Masaharu Kotani

ALK receptor tyrosine kinase promotes cell growth and neurite outgrowth.

Anaplastic lymphoma kinase (ALK) is a receptor-type protein tyrosine kinase that is expressed preferentially in neurons of the central and peripheral nervous systems at late embryonic stages. To elucidate the role of ALK in neurons, we developed an agonist monoclonal antibody (mAb) against the extracellular domain of ALK. Here we show that mAb16-39 elicits tyrosine phosphorylation of endogenously expressed ALK in human neuroblastoma (SK-N-SH) cells. Stimulation of these cells with mAb16-39 markedly induces the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), Shc, and c-Cbl and also their interaction with ALK and activation of ERK1/2. Furthermore, we show that continuous incubation with mAb16-39 induces the cell growth and neurite outgrowth of SK-N-SH cells. These responses are completely blocked by MEK inhibitor PD98059 but not by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin, indicating an essential role of the mitogen-activated protein kinase (MAP kinase) signaling cascade in ALK-mediated growth and differentiation of neurons.

Comparison of the effects of agalsidase alfa and agalsidase beta on cultured human Fabry fibroblasts and Fabry mice

AbstractWe compared two recombinant α-galactosidases developed for enzyme replacement therapy for Fabry disease, agalsidase alfa and agalsidase beta, as to specific α-galactosidase activity, stability in plasma, mannose 6-phosphate (M6P) residue content, and effects on cultured human Fabry fibroblasts and Fabry mice. The specific enzyme activities of agalsidase alfa and agalsidase beta were 1.70 and 3.24 mmol h−1 mg protein−1, respectively, and there was no difference in stability in plasma between them. The M6P content of agalsidase beta (3.6 mol/mol protein) was higher than that of agalsidase alfa (1.3 mol/mol protein). The administration of both enzymes resulted in marked increases in α-galactosidase activity in cultured human Fabry fibroblasts, and Fabry mouse kidneys, heart, spleen and liver. However, the increase in enzyme activity in cultured fibroblasts, kidneys, heart and spleen was higher when agalsidase beta was used. An immunocytochemical analysis revealed that the incorporated recombinant enzyme degraded the globotriaosyl ceramide accumulated in cultured Fabry fibroblasts in a dose-dependent manner, with the effect being maintained for at least 7 days. Repeated administration of agalsidase beta apparently decreased the number of accumulated lamellar inclusion bodies in renal tubular cells of Fabry mice.

Developmental changes of ganglioside expressions in postnatal rat cerebellar cortex.

We previously described the differential distribution of gangliosides in adult rat brain as detected by specific antibodies. We report here the distribution of gangliosides during the development of postnatal rat cerebellum by an immunofluorescence technique with mouse monoclonal antibodies (mAbs). Eleven mAbs that specifically recognize each ganglioside were used. Our study revealed that the expression of each ganglioside changed dramatically during the development. GD3 and O-Ac-GD3 were expressed intensely in the external granular layer at 1, 5, and 10 days, whereas GD2 was firstly detected in the internal granular layer at 5 days and GD1b WAS diffusely detected throughout all layers of the cerebellar cortex at early postnatal days. GD2 and GD1b were more intensely expressed in the granular layer at 20, 30, and 80 days, suggesting that premature granule cells expressed GD3 and its derivative, O-Ac-GD3, whereas mature granule cells express GD2 and GD1B intensely. On the other hand, GM1 was exclusively detected in the external granular layer and the molecular layer at 1 and 5 days. The staining sites spread gradually from these outer layers into the internal granular layer and the white matter after 10 days. The positive cells in the external granular layer and the molecular layer appeared to be Bergmann glial cells and their radially ascending cytoplasmic processes. The intensity of the staining in these specialized astroglial cells decreased gradually during postnatal days. In contrast, the expression of GQ1b was very faint at birth, but gradually increased during the development and was detected intensely in the internal granular layer, particularly in the cerebellar glomeruli in adulthood, suggesting that GQ1b expression may be associated with synapse-related structures. The developmental changes of the expression of other gangliosides were also recognized in the postnatal rat cerebellum. These results suggest that specific gangliosides may play an important role in regulating the early events responsible for the orderly formation of the cerebellar cortex.

