A genetic analysis of hermaphrodite, a pleiotropic sex determination gene in Drosophila melanogaster.

Sex determination in Drosophila is controlled by a cascade of regulatory genes. Here we describe hermaphrodite (her), a new component of this regulatory cascade with pleiotropic zygotic and maternal functions. Zygotically, her+ function is required for female sexual differentiation: when zygotic her+ function is lacking, females are transformed to intersexes. Zygotic her+ function may also play a role in male sexual differentiation. Maternally, her+ function is needed to ensure the viability of female progeny: a partial loss of her+ function preferentially kills daughters. In addition, her has both zygotic and maternal functions required for viability in both sexes. Temperature sensitivity prevails for all known her alleles and for all of the her phenotypes described above, suggesting that her may participate in an intrinsically temperature-sensitive process. This analysis of four her alleles also indicates that the zygotic and maternal components of of her function are differentially mutable. We have localized her cytologically to 36A3-36A11.

Occurrence of prosaposin as a neuronal surface membrane component.

Prosaposin is a precursor of four saposins that are required for the lysosomal hydrolysis of sphingolipids by specific hydrolases. Besides its precursor role, prosaposin also exists as a secreted protein. The present investigation reveals that prosaposin also exists as an integral component of the surface membranes of neuronal cells. Subcellular fractionation studies demonstrate that the membrane-bound prosaposin occurs specifically in plasma membranes of NS20Y rat neuroblastoma cells. An immunohistochemical study of the neuroblastoma cells using rat prosaposin-specific antibodies also showed that a portion of prosaposin is located on the surface of neurites as well as on cell bodies. Similar histochemical studies with antibodies that specifically recognized human prosaposin revealed the presence of prosaposin in dendrites, axons, and cell bodies of subcortical and spinal cord neurons in both human adult brain and in fetal brain (24-wk gestation). These findings suggest an important role of prosaposin in neuronal development.

Structure and sequence of the human alpha-L-fucosidase gene and pseudogene.

Fucosidosis is a rare lysosomal storage disease resulting from a nearly complete deficiency of alpha-L-fucosidase enzyme activity. Previously, cDNA encoding human fucosidase was cloned and sequenced. Here we report the determination of the human fucosidase gene structure and sequence as well as the sequence of the fucosidase pseudogene. The gene encoding fucosidase is composed of eight exons spanning 23 kb of DNA. Analysis of the sequence 5' of the open reading frame indicates the presence of multiple transcription factor binding sites but no TATA box. Northern blot analysis has confirmed an mRNA size of 2.3 kb in human lymphoblasts, testis, and epithelial cells. We have also sequenced the processed pseudogene of fucosidase. The sequence of the pseudogene is 80% identical to that of fucosidase cDNA but does not contain an open reading frame.

Identification of prosaposin as a neurotrophic factor.

Prosaposin was identified as a neurotrophic factor stimulating neurite outgrowth in murine neuroblastoma (NS20Y) cells and choline acetyltransferase (ChAT) activity in human neuroblastoma (SK-N-MC) cells. The four naturally occurring saposins, which are derived by proteolytic processing of prosaposin, were tested for activity. Saposin C was found to be active, whereas saposins A, B, and D were inactive as neurotrophic factors. Dose-response curves demonstrated that nanomolar concentrations of prosaposin and saposin C stimulated neurite outgrowth and increased ChAT activity. Prosaposin and saposin C exerted activity by a mechanism independent of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3. Binding assays utilizing saposin C as a ligand gave two saturable binding constants, a high-affinity (Kd = 19 pM) and a low-affinity (Kd = 1 nM) constant, with 2000 and 15,000 sites per NS20Y cell, respectively. Phosphorylation stimulation experiments demonstrated that brief treatment with prosaposin or saposin C enhanced phosphorylation of a variety of proteins, some of which contained phosphorylated tyrosine(s). Since both cell lines were also stimulated by ciliary neurotrophic factor (CNTF) as well as prosaposin, inhibition was tested by utilizing an anti-gp130 monoclonal antibody, which specifically inhibited CNTF stimulation; this antibody did not inhibit prosaposin or saposin C stimulation. These results indicate that prosaposin and saposin C are neurotrophic factors which initiate signal transduction by binding to a high-affinity receptor that induces protein phosphorylation.

Prosaposin, a neurotrophic factor: presence and properties in milk.

