Masao Hiraiwa

Changes of growth inhibitory factor after stab wounds in rat brain

The growth inhibitory factor (GIF) is a new metallothionein (MT)-like protein that is downregulated in Alzheimers disease (AD) brain. The biological function of GIF has not been fully clarified yet. We have raised an antibody to the synthetic polypeptide that is specific for rat GIF. The purified antibody reacted to recombinant GIF and native rat GIF but not to MT or maltose-binding protein. Using the antibody and GIF cDNA probe, we investigated changes of GIF and GIF mRNA by Western and Northern blotting techniques in rat brains after stab wounds. The levels of GIF and GIF mRNA began to increase 4 days postoperation, reached a maximum at 14-21 days and sustained the increased level at least through 28 days. While both glial fibrillary acidic protein (GFAP) and GIF were recognized in astrocytes, the increases of these 2 proteins after stab wounds showed different patterns. The results indicated that GIF could play an important role in the repair after brain damage and also produce new insights into the mechanism of gliosis investigated mainly from the viewpoint of GFAP.

Prosaposin: A myelinotrophic protein that promotes expression of myelin constituents and is secreted after nerve injury

Recently, we demonstrated that prosaposin and prosaptides (peptides encompassing the neurotrophic sequence in prosaposin) prevent cell death and increase extracellular regulated kinase (ERK) phosphorylation and sulfatide content in primary Schwann cells or oligodendrocytes (Hiraiwa et al., 1997a). Here, we examine the effect of prosaptide on other myelin constituents, on Schwann cell morphology and proliferation, and characterize the time course of expression of prosaposin protein after sciatic nerve injury. After 24 h of treatment with 10 nM TX14(A), a 14‐mer prosaptide, the specific activity of UDP‐galactose:ceramide galactosyltransferase (GalT) in primary Schwann cells was increased by 150% over controls. Under the same conditions, the maximum content of sulfatide increased 3‐fold over controls after 48 h of treatment. Northern blot analysis, probed with oligonucleotide sequences from the GalT and P0 cDNAs, revealed that the mRNA levels of GalT and P0 protein were elevated about 30 and 200%, respectively, over controls after 24 h of treatment with TX14(A). Treatment of primary Schwann cells with TX14(A) also induced a morphological change at 10 nM; the peptide‐treated cells had a bipolar (spindle‐shaped) appearance after 48 h of treatment, compared to control cells which were irregular and multipolar. TX14(A) did not induce cell proliferation, indicating that TX14(A), unlike IGF‐I, is not mitogenic. After sciatic nerve transection, Western blot analysis demonstrated the presence of intact prosaposin in tubular fluid in a silicon chamber into which the proximal and distal nerve stumps were sutured. The concentration of prosaposin in the fluid was maximum after 9 days post‐surgery and returned to normal after 28 days post‐surgery. In uninjured and injured nerve, prosaposin immunolocalized to the smooth muscle of epineurial and endoneurial vessels. These findings indicated that sciatic nerve secreted prosaposin after injury and that prosaposin is a naturally occurring injury‐repair protein which acts to prevent degeneration and to promote regeneration of peripheral nerves. GLIA 26:353–360, 1999.

The crystal structure of sphingosine-1-phosphate in complex with a Fab fragment reveals metal bridging of an antibody and its antigen

The pleiotropic signaling lipid sphingosine-1-phosphate (S1P) plays significant roles in angiogenesis, heart disease, and cancer. LT1009 (also known as sonepcizumab) is a humanized monoclonal antibody that binds S1P with high affinity and specificity. Because the antibody is currently in clinical trials, it is important to confirm by structural and biochemical analyses that it binds its target in a predictable manner. Therefore, we determined the structure of a complex between the LT1009 antibody Fab fragment and S1P refined to 1.90 Å resolution. The antibody employs unique and diverse strategies to recognize its antigen. Two metal ions bridge complementarity determining regions from the antibody light chain and S1P. The coordination geometry, inductively coupled plasma spectroscopy, surface plasmon resonance spectroscopy, and biochemical assays suggest that these are Ca2+. The amino alcohol head group of the sphingosine backbone is recognized through hydrogen bonding interactions from 1 aa side chain and polypeptide backbone atoms of the antibody light and heavy chains. The S1P hydrophobic tail is almost completely enclosed within a hydrophobic channel formed primarily by the heavy chain. Both treatment of the complex with metal chelators and mutation of amino acids in the light chain that coordinate the metal atoms or directly contact the polar head group abrogate binding, while mutations within the hydrophobic cavity also decrease S1P binding affinity. The structure suggests mechanistic details for recognition of a signaling lipid by a therapeutic antibody candidate. Moreover, this study provides direct structural evidence that antibodies are capable of using metals to bridge antigen:antibody complexes.

