physiological roles in cardiovascular biology.
Asunto(s)
Plaquetas/metabolismo , Proteínas de la Membrana/sangre , Animales , Anticuerpos Monoclonales/inmunología , Aterosclerosis/sangre , Western Blotting , Proteínas de Unión al Calcio , Expresión Génica , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/farmacología , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Activación Plaquetaria/fisiología , Adhesividad Plaquetaria/fisiología , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/fisiología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Ristocetina/farmacologíaRESUMEN
We have previously identified a novel family of secreted, cell-surface proteins expressed in human vascular endothelium. To date, two family members have been described, sharing a characteristic domain structure including an amino-terminal signal peptide followed by multiple copies of epidermal growth factor (EGF)-like repeats, a spacer region, and one CUB domain at the carboxyl terminus. Thus, this family was termed SCUBE for signal peptide-CUB-EGF-like domain containing proteins. Here we described the identification and characterization of one additional member of the SCUBE family named SCUBE3 in humans, sharing an overall 60% protein identity and a similar domain structure with other family members. Real-time quantitative reverse transcriptase-PCR and Northern blot analyses revealed that this gene is highly expressed in primary osteoblasts and the long bones (humerus and femur), followed by a low level of expression in human umbilical vein endothelial cells and in heart. When overexpressed in human embryonic kidney 293T cells, the recombinant hSCUBE3 protein is a secreted glycoprotein that can form oligomers tethered to the cell surface. Interestingly, the secreted hSCUBE3 protein can be further proteolytically processed by a serum-associated protease to release the EGF-like repeats from the CUB domain. The SCUBE3 gene is mapped to human chromosome 6p21.3, a region that has been linked with the locus for a rare form of metabolic bone disease, Paget's disease of bone 1. Together, this novel secreted, cell-surface osteoblast protein may act locally and/or distantly through a proteolytic mechanism, and may play an important role in bone cell biology.
Asunto(s)
Huesos/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Osteoblastos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Unión al Calcio , Células Cultivadas , Clonación Molecular , Cartilla de ADN , Factor de Crecimiento Epidérmico/química , Glicoproteínas/química , Glicoproteínas/genética , Glicosilación , Humanos , Masculino , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , ARN Mensajero/genéticaRESUMEN
We have identified a novel membrane form of guanylate cyclase (GC) from a mouse testis cDNA library and termed it mGC-G (mouse GC-G) based on its high sequence homology to rat GC-G. It encodes a potential type I transmembrane receptor, with the characteristic domain structure common to all members of the family of membrane GCs, including an extracellular, putative ligand-binding domain, a single membrane-spanning segment and cytoplasmic protein kinase-like and cyclase catalytic domains. Real-time quantitative reverse transcriptase--PCR and Northern-blot analyses showed that mGC-G is highly and selectively expressed in mouse testis. Phylogenetic analysis based on the extracellular protein sequence revealed that mGC-G is closely related to members of the subfamily of natriuretic peptide receptor GCs. When overexpressed in HEK-293T cells (human embryonic kidney 293T cells) or COS-7 cells, mGC-G manifests as a membrane-bound glycoprotein, which can form either homomeric or heteromeric complexes with the natriuretic peptide receptor GC-A. It exhibits marked cGMP-generating GC activity; however, notably, all ligands known to activate other receptor GCs failed to stimulate enzymic activity. The unique testis-enriched expression of mGC-G, which is completely different from the broader tissue distribution of rat GC-G, suggests the existence of as-yet-unidentified ligands and unappreciated species-specific physiological functions mediated through mGC-G/cGMP signalling in the testis.