Prostasin is a glycosylphosphatidylinositol-anchored active serine protease.

A recombinant human prostasin serine protease was expressed in several human cell lines. Subcellular fractionation showed that this serine protease is synthesized as a membrane-bound protein while a free-form prostasin is secreted into the culture medium. Prostasin was identified in nuclear and membrane fractions. Membrane-bound prostasin can be released by phosphatidylinositol-specific phospholipase C treatment, or labeled by [(3)H]ethanolamine, indicating a glycosylphosphatidylinositol anchorage. A prostasin-binding protein was identified in mouse and human seminal vesicle fluid. Both the secreted and the membrane-bound prostasin were able to form a covalently linked 82-kDa complex when incubated with seminal vesicle fluid. The complex formation between prostasin and the prostasin-binding protein was inhibited by a prostasin antibody, heparin, and serine protease inhibitors. Prostasin's serine protease activity was inhibited when bound to the prostasin-binding protein in mouse seminal vesicle fluid. This study indicates that prostasin is an active serine protease in its membrane-bound form.

Defining oligosaccharide specificity for initial sperm-zona pellucida adhesion in the mouse.

The identity of the sperm surface protein(s) responsible for sperm-zona pellucida binding in the mouse, as well as the characteristics of the oligosaccharide groups on zona pellucida glycoprotein 3 (ZP3) having ligand activity toward this receptor, remain controversial. Conflicting results from several groups have made interpretation of the current data difficult. By developing a quantitative binding assay to evaluate the molecular interactions between mammalian sperm and the zona pellucida during initial gamete interactions, we directly quantified sperm-ZP binding interactions at the molecular level for the first time. The ZP binding assay demonstrated that live, capacitated mouse sperm bind solubilized 125I-labeled ZP glycoproteins in a concentration-dependent manner characterized by a rapid forward rate constant of 3.0 x 10 (7)M-1 min-1. Following the initial characterization, the binding assay was used to examine the roles of the sperm surface enzymes galactosyltransferase (GalTase) and fucosyltransferase (FucTase) in sperm-zone pellucida binding in the mouse. These data indicate that substrates for FucTase, but not for GalTase, inhibit sperm-ZP binding, in contrast to earlier reports in which GalTase substrates significantly inhibited sperm binding to intact ZPs. A model is presented which resolves conflicting results between assays using intact ZPs and the results obtained here using soluble 125I-ZPs. Assuming a complex binding/recognition site, monosaccharides that could occupy part of the binding site would have a dramatic effect on sperm-ZP binding to the intact ZP, since they need only occupy the binding sites for a short time (approximately 100 msec) to disrupt binding. The current results suggest that the sperm ZP3 receptor binding site minimally recognizes the gal beta 1, 3-GlcNAc moiety also recognized by FucTases. The current data do not exclude the possibility that additional sugar residues form part of the ligand oligosaccharide group and are recognized by a yet-to-be-identified sperm surface protein which serves as the ZP3 receptor.

The initial molecular interaction between mouse sperm and the zona pellucida is a complex binding event.

Prior to fertilization, mammalian sperm must first bind to the zona pellucida (ZP), a glycoprotein matrix surrounding the egg. Sperm specifically bind to ZP3, an 83-kDa glycoprotein which functions as both an adhesion molecule and as a secretagogue for acrosomal exocytosis (Litscher, E. S., and Wassarman, P. M. (1993) Trends Glycosci. Glycotechnol. 5, 369-388). We used acid solubilized, 125I-labeled ZPs to quantify the initial binding event on mouse spermatozoa. Live sperm could not be used since solubilized ZPs rapidly initiated exocytosis. Instead, acrosome intact mouse sperm were briefly fixed in 1% glutaraldehyde for binding studies using a standard filtration assay. The fixed sperm are suitable for sperm-zona binding assays based on two experiments: 1) incubating either live or fixed sperm in low concentrations of 125I-ZPs not sufficient to induce acrosomal exocytosis revealed no differences in binding up to 15 min and 2) solubilized, unlabeled ZPs competed for 125I-ZPs with an KI of approximately 3.78 nm. Sperm-125I-ZP binding reached equilibrium with a tau1/2 of approximately 22 min at 37 degrees C. Affinity parameters were calculated using the well substantiated assumption that only ZP3 binds intact mouse sperm. The on-rate constant for association of 125I-ZP binding to the mouse sperm surface was calculated to be 3.2 x 10(6) M-1 min-1. The saturation binding isotherm revealed that there are approximately 30,000 binding sites, ascribed to ZP3, with an EC50 of 1.29 nM. Further analysis indicated that this binding is complex (Hill coefficient = 1.72), suggesting involvement of multiple receptors on the sperm surface and/or multiple ligand moieties. High and low affinity ZP binding sites on the sperm surface were confirmed by dissociation experiments. 125I-ZP dissociation was clearly biphasic, and kinetic off-rate constants of 0.161 min-1 and 0.0023 min-1 were calculated for the low and high affinity sites, respectively. Apparent affinities (Kd values) of 50 nM for the low affinity and 0.72 nM for the high affinity interaction were calculated from the rate constants. These data demonstrate that the initial adhesion event between mouse sperm and the zona pellucida is a high affinity event which is sufficient to tether a sperm to the extracellular matrix prior to the induction of acrosomal exocytosis.