Saposins (sap) A and C activate the degradation of galactosylceramide in living cells

In loading tests using galactosylceramide which had been labelled with tritium in the ceramide moiety, living skin fibroblast lines derived from the original prosaposin‐deficient patients had a markedly reduced capacity to degrade galactosylceramide. The hydrolysis of galactosylceramide could be partially restored in these cells, up to about half the normal rate, by adding pure saposin A, pure saposin C, or a mixture of these saposins to the culture medium. By contrast, saposins B and D had little effect on galactosylceramide hydrolysis in the prosaposin‐deficient cells. Cells from β‐galactocerebrosidase‐deficient (Krabbe) patients had a relatively high residual galactosylceramide degradation, which was similar to the rate observed for prosaposin‐deficient cells in the presence of saposin A or C. An SV40‐transformed fibroblast line from the original saposin C‐deficient patient, where saposin A is not affected, showed normal degradation of galactosylceramide. The findings support the hypothesis, which was deduced originally from in vitro experiments, that saposins A and C are the in vivo activators of galactosylceramide degradation. Although the results with saposin C‐deficient fibroblasts suggest that the presence of only saposin A allows galactosylceramide breakdown to proceed at a normal rate in fibroblasts, it remains to be determined whether saposins A and C can substitute for each other with respect to their effects on galactosylceramide metabolism in the whole organism.

Amplification and overexpression of prosaposin in prostate cancer.

We identified prosaposin (PSAP) as a secreted protein expressed in androgen‐independent (AI) prostate cancer cells by cloning/sequencing, after probing a PC‐3 cDNA library expressed in the λTriplEx phagemid expression vector with a polyclonal rabbit antibody generated against pooled human seminal plasma. PSAP is a neurotrophic molecule; its deficiency or inactivation has proved to be lethal in man and mice, and in mice, it leads to abnormal development and atrophy of the prostate gland, despite normal testosterone levels. We used Southern hybridization, quantitative real‐time polymerase chain reaction, and/or single nucleotide polymorphism (SNP) array analysis, and we now report the genomic amplification of PSAP in the metastatic AI prostate cancer cell lines, PC‐3, DU‐145, MDA‐PCa 2b, M‐12, and NCI‐H660. In addition, by using SNP arrays and a set of 25 punch biopsy samples of human prostate cancer xenografts (LAPC3, LuCaP 23.1, 35, 49, 58, 73, 77, 81, 86.2, 92.1, 93, 96, 105, and 115), lymph nodes, and visceral‐organ metastases, we detected amplification of the PSAP locus (10q22.1) in LuCaP 58 and 96 xenografts and two lymph node metastases. In addition, AI metastatic prostate cancer cell lines C4‐2B and IA8‐ARCaP over‐expressed PSAP mRNA without evidence of genomic amplification. Taken together with prior data that demonstrated the growth‐, migration‐, and invasion‐promoting activities, the activation of multiple signal transduction pathways, and the antiapoptotic effect of PSAP (or one of its active domains, saposin C) in prostate cancer cells, our current observation of PSAP amplification or overexpression in prostate cancer suggests, for the first time, a role for this molecule in the process of carcinogenesis or cancer progression in the prostate.