Monoclonal antibody Rip specifically recognizes 2′,3′-cyclic nucleotide 3′-phosphodiesterase in oligodendrocytes

The antigen recognized with monoclonal antibody (mAb) Rip (Rip‐antigen) has been long used as a marker of oligodendrocytes and myelin sheaths. However, the identity of Rip‐antigen has yet to be elucidated. We herein identified the Rip‐antigen. No signal recognized by mAb‐Rip was detected by immunoblot analyses in the rat brain, cultured rat oligodendrocytes, or the oligodendrocyte cell line CG‐4. As this antibody worked very well on immunocytochemistry and immunohistochemistry, Rip‐antigen was immunopurified with mAb‐Rip from the differentiated CG‐4 cells. Eight strong‐intensity bands thus appeared on 5–20% SDS‐PAGE with SYPRO ruby fluorescence staining. To identify these molecules, each band extracted from the gel was analyzed by MALDI‐QIT/TOF mass spectrometry. We found an interesting molecule in the oligodendrocytes from an approximately 44‐kDa band as 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP). To test whether CNP was recognized by mAb‐Rip, double‐immunofluorescence staining was performed by using Alexa Fluor 488‐conjugated mAb‐Rip and Alexa Fluor 568‐conjugated mAb‐CNP in the rat cerebellum, mouse cerebellum, cultured rat oligodendrocytes, and CG‐4 cells. The Rip‐antigen was colocalized with CNP in these cells and tissues. To provide direct evidence that CNP was recognized by mAb‐Rip, rat Cnp1‐transfected HEK293T cells were used for double‐immunofluorescence staining with mAb‐Rip and mAb‐CNP. The Rip‐antigen was colocalized with CNP in rat Cnp1‐transfected HEK293T cells, but the antigen was not detected by mAb‐Rip and mAb‐CNP in mock‐transfected HEK293T cells. Overall, we have demonstrated that the antigen labeled with mAb‐Rip is CNP in the oligodendrocytes.

Distal Myopathy with Rimmed Vacuoles : Impaired O-Glycan Formation in Muscular Glycoproteins

Distal myopathy with rimmed vacuoles (DMRV), is an autosomal recessive disorder with early adult onset, displays distal dominant muscular involvement and is characterized by the presence of numerous rimmed vacuoles in the affected muscle fibers. The pathophysiology of DMRV has not been clarified yet, although the responsible gene was identified as that encoding UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase involved in the biosynthesis of sialic acids. To identify defective carbohydrate moieties of muscular glycoproteins from DMRV patients, frozen skeletal muscle sections from seven patients with DMRV, as well as normal and pathological controls, were treated with or without sialidase or N-glycosidase F followed by lectin staining and lectin blotting analysis. The sialic acid contents of the O-glycans in the skeletal muscle specimens from the DMRV patients were also measured. We found that Arachis hypogaea agglutinin (PNA) lectin reacted strongly with sarcolemmal glycoproteins in the DMRV patients but not with those in control subjects. alpha-Dystroglycan from the DMRV patients strongly associated with PNA lectin, although that from controls did not. The sialic acid level of the O-glycans in the DMRV muscular glycoproteins with molecular weights of 30 to 200 kd was reduced to 60 to 80% of the control level. The results show that impaired sialyl O-glycan formation in muscular glycoproteins, including alpha-dystroglycan, occurs in DMRV.

Structural and immunocytochemical studies on α-N-acetylgalactosaminidase deficiency (Schindler/Kanzaki disease)

Abstractα-N-Acetylgalactosaminidase (α-NAGA) deficiency (Schindler/Kanzaki disease) is a clinically and pathologically heterogeneous genetic disease with a wide spectrum including an early onset neuroaxonal dystrophy (Schindler disease) and late onset angiokeratoma corporis diffusum (Kanzaki disease). In α-NAGA deficiency, there are discrepancies between the genotype and phenotype, and also between urinary excretion products (sialyl glycoconjugates) and a theoretical accumulated material (Tn-antigen; Gal NAcα1-O-Ser/Thr) resulting from a defect in α-NAGA. As for the former issue, previously reported genetic, biochemical and pathological data raise the question whether or not E325K mutation found in Schindler disease patients really leads to the severe phenotype of α-NAGA deficiency. The latter issue leads to the question of whether α-NAGA deficiency is associated with the basic pathogenesis of this disease. To clarify the pathogenesis of this disease, we performed structural and immunocytochemical studies. The structure of human α-NAGA deduced on homology modeling is composed of two domains, domain I, including the active site, and domain II. R329W/Q, identified in patients with Kanzaki disease have been deduced to cause drastic changes at the interface between domains I and II. The structural change caused by E325K found in patients with Schindler disease is localized on the N-terminal side of the tenth β-strand in domain II and is smaller than those caused by R329W/Q. Immunocytochemical analysis revealed that the main lysosomal accumulated material in cultured fibroblasts from patients with Kanzaki disease is Tn-antigen. These data suggest that a prototype of α-NAGA deficiency in Kanzaki disease and factors other than the defect of α-NAGA may contribute to severe neurological disorders, and Kanzaki disease is thought to be caused by a single enzyme deficiency.