Microtubules and microtubule motors: mechanisms of regulation.

Microtubule-based motility is precisely regulated, and the targets of regulation may be the motor proteins, the microtubules, or both components of this intricately controlled system. Regulation of microtubule behavior can be mediated by cell cycle-dependent changes in centrosomal microtubule nucleating ability and by cell-specific, microtubule-associated proteins (MAPs). Changes in microtubule organization and dynamics have been correlated with changes in phosphorylation. Regulation of motor proteins may be required both to initiate movement and to dictate its direction. Axonemal and cytoplasmic dyneins as well as kinesin can be phosphorylated and this modification may affect the motor activities of these enzymes or their ability to interact with organelles. A more complete understanding of how motors can be modulated by phosphorylation, either of the motor proteins or of other associated substrates, will be necessary in order to understand how bidirectional transport is regulated.

Biochemical characterization of a glycosylphosphatidylinositol-linked hyaluronidase on mouse sperm.

On the basis of DNA homology to bee venom hyaluronidase, it was recently suggested that the GPI-linked mammalian sperm antigen, PH-20, may function as a cell surface hyaluronidase [Gmachl, M., & Kreil, G. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3569-3573]. We have quantified the activity of the soluble acrosomal hyaluronidase of mouse sperm and further demonstrate the existence of a membrane-bound hyaluronidase, detected on both acrosome-intact and acrosome-reacted mouse sperm, distinct from the soluble form of the enzyme. The membrane-bound hyaluronidase was specifically released by PI-PLC, indicating that it is GPI linked. Acrosome-intact and acrosome-reacted sperm released several polypeptides (68, 44, 39, 34, 17, and 15 kDa) when treated with PI-PLC. In addition, GPI-linked polypeptides unique to acrosome-intact or to acrosome-reacted sperm were identified. Fractionation of the PI-PLC-released components from acrosome-reacted sperm using size exclusion chromatography revealed a single peak of hyaluronidase activity which comigrates with a 68 kDa GPI-linked protein present in these fractions. Taken together, these data demonstrate the existence of at least two isoforms of hyaluronidase: a soluble form within the acrosomal vesicle which is released during acrosomal exocytosis and a GPI-linked form which is present on the surface of both acrosome-intact and acrosome-reacted sperm. Both forms may be necessary for successful penetration of the extracellular vestments that surround the egg prior to fertilization.

Leukemia inhibitory factor and neurotrophins support overlapping populations of rat nodose sensory neurons in culture.

Recent studies in this and other laboratories demonstrated that leukemia inhibitory factor (LIF) can regulate differentiation and survival of nerve growth factor (NGF)-dependent primary sensory neurons. To determine whether development of NGF-independent sensory neurons could be similarly regulated, the present study examined the effects of LIF and the related cytokine-like growth factor, ciliary neurotrophic factor (CNTF), on survival of rat nodose ganglion (NG) cells in culture. In addition, survival in LIF-treated cultures was compared in the presence and absence of the neurotrophins brain-derived neurotrophic factor (BDNF), NT-3, and NT-4, factors previously shown to target NG sensory neurons, to determine whether the cytokine-like factors and neurotrophins act on the same population of ganglion cells. Treatment of dissociate cultures of Embryonic Day (E) 16.5 NG with LIF or CNTF (10 ng/ml) resulted in a four- to fivefold increase in neuronal survival; the number of neurons supported by either factor represented approximately 50% of the total NG population. In contrast, less than 10% of newborn NG neurons survived in the presence of LIF or CNTF, suggesting a loss in responsiveness to these factors during fetal development in vivo. In cultures of E16.5 ganglia, BDNF, NT-3, or NT-4 alone each increased cell survival to varying degrees (BDNF > NT-4 = LIF > NT-3); NGF, on the other hand, had no effect. Combining LIF with BDNF, NT-3, or NT-4 had only partially additive effects on NG neuron number (LIF + BDNF, 36% greater than BDNF alone; LIF + NT-3, 21% greater than LIF alone; LIF + NT-4, 49% greater than NT-4 or LIF alone; P < 0.05), indicating that LIF and these neurotrophins support survival of overlapping subsets of NG neurons in culture. To begin defining whether NG neurons were capable of responding to CNTF and LIF in vivo, Northern blots were used to examine expression of mRNAs coding for CNTF and LIF receptor components (CNTFR alpha, gp130, and LIFR beta) in the intact ganglion. mRNAs for all three receptor components were present in both E16.5 and newborn ganglia, suggesting that LIF and CNTF receptors are expressed in the NG in vivo. Taken together, these data demonstrate that LIF and CNTF can support survival of NGF-independent sensory neurons in culture and indicate that cytokine-like growth factors may play a role in regulating visceral sensory development in vivo.