Immunoreactivity of growth inhibitory factor in normal rat brain and after stab wounds — an immunocytochemical study using confocal laser scan microscope

The growth inhibitor factor (GIF) is a new member of the metallothionein family that is downregulated in Alzheimers disease brain. Using a confocal laser scan microscope with polyclonal and monoclonal antibodies to GIF, and monoclonal antibodies to glial fibrillary acidic protein (GFAP) and MAP-2, we demonstrated that GIF immunoreactivity was expressed primarily in astrocytes and much less in neurons. In astrocytes of normal rat brain GIF immunoreactivity was detected mainly in the cell bodies, while GFAP immunoreactivity was detected mainly in the processes. GIF immunoreactivity was more strongly expressed in reactive astrocytes. These findings were confirmed with both polyclonal and monoclonal antibodies. Following stab wounds, a number of GIF-positive reactive astrocytes were detected around the wounds at 3 days postoperation. After 7 days GIF immunoreactivity was detected in cell bodies and processes of reactive astrocytes. The number of GIF-positive astrocytes and the intensity of the immunoreactivity remained elevated over the control levels at least through 28 days. These immunocytochemical findings correlated well with changes in GIF protein and mRNA levels. Not only changes in GIF protein and mRNA levels but also intracellular localization of GIF in normal rat brain and after stab wounds in rat brain were different from those of GFAP. These results support the concept that GIF plays an important role in the processing of reconstruction after brain damage.

Colocalization and Complex Formation Between Prosaposin and Monosialoganglioside GM3 in Neural Cells

Abstract: Prosaposin, the precursor of saposins A, B, C, and D, was recently identified as a neurotrophic factor in vitro as well as in vivo. Its neurotrophic activity has been localized to a linear 12‐amino acid sequence located in the NH2‐terminal portion of the saposin C domain. In this study, we show the colocalization of prosaposin and ganglioside GM3 on NS20Y cell plasma membrane by scanning confocal microscopy. Also, TLC and western blot analyses showed that GM3 was specifically associated with prosaposin in immunoprecipitates; this binding was Ca2+‐independent and not disassociated during sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The association of prosaposin‐GM3 complexes on the cell surface appeared to be functionally important, as determined by differentiation assays. Neurite sprouting, induced by GM3, was inhibited by antibodies raised against a 22‐mer peptide, prosaptide 769, containing the neurotrophic sequence of prosaposin. In addition, pertussis toxin inhibited prosaptide‐induced neurite outgrowth, as well as prosaptide‐enhanced ganglioside concentrations in NS20Y cells, suggesting that prosaposin acted via a G protein‐mediated pathway, affecting both ganglioside content and neuronal differentiation. Our findings revealed a direct and right GM3‐prosaposin association on NS20Y plasma membranes. We suggest that ganglioside‐protein complexes are structural components of the prosaposin receptor involved in cell differentiation.

Prosaposin prevents programmed cell death of rat cerebellar granule neurons in culture

Prosaposin, the precursor of sphingolipid activator proteins (saposin A-D), has been reported to be a neurotrophic factor in vitro and in vivo. Prosaposin mRNA is transiently expressed at a high level in developing cerebellum during the period of granule cell proliferation and maturation, suggesting its significance during development of cerebellum. Here we investigated the neuroprotective effect of prosaposin on cerebellar granule neurons, exposing primary cerebellar granule cells to low K+ which induced programmed cell death. Prosaposin rescued mature cerebellar granule neurons in a bimodal manner. A similar neuroprotective effect was obtained using TX14(A), a 14-mer neurotrophic peptide derivative of prosaposin. An additive neuroprotective effect was observed between BDNF and TX14(A), but not between IGF-1 and TX14(A). Prosaposin rescued 60% of nifedipine sensitive cerebellar granule neurons as well as IGF-1, while BDNF did not. Furthermore, the neuroprotective action of prosaposin was inhibited by LY294002, a specific inhibitor of PI 3-kinase. These findings indicated that prosaposin had a trophic effect upon newborn cerebellar granule cells and that the neuroprotective action was similar to that of IGF-1 rather than BDNF. Prosaposin may play a role in cerebellar development during programmed cell death of cerebellar neurons.

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 recognize 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.

Molecular cloning of a full-length cDNA for human α-N-acetylgalactosaminidase (α-galactosidase B)

Abstract In the process of molecular cloning of cDNA for proteins associated with a purified human placental sialidase fraction, we discovered one of the proteins with apparent molecular weight of 46 kDa is in reality α-N-acetylgalactosaminidase. The full lengh cDNA, pcD-HS1204, codes for 358 amino acids with the first 17 residues representing a putative signal peptide. The predicted amino acid sequence shows striking homology with human α-galactosidase A and yeast α-galactosidase. The substrate specificities as well as the behavior of the 46 kDa protein on hydroxylapatite chromatography confirmed that the 46 kDa protein is in reality α-N-acetylgalactosaminidase.