Molecular basis for oviductin‐mediated processing from gp43 to gp41, the predominant glycoproteins of Xenopus egg envelopes

Acquisition of fertilizability in Xenopus coelomic eggs is correlated with the conversion from coelomic to vitelline envelope during passage of the eggs through the pars recta portion of oviduct. The conversion includes processing of a major envelope constituent gp43 of coelomic envelopes to gp41 of vitelline envelopes by a trypsin-type protease, oviductin, which is secreted from the pars recta. Our recent sequencing analyses [Kubo et al., (1997): Dev Growth Diff 39:405-411] strongly suggested that the N-terminal portion of gp41 is exposed as a result of oviductin digestion. In this study, a monoclonal antibody specific to the predicted N-terminus of gp41 was raised by immunizing mice with a synthetic N-terminal hexapeptide (QLPVSP) coupled to keyhole limpet hemocyanin. The antibody specifically reacted to gp41, but not to gp43, indicating that Gln62 is exposed as the N-terminal amino acid of gp41 by oviductin-mediated cleavage of gp43 at Arg61 in GSR61. The C-terminal sequencing of gp43 and gp41 indicated that Arg373 in GSR373 as the C-terminus of gp41 is generated by cleavage of three amino acid (WNQ) residues from the C-terminus of gp43. The resulting polypeptide moiety of gp41 has a molecular mass of 33900 Da with 312 amino acid residues. We propose that oviductin possessing the substrate specificity of GSR simultaneously digests gp43 at Arg residues in GSR61 and GSR373 to generate the N- and C-terminus of gp41, respectively.

Generation of a monoclonal antibody specific for a new class of minor ganglioside antigens, GQ1bα and GT1aα : its binding to dorsal and lateral horn of human thoracic cord

Abstract We have established a monoclonal antibody, GGR41, specific for a new class of minor gangliosides, such as GQ1bα and GT1aα, by immunizing mice with a GQ1b-rich ganglioside fraction extracted from bovine brain. Each of those minor gangliosides has been reported to be one of the cholinergic-specific gangliosides (Chol-1). Careful examination of binding specificity of the antibody by both an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatograms showed that the antibody recognizes three sialyl residues separately attaching to the gangliotetraosyl backbone structure. Immunohistochemical analysis revealed that GGR41 immunostained lamina I and III of dorsal horn and lateral horn of human thoracic cord but motor neurons were not immunostained. Except for negative staining of motor neurons, this distribution is similar to the distribution pattern of staining as reported in rats and humans using a polyclonal antibody against Chol-1. Thus, the antibody obtained in this study should be a useful reagent to study the function of a unique new class of the minor gangliosides.

Identification of neuronal cell lineage-specific molecules in the neuronal differentiation of P19 EC cells and mouse central nervous system

P19 embryonic carcinoma (EC) cells are one of the simplest systems for analyzing the neuronal differentiation. To identify the membrane‐associated molecules on the neuronal cells involved in the early neuronal differentiation in mice, we generated two monoclonal antibodies, SKY‐1 and SKY‐2, by immunizing rats with a membrane fraction of the neuronally committed P19 EC cells as an antigen. SKY‐1 and SKY‐2 recognized the carbohydrate moiety of a 90 kDa protein (RANDAM‐1) and the polypeptide core of a 40 kDa protein (RANDAM‐2), respectively. In the P19 EC cells, the expression of RANDAM‐1 was colocalized to a part of Nestin‐positive cells, whereas that of RANDAM‐2 was observed in most Nestin‐positive cells as well as β‐III‐tubulin positive neurons. In the embryonic and adult brain of mice, RANDAM‐1 was expressed at embryonic day 8.5 (E8.5), and the localization of antigen was restricted on the neuroepithelium and choroid plexus. The RANDAM‐2 expression commenced at E6.0, and the antigen was distributed not only on the neuroepithelium of embryonic brain but on the neurons of adult brain. Collectively, it was concluded that RANDAM‐1 is a stage specific antigen to express on the neural stem cells, and RANDAM‐2 is constitutively expressed on both the neural stem cells and differentiated neuronal cells in mouse central nervous system (CNS).

Corrective effect on Fabry mice of yeast recombinant human α-galactosidase with N -linked sugar chains suitable for lysosomal delivery

AbstractWe have previously reported the production of a recombinant α-galactosidase with engineered N-linked sugar chains facilitating uptake and transport to lysosomes in a Saccharomyces cerevisiae mutant. In this study, we improved the purification procedure, allowing us to obtain a large amount of highly purified enzyme protein with mannose-6-phosphate residues at the non-reducing ends of sugar chains. The products were incorporated into cultured fibroblasts derived from a patient with Fabry disease via mannose-6-phosphate receptors. The ceramide trihexoside (CTH) accumulated in lysosomes was cleaved dose-dependently, and the disappearance of deposited CTH was maintained for at least 7 days after administration. We next examined the effect of the recombinant α-galactosidase on Fabry mice. Repeated intravascular administration of the enzyme led to successful degradation of CTH accumulated in the liver, kidneys, heart, and spleen. However, cleavage of the accumulated CTH in the dorsal root ganglia was insufficient. As the culture of yeast cells is easy and economical, and does not require fetal calf serum, the recombinant α-galactosidase produced in yeast cells is highly promising as an enzyme source for enzyme replacement therapy in Fabry disease.