Structural Prediction and In Silico Physicochemical Characterization for Mouse Caltrin I and Bovine Caltrin Proteins.

It is known that caltrin (calcium transport inhibitor) protein binds to sperm cells during ejaculation and inhibits extracellular Ca2+ uptake. Although the sequence and some biological features of mouse caltrin I and bovine caltrin are known, their physicochemical properties and tertiary structure are mainly unknown. We predicted the 3D structures of mouse caltrin I and bovine caltrin by molecular homology modeling and threading. Surface electrostatic potentials and electric fields were calculated using the Poisson-Boltzmann equation. Several different bioinformatics tools and available web servers were used to thoroughly analyze the physicochemical characteristics of both proteins, such as their Kyte and Doolittle hydropathy scores and helical wheel projections. The results presented in this work significantly aid further understanding of the molecular mechanisms of caltrin proteins modulating physiological processes associated with fertilization.

Rat caltrin protein modulates the acrosomal exocytosis during sperm capacitation.

Caltrin is a small and basic protein of the seminal vesicle secretion that inhibits sperm calcium uptake. The influence of rat caltrin on sperm physiological processes related to fertilizing competence was studied by examining its effect on 1) spontaneous acrosomal exocytosis, 2) protein tyrosine phosphorylation, and 3) sperm-egg interaction. Results show that the presence of caltrin during in vitro capacitation both reduced the rate of spontaneous acrosomal exocytosis without altering the pattern of protein tyrosine phosphorylation, and enhanced the sperm ability to bind to the zona pellucida (ZP). The significantly higher proportion of sperm with intact acrosome observed in the presence of caltrin was accompanied by a strong inhibition in the acrosomal hyaluronidase release. Enhancement of sperm-ZP binding was evident by the increase in the percentage of eggs with bound spermatozoa as well as in the number of bound sperm per egg. Similar results were obtained when the assays were performed using spermatozoa preincubated with caltrin and then washed to remove the unbound protein, indicating that the sperm-bound caltrin was the one involved in both acrosomal exocytosis inhibition and sperm-ZP binding enhancement. Caltrin bound to the sperm head was partially released during the acrosomal exocytosis induced by Ca-ionophore A23187. Indirect immunofluorescence and immunoelectron microscopy studies revealed that caltrin molecules distributed on the dorsal sperm surface disappeared after ionophore exposure, whereas those on the ventral region remained in this localization after the treatment. The present data suggest that rat caltrin molecules bound to the sperm head during ejaculation prevent the occurrence of the spontaneous acrosomal exocytosis along the female reproductive tract. Consequently, more competent spermatozoa with intact and functional acrosome would be available in the oviduct to participate in fertilization.

beta-Microseminoprotein in human spermatozoa and its potential role in male fertility.

beta-Microseminoprotein (MSMB) is one of the most abundant proteins in human seminal plasma. The objectives of this study were: (1) to purify MSMB from seminal plasma (SP) and generate antibodies against the pure protein; (2) to investigate the interaction of MSMB with ejaculated spermatozoa and its possible effect on the spontaneous acrosome reaction (AR); and (3) to quantify MSMB content in SP and examine its relationship with the clinical sperm parameters. MSMB was purified from SP and its presence on the sperm surface was examined by indirect immunofluorescence using a specific polyclonal antibody. The effect of MSMB on the AR was evaluated using guinea pig epididymal spermatozoa as a model. MSMB quantification assay was performed with a two-site binding ELISA using two polyclonal antibodies against MSMB. MSMB was assessed in semen samples from fertile donors (controls) and subfertile patients according to World Health Organization criteria. MSMB was detected on the sperm surface and mainly localized to the acrosomal region of the head and neck. A significant spontaneous AR inhibition was observed when guinea pig epididymal spermatozoa were preincubated with MSMB. Finally, MSMB was significantly increased in subfertile patients when compared with fertile controls (P<0.02). The association of MSMB to the sperm surface, the inhibitor effect on the spontaneous AR and the increased MSMB levels found in SP in subfertile men suggests a relationship between this protein and semen quality and a possible role in the process of fertilization.

The activity of a developmentally regulated cysteine proteinase is required for cyst wall formation in the primitive eukaryote Giardia lamblia.

Giardia is an intestinal parasite that belongs to the earliest diverging branch of the eukaryotic lineage of descent. Giardia undergoes adaptation for survival outside the host's intestine by differentiating into infective cysts. Encystation involves the synthesis and transport of cyst wall constituents to the plasma membrane for release and extracellular organization. Nevertheless, little is known about the molecular events related to cyst wall biogenesis in Giardia. Among the components of the cyst wall there are two proteins that we have previously identified and characterized: CWP1 (26 kDa) and CWP2 (39 kDa). Expression of these proteins is coordinately induced, and both concentrated within encystation-specific secretory vesicles before their extracellular polymerization. Although highly similar to each other at the amino terminus, CWP2 includes a COOH-terminal 121-amino acid extension. Here, we show that this extension, rich in basic residues, is cleaved from CWP2 before cyst wall formation by an intracellular cysteine proteinase activity, which is induced during encystation like CWPs. Specific inhibitors prevent release of cyst wall materials, abolishing cyst wall formation. We also report the purification, cloning, and characterization of the encystation-specific cysteine proteinase responsible for the proteolytic processing of CWP2, which is homologue to lysosomal cathepsin C. Encystation-specific cysteine proteinase ESCP possesses unique characteristics compared with cathepsins from higher eukaryotes, such as a transmembrane domain and a short cytoplasmic tail. These features make this enzyme the most divergent cathepsin C identified to date and provide new insights regarding cyst wall formation in Giardia.