The presence of prosaposin, the precursor of the sphingolipid activator proteins (saposins A, B, C, and D), was investigated in bovine milk. The milk proteins were resolved by SDS-PAGE, blotted onto nitrocellulose sheets, and immunostained. Each of three appropriate antibodies defined a band from milk that matched in mobility the reference prosaposin from human milk at a relative molecular mass of 66,000. Evidence of mature saposins was not found. Prosaposin was detected in milk of other species chimpanzee, rhesus, goat, and rat) and was consistently observed in samples of retail milk and from individual cows. Prosaposin was not associated with particulate matter (fat globules, casein micelles, membrane fragments, and somatic cells) in either human or bovine milk. Rather, prosaposin was located exclusively in the milk serum (whey), existing in monomeric form, as revealed by nondenaturing PAGE. A commercial whey protein concentrate (75% protein) appeared to retain milk prosaposin quantitatively. Properties that were useful in the isolation of prosaposin from milk were its binding to concanavalin A, retention by anion-exchange cellulose, and resistance to precipitation by heating. The possibility that bovine milk prosaposin nutritionally benefits the humans who consume it is enhanced by the fact that only part of its saposin C segment is required for neurotrophic activity.

Prosaposin and prosaptide, a peptide from prosaposin, induce an increase in ganglioside content on NS20Y neuroblastoma cells.

Prosaposin has been recently identified as a neurotrophic factor eliciting differentiation in neuronal cultured cells (NS20Y). In this paper we investigate whether prosaposin and its active peptide (prosaptide) may modify the ganglioside pattern in neuroblastoma cells. The analysis by high performance thin layer chromatography did not reveal qualitative changes in the ganglioside pattern of NS20Y cells incubated in the presence of prosaposin, compared to control cells, but it did reveal an increase of the content of all three major resorcinol positive bands (GM3, GM2, GD1a). Cytofluorimetric and immunofluorescence microscopic analysis revealed that the increase of the ganglioside content was at the plasma membrane level. These findings suggest that the neurotrophic activity of prosaposin on NS20Y neuroblastoma cells might be mediated in part by the increase of cell surface gangliosides.

Characterization of a mutation in a family with saposin B deficiency: a glycosylation site defect.

Saposins are small, heat-stable glycoproteins required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. Saposins A, B, C, and D are derived by proteolytic processing from a single precursor protein named prosaposin. Saposin B, previously known as SAP-1 and sulfatide activator, stimulates the hydrolysis of a wide variety of substrates including cerebroside sulfate, GM1 ganglioside, and globotriaosylceramide by arylsulfatase A, acid beta-galactosidase, and alpha-galactosidase, respectively. Human saposin B deficiency, transmitted as an autosomal recessive trait, results in tissue accumulation of cerebroside sulfate and a clinical picture resembling metachromatic leukodystrophy (activator-deficient metachromatic leukodystrophy). We have examined transformed lymphoblasts from the initially reported saposin B-deficient patient and found normal amounts of saposins A, C, and D. After preparing first-strand cDNA from lymphoblast total RNA, we used the polymerase chain reaction to amplify the prosaposin cDNA. The patient's mRNA differed from the normal sequence by only one C----T transition in the 23rd codon of saposin B, resulting in a threonine to isoleucine amino acid substitution. An affected male sibling has the same mutation as the proband and their heterozygous mother carries both the normal and mutant sequences, providing additional evidence that this base change is the disease-causing mutation. This base change results in the replacement of a polar amino acid (threonine) with a nonpolar amino acid (isoleucine) and, more importantly, eliminates the glycosylation signal in this activator protein. One explanation for the deficiency of saposin B in this disease is that the mutation may increase the degradation of saposin B by exposing a potential proteolytic cleavage site (arginine) two amino acids to the amino-terminal side of the glycosylation site when the carbohydrate side chain is absent.

Identification of the neurotrophic factor sequence of prosaposin.

Prosaposin, recently identified as a neurotrophic factor (1), is the precursor of saposins A, B, C, and D. The neurotrophic activity of prosaposin resides in the saposin C domain. We have pinpointed the active sequence to a linear 12-mer located in the NH2-terminal sequence of saposin C (LIDNNKTEKEIL). Nanomolar concentrations of a 22-mer peptide encompassing this region stimulated neurite outgrowth and choline acetyltransferase activity, and prevented cell death in neuroblastoma cells. In primary cerebellar granule cells, the 22-mer also stimulated neurite outgroth. Studies of the neuroblastoma line NS20Y using a radiolabeled 18-mer from the neurotrophic region identified a high-affinity (Kd = 70 pM) binding site indicative of receptor-ligand interaction. The 22-mer stimulated protein phosphorylation of several proteins, some of which were tyrosine-phosphorylated after brief exposure similar to saposin C. Circular dichroism studies demonstrated that the 22-mer was converted from a random to a helical structure by addition of ganglioside GM1. The results are consistent with receptor-ligand binding by the peptide initiating a signal transduction cascade and resulting in neuronal differentiation.