Control of organelle transport in melanophores: regulation of Ca2+ and cAMP levels.

Melanophores of the cichlid Tilapia mossambica can be induced to aggregate pigment by addition of epinephrine to the medium, suggesting adrenergic control of this transport. The melanophore response to adrenergic stimulation was examined using agonists and antagonists that are highly specific for each alpha-adrenoceptor subclass. The signal transduction mechanism of each subclass is unique: stimulation of alpha 1 receptors results in a rise in intracellular free Ca2+, while alpha 2 stimulation results in decreased cAMP levels [Exton, 1985: Am. J. Physiol. 248:E633-E647]. Each alpha 1 or alpha 2 specific agonist tested showed a dose dependent ability to induce aggregation and each was able to effect complete aggregation of pigment, suggesting that aggregation can be mediated either by elevating Ca2+ or by lowering cAMP. However, in the presence of either an alpha 1 or an alpha 2 receptor antagonist, none of the agonists were able to induce significant aggregation, suggesting that changes in levels of both messengers are required for pigment aggregation in the melanophores. Moreover, experiments in which intracellular levels of Ca2+ or cAMP were perturbed, using BAPTA and forskolin, respectively, indicated that elevating Ca2+ in the presence of high cAMP is not sufficient to induce aggregation and, conversely, that lowering cAMP levels in the presence of reduced Ca2+ is not sufficient to induce pigment aggregation. These data indicate that the concentrations of both cAMP and Ca2+ are important in regulating pigment aggregation in teleost melanophores, and suggest that maximal aggregation of pigment requires altering the levels of both messengers.

Regulation of organelle transport in melanophores by calcineurin.

Previous studies have shown that pigment granule dispersion and aggregation in melanophores of the African cichlid, Tilapia mossambica, are regulated by protein phosphorylation and dephosphorylation, respectively (Rozdzial, M. M., and L. T. Haimo. 1986. Cell. 47:1061-1070). The present studies suggest that calcineurin, a Ca2+/calmodulin-stimulated phosphatase, is the endogenous phosphatase that mediates pigment aggregation in melanophores. Aggregation, but not dispersion, is inhibited by okadaic acid at concentrations consistent with an inhibition of calcineurin activity. Inhibition of aggregation in melanophores that have been BAPTA loaded or treated with calmodulin antagonists implicate Ca2+ and calmodulin, respectively, in this process. Moreover, addition of calcineurin rescues aggregation in lysed melanophores which are otherwise incapable of aggregating pigment. Immunoblotting with an anticalcineurin IgG reveals that calcineurin is a component of the dermis, which contains the melanophores, and indirect immunofluorescence localizes calcineurin specifically to the melanophores. Finally, this antibody, which inhibits calcineurin's phosphatase activity (Tash, J. S., M. Krinks, J. Patel, R. L. Means, C. B. Klee, and A. R. Means. 1988. J. Cell Biol. 106:1625-1633), inhibits aggregation but has no effect on pigment granule dispersion. Together these studies indicate that retrograde transport of pigment granules to the melanophore cell center depends upon the participation of calcineurin.

Comparison of the NES and CNS/B neuropsychological screening batteries.

Computer and hand administered neuropsychological tests are utilized in the evaluation of central nervous system changes associated with environmental or occupational exposure to neurotoxicants. This study compares the computerized Neurobehavioral Evaluation System (NES) developed by Baker et al., 1985, with the hand administered California Neuropsychological Screening Battery (CNS/B) developed by Bowler et al., 1986. Both batteries were designed for screening of the effects of neurotoxicants on the central nervous system and require less than one hr to administer. Both screening batteries consist of tests of: mood, word knowledge, attention, concentration, learning and memory and psychomotor and visuomotor and visuospatial abilities. They were administered in a standardized fashion to 106 subjects. Results indicate strong positive correlations for tests of word knowledge, moderate correlations for attention and concentration, while weaker correlations were obtained for tests of memory, psychomotor and visuomotor and visuospatial ability. The NES may be more useful for large epidemiological studies while the CNS/B appears more useful for individual screening and clinical